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BIOTECHNOLOGY (227 journals)                  1 2 | Last

Showing 1 - 200 of 227 Journals sorted alphabetically
3 Biotech     Open Access   (Followers: 7)
Advances in Bioscience and Biotechnology     Open Access   (Followers: 14)
Advances in Genetic Engineering & Biotechnology     Hybrid Journal   (Followers: 7)
African Journal of Biotechnology     Open Access   (Followers: 6)
Algal Research     Partially Free   (Followers: 9)
American Journal of Biochemistry and Biotechnology     Open Access   (Followers: 69)
American Journal of Bioinformatics Research     Open Access   (Followers: 8)
American Journal of Polymer Science     Open Access   (Followers: 30)
Animal Biotechnology     Hybrid Journal   (Followers: 9)
Annales des Sciences Agronomiques     Full-text available via subscription  
Applied Biochemistry and Biotechnology     Hybrid Journal   (Followers: 42)
Applied Bioenergy     Open Access  
Applied Biosafety     Hybrid Journal  
Applied Microbiology and Biotechnology     Hybrid Journal   (Followers: 62)
Applied Mycology and Biotechnology     Full-text available via subscription   (Followers: 5)
Arthroplasty Today     Open Access   (Followers: 1)
Artificial Cells, Nanomedicine and Biotechnology     Hybrid Journal   (Followers: 2)
Asia Pacific Biotech News     Hybrid Journal   (Followers: 2)
Asian Journal of Biotechnology     Open Access   (Followers: 8)
Asian Pacific Journal of Tropical Biomedicine     Open Access   (Followers: 2)
Australasian Biotechnology     Full-text available via subscription   (Followers: 1)
Banat's Journal of Biotechnology     Open Access  
BBR : Biochemistry and Biotechnology Reports     Open Access   (Followers: 4)
Bio-Algorithms and Med-Systems     Hybrid Journal   (Followers: 1)
Bio-Research     Full-text available via subscription   (Followers: 2)
Bioactive Materials     Open Access   (Followers: 1)
Biocatalysis and Agricultural Biotechnology     Hybrid Journal   (Followers: 4)
Biocybernetics and Biological Engineering     Full-text available via subscription   (Followers: 5)
Bioethics UPdate     Hybrid Journal  
Biofuels     Hybrid Journal   (Followers: 11)
Biofuels Engineering     Open Access   (Followers: 1)
Biological & Pharmaceutical Bulletin     Full-text available via subscription   (Followers: 5)
Biological Cybernetics     Hybrid Journal   (Followers: 10)
Biomarkers and Genomic Medicine     Open Access   (Followers: 5)
Biomarkers in Drug Development     Partially Free   (Followers: 1)
Biomaterials Research     Open Access   (Followers: 4)
BioMed Research International     Open Access   (Followers: 6)
Biomédica     Open Access  
Biomedical Engineering Research     Open Access   (Followers: 7)
Biomedical glasses     Open Access  
Biomedical Reports     Full-text available via subscription  
BioMedicine     Open Access  
Bioprinting     Hybrid Journal  
Bioresource Technology Reports     Hybrid Journal  
Bioscience, Biotechnology, and Biochemistry     Hybrid Journal   (Followers: 22)
Biosimilars     Open Access   (Followers: 1)
Biosurface and Biotribology     Open Access  
Biotechnic and Histochemistry     Hybrid Journal   (Followers: 2)
BioTechniques : The International Journal of Life Science Methods     Full-text available via subscription   (Followers: 28)
Biotechnologia Acta     Open Access   (Followers: 1)
Biotechnologie, Agronomie, Société et Environnement     Open Access   (Followers: 2)
Biotechnology     Open Access   (Followers: 6)
Biotechnology & Biotechnological Equipment     Open Access   (Followers: 5)
Biotechnology Advances     Hybrid Journal   (Followers: 33)
Biotechnology and Applied Biochemistry     Hybrid Journal   (Followers: 44)
Biotechnology and Bioengineering     Hybrid Journal   (Followers: 160)
Biotechnology and Bioprocess Engineering     Hybrid Journal   (Followers: 6)
Biotechnology and Genetic Engineering Reviews     Hybrid Journal   (Followers: 14)
Biotechnology and Health Sciences     Open Access   (Followers: 1)
Biotechnology and Molecular Biology Reviews     Open Access   (Followers: 1)
Biotechnology Annual Review     Full-text available via subscription   (Followers: 7)
Biotechnology for Biofuels     Open Access   (Followers: 10)
Biotechnology Frontier     Open Access   (Followers: 2)
Biotechnology Journal     Hybrid Journal   (Followers: 15)
Biotechnology Law Report     Hybrid Journal   (Followers: 4)
Biotechnology Letters     Hybrid Journal   (Followers: 33)
Biotechnology Progress     Hybrid Journal   (Followers: 39)
Biotechnology Reports     Open Access  
Biotechnology Research International     Open Access   (Followers: 2)
Biotechnology Techniques     Hybrid Journal   (Followers: 10)
Biotecnología Aplicada     Open Access  
Biotribology     Hybrid Journal  
BMC Biotechnology     Open Access   (Followers: 15)
Chinese Journal of Agricultural Biotechnology     Full-text available via subscription   (Followers: 3)
Communications in Mathematical Biology and Neuroscience     Open Access  
Computational and Structural Biotechnology Journal     Open Access   (Followers: 2)
Computer Methods and Programs in Biomedicine     Hybrid Journal   (Followers: 8)
Contributions to Tobacco Research     Open Access   (Followers: 3)
Copernican Letters     Open Access   (Followers: 1)
Critical Reviews in Biotechnology     Hybrid Journal   (Followers: 20)
Crop Breeding and Applied Biotechnology     Open Access   (Followers: 4)
Current Bionanotechnology     Hybrid Journal  
Current Biotechnology     Hybrid Journal   (Followers: 3)
Current Opinion in Biomedical Engineering     Hybrid Journal   (Followers: 1)
Current Opinion in Biotechnology     Hybrid Journal   (Followers: 55)
Current Pharmaceutical Biotechnology     Hybrid Journal   (Followers: 9)
Current Research in Bioinformatics     Open Access   (Followers: 14)
Current trends in Biotechnology and Pharmacy     Open Access   (Followers: 9)
EBioMedicine     Open Access  
Electronic Journal of Biotechnology     Open Access   (Followers: 1)
Entomologia Generalis     Full-text available via subscription  
Environmental Science : Processes & Impacts     Full-text available via subscription   (Followers: 4)
Experimental Biology and Medicine     Hybrid Journal   (Followers: 3)
Folia Medica Indonesiana     Open Access  
Food Bioscience     Hybrid Journal  
Food Biotechnology     Hybrid Journal   (Followers: 12)
Food Science and Biotechnology     Hybrid Journal   (Followers: 9)
Frontiers in Bioengineering and Biotechnology     Open Access   (Followers: 6)
Frontiers in Systems Biology     Open Access   (Followers: 2)
Fungal Biology and Biotechnology     Open Access   (Followers: 1)
GM Crops and Food: Biotechnology in Agriculture and the Food Chain     Full-text available via subscription   (Followers: 1)
GSTF Journal of BioSciences     Open Access  
HAYATI Journal of Biosciences     Open Access  
Horticulture, Environment, and Biotechnology     Hybrid Journal   (Followers: 11)
IEEE Transactions on Molecular, Biological and Multi-Scale Communications     Hybrid Journal   (Followers: 1)
IET Nanobiotechnology     Hybrid Journal   (Followers: 2)
IIOAB Letters     Open Access  
IN VIVO     Full-text available via subscription   (Followers: 4)
Indian Journal of Biotechnology (IJBT)     Open Access   (Followers: 2)
Indonesia Journal of Biomedical Science     Open Access   (Followers: 1)
Indonesian Journal of Biotechnology     Open Access   (Followers: 1)
Industrial Biotechnology     Hybrid Journal   (Followers: 18)
International Biomechanics     Open Access  
International Journal of Bioinformatics Research and Applications     Hybrid Journal   (Followers: 15)
International Journal of Biomechatronics and Biomedical Robotics     Hybrid Journal   (Followers: 4)
International Journal of Biomedical Research     Open Access   (Followers: 2)
International Journal of Biotechnology     Hybrid Journal   (Followers: 5)
International Journal of Biotechnology and Molecular Biology Research     Open Access   (Followers: 2)
International Journal of Biotechnology for Wellness Industries     Partially Free   (Followers: 1)
International Journal of Environment, Agriculture and Biotechnology     Open Access   (Followers: 5)
International Journal of Functional Informatics and Personalised Medicine     Hybrid Journal   (Followers: 4)
International Journal of Medicine and Biomedical Research     Open Access   (Followers: 1)
International Journal of Nanotechnology and Molecular Computation     Full-text available via subscription   (Followers: 3)
International Journal of Radiation Biology     Hybrid Journal   (Followers: 4)
Iranian Journal of Biotechnology     Open Access  
ISABB Journal of Biotechnology and Bioinformatics     Open Access  
Italian Journal of Food Science     Open Access   (Followers: 1)
Journal of Biometrics & Biostatistics     Open Access   (Followers: 3)
Journal of Bioterrorism & Biodefense     Open Access   (Followers: 6)
Journal of Petroleum & Environmental Biotechnology     Open Access   (Followers: 2)
Journal of Advanced Therapies and Medical Innovation Sciences     Open Access  
Journal of Advances in Biotechnology     Open Access   (Followers: 5)
Journal Of Agrobiotechnology     Open Access  
Journal of Analytical & Bioanalytical Techniques     Open Access   (Followers: 7)
Journal of Animal Science and Biotechnology     Open Access   (Followers: 6)
Journal of Applied Biomedicine     Open Access   (Followers: 3)
Journal of Applied Biotechnology     Open Access   (Followers: 2)
Journal of Applied Biotechnology Reports     Open Access   (Followers: 2)
Journal of Applied Mathematics & Bioinformatics     Open Access   (Followers: 5)
Journal of Biologically Active Products from Nature     Hybrid Journal   (Followers: 1)
Journal of Biomaterials and Nanobiotechnology     Open Access   (Followers: 6)
Journal of Biomedical Photonics & Engineering     Open Access  
Journal of Biomedical Practitioners     Open Access  
Journal of Bioprocess Engineering and Biorefinery     Full-text available via subscription  
Journal of Bioprocessing & Biotechniques     Open Access  
Journal of Biosecurity, Biosafety and Biodefense Law     Hybrid Journal   (Followers: 3)
Journal of Biotechnology     Hybrid Journal   (Followers: 68)
Journal of Chemical and Biological Interfaces     Full-text available via subscription   (Followers: 1)
Journal of Chemical Technology & Biotechnology     Hybrid Journal   (Followers: 10)
Journal of Chitin and Chitosan Science     Full-text available via subscription  
Journal of Colloid Science and Biotechnology     Full-text available via subscription  
Journal of Commercial Biotechnology     Full-text available via subscription   (Followers: 6)
Journal of Crop Science and Biotechnology     Hybrid Journal   (Followers: 7)
Journal of Essential Oil Research     Hybrid Journal   (Followers: 3)
Journal of Experimental Biology     Full-text available via subscription   (Followers: 25)
Journal of Genetic Engineering and Biotechnology     Open Access   (Followers: 5)
Journal of Ginseng Research     Open Access  
Journal of Industrial Microbiology and Biotechnology     Hybrid Journal   (Followers: 16)
Journal of Integrative Bioinformatics     Open Access  
Journal of International Biotechnology Law     Hybrid Journal   (Followers: 3)
Journal of Medical Imaging and Health Informatics     Full-text available via subscription  
Journal of Molecular Microbiology and Biotechnology     Full-text available via subscription   (Followers: 14)
Journal of Nano Education     Full-text available via subscription  
Journal of Nanobiotechnology     Open Access   (Followers: 4)
Journal of Nanofluids     Full-text available via subscription   (Followers: 2)
Journal of Organic and Biomolecular Simulations     Open Access  
Journal of Plant Biochemistry and Biotechnology     Hybrid Journal   (Followers: 6)
Journal of Science and Applications : Biomedicine     Open Access  
Journal of the Mechanical Behavior of Biomedical Materials     Hybrid Journal   (Followers: 11)
Journal of Trace Elements in Medicine and Biology     Hybrid Journal   (Followers: 1)
Journal of Tropical Microbiology and Biotechnology     Full-text available via subscription  
Journal of Yeast and Fungal Research     Open Access   (Followers: 1)
Marine Biotechnology     Hybrid Journal   (Followers: 5)
Messenger     Full-text available via subscription  
Metabolic Engineering Communications     Open Access   (Followers: 4)
Metalloproteinases In Medicine     Open Access  
Microalgae Biotechnology     Open Access   (Followers: 2)
Microbial Biotechnology     Open Access   (Followers: 9)
MicroMedicine     Open Access   (Followers: 3)
Molecular and Cellular Biomedical Sciences     Open Access  
Molecular Biotechnology     Hybrid Journal   (Followers: 16)
Molecular Genetics and Metabolism Reports     Open Access   (Followers: 3)
Nanobiomedicine     Open Access  
Nanobiotechnology     Hybrid Journal   (Followers: 3)
Nanomaterials and Nanotechnology     Open Access  
Nanomaterials and Tissue Regeneration     Open Access  
Nanomedicine and Nanobiology     Full-text available via subscription  
Nanomedicine Research Journal     Open Access  
Nanotechnology Reviews     Hybrid Journal   (Followers: 5)
Nature Biotechnology     Full-text available via subscription   (Followers: 521)
Network Modeling and Analysis in Health Informatics and Bioinformatics     Hybrid Journal   (Followers: 3)
New Biotechnology     Hybrid Journal   (Followers: 4)
Nigerian Journal of Biotechnology     Open Access  
Nova Biotechnologica et Chimica     Open Access  
NPG Asia Materials     Open Access  
npj Biofilms and Microbiomes     Open Access  
OA Biotechnology     Open Access  
Plant Biotechnology Journal     Open Access   (Followers: 10)
Plant Biotechnology Reports     Hybrid Journal   (Followers: 4)
Preparative Biochemistry and Biotechnology     Hybrid Journal   (Followers: 4)

        1 2 | Last

Journal Cover Metabolic Engineering Communications
  [SJR: 1.215]   [H-I: 3]   [4 followers]  Follow
  This is an Open Access Journal Open Access journal
   ISSN (Print) 2214-0301
   Published by Elsevier Homepage  [3177 journals]
  • Engineering Escherichia coli for the production of terpene mixture
           enriched in caryophyllene and caryophyllene alcohol as potential aviation
           fuel compounds

    • Authors: Weihua Wu; Fang Liu; Ryan W. Davis
      Pages: 13 - 21
      Abstract: Publication date: June 2018
      Source:Metabolic Engineering Communications, Volume 6
      Author(s): Weihua Wu, Fang Liu, Ryan W. Davis
      Recent studies have revealed that caryophyllene and its stereoisomers not only exhibit multiple biological activities but also have desired properties as renewable candidates for ground transportation and jet fuel applications. This study presents the first significant production of caryophyllene and caryolan-1-ol by an engineered E. coli with heterologous expression of mevalonate pathway genes with a caryophyllene synthase and a caryolan-1-ol synthase. By optimizing metabolic flux and fermentation parameters, the engineered strains yielded 449mg/L of total terpene, including 406mg/L sesquiterpene with 100mg/L caryophyllene and 10mg/L caryolan-1-ol. Furthermore, a marine microalgae hydrolysate was used as the sole carbon source for the production of caryophyllene and other terpene compounds. Under the optimal fermentation conditions, 360mg/L of total terpene, 322mg/L of sesquiterpene, and 75mg/L caryophyllene were obtained from the pretreated algae hydrolysates. The highest yields achieved on the biomass basis were 48mg total terpene/g algae and 10mg caryophyllene/g algae and the caryophyllene yield is approximately ten times higher than that from plant tissues by solvent extraction. The study provides a sustainable alternative for production of caryophyllene and its alcohol from microalgae biomass as potential candidates for next generation aviation fuels.

      PubDate: 2018-01-10T11:49:54Z
      DOI: 10.1016/j.meteno.2018.01.001
      Issue No: Vol. 6 (2018)
  • Metabolic engineering of Saccharomyces cerevisiae for overproduction of

    • Authors: Raphael Ferreira; Paulo Gonçalves Teixeira; Michael Gossing; Florian David; Verena Siewers; Jens Nielsen
      Pages: 22 - 27
      Abstract: Publication date: June 2018
      Source:Metabolic Engineering Communications, Volume 6
      Author(s): Raphael Ferreira, Paulo Gonçalves Teixeira, Michael Gossing, Florian David, Verena Siewers, Jens Nielsen
      Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1 S659A/S1157A (ACC1**), as well as the last two steps of TAG formation: phosphatidic phosphatase (PAH1) and diacylglycerol acyltransferase (DGA1), ultimately leading to 129 mg∙gCDW−1 of TAGs. Disruption of TAG lipase genes TGL3, TGL4, TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW−1. Further disruption of the beta-oxidation by deletion of POX1, as well as glycerol-3-phosphate utilization through deletion of GUT2, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW−1. The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.

      PubDate: 2018-02-06T09:54:26Z
      DOI: 10.1016/j.meteno.2018.01.002
      Issue No: Vol. 6 (2018)
  • Biocatalytic Production of Adipic Acid from Glucose Using Engineered
           Saccharomyces cerevisiae

    • Authors: Kaushik Raj; Siavash Partow; Kevin Correia; Anna N. Khusnutdinova; Alexander F. Yakunin; Radhakrishnan Mahadevan
      Abstract: Publication date: Available online 3 February 2018
      Source:Metabolic Engineering Communications
      Author(s): Kaushik Raj, Siavash Partow, Kevin Correia, Anna N. Khusnutdinova, Alexander F. Yakunin, Radhakrishnan Mahadevan
      Adipic acid is an important industrial chemical used in the synthesis of nylon-6,6. The commercial synthesis of adipic acid uses petroleum-derived benzene and releases significant quantities of greenhouse gases. Biocatalytic production of adipic acid from renewable feedstocks could potentially reduce the environmental damage and eliminate the need for fossil fuel precursors. Recently, we have demonstrated the first enzymatic hydrogenation of muconic acid to adipic acid using microbial enoate reductases (ERs) - complex iron-sulfur and flavin containing enzymes. In this work, we successfully expressed the Bacillus coagulans ER in a Saccharomyces cerevisiae strain producing muconic acid and developed a three-stage fermentation process enabling the synthesis of adipic acid from glucose. The ability to express active ERs and significant acid tolerance of S. cerevisiae highlight the applicability of the developed yeast strain for the biocatalytic production of adipic acid from renewable feedstocks.

      PubDate: 2018-02-06T09:54:26Z
      DOI: 10.1016/j.meteno.2018.02.001
  • A comprehensive evaluation of constraining amino acid biosynthesis in
           compartmented models for metabolic flux analysis

    • Authors: Mathias Lehnen; Birgitta E. Ebert; Lars M. Blank
      Pages: 34 - 44
      Abstract: Publication date: December 2017
      Source:Metabolic Engineering Communications, Volume 5
      Author(s): Mathias Lehnen, Birgitta E. Ebert, Lars M. Blank
      Recent advances in the availability and applicability of genetic tools for non-conventional yeasts have raised high hopes regarding the industrial applications of such yeasts; however, quantitative physiological data on these yeasts, including intracellular flux distributions, are scarce and have rarely aided in the development of novel yeast applications. The compartmentation of eukaryotic cells adds to model complexity. Model constraints are ideally based on biochemical evidence, which is rarely available for non-conventional yeast and eukaryotic cells. A small-scale model for 13C-based metabolic flux analysis of central yeast carbon metabolism was developed that is universally valid and does not depend on localization information regarding amino acid anabolism. The variable compartmental origin of traced metabolites is a feature that allows application of the model to yeasts with uncertain genomic and transcriptional backgrounds. The presented test case includes the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha. Highly similar flux solutions were computed using either a model with undefined pathway localization or a model with constraints based on curated (S. cerevisiae) or computationally predicted (H. polymorpha) localization information, while false solutions were found with incorrect localization constraints. These results indicate a potentially adverse effect of universally assuming Saccharomyces-like constraints on amino acid biosynthesis for non-conventional yeasts and verify the validity of neglecting compartmentation constraints using a small-scale metabolic model. The model was specifically designed to investigate the intracellular metabolism of wild-type yeasts under various growth conditions but is also expected to be useful for computing fluxes of other eukaryotic cells.

      PubDate: 2017-07-19T07:51:10Z
      DOI: 10.1016/j.meteno.2017.07.001
      Issue No: Vol. 5 (2017)
  • Isobutanol production in Synechocystis PCC 6803 using heterologous and
           endogenous alcohol dehydrogenases

    • Authors: Rui Miao; Xufeng Liu; Elias Englund; Pia Lindberg; Peter Lindblad
      Pages: 45 - 53
      Abstract: Publication date: Available online 29 July 2017
      Source:Metabolic Engineering Communications
      Author(s): Rui Miao, Xufeng Liu, Elias Englund, Pia Lindberg, Peter Lindblad
      Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3mgL−1 OD750 −1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8mgL−1 isobutanol was produced after 24hours when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

      PubDate: 2017-08-04T09:57:16Z
      DOI: 10.1016/j.meteno.2017.07.003
      Issue No: Vol. 5 (2017)
  • Genome sequence and analysis of Escherichia coli production strain LS5218

    • Authors: Jacqueline M. Rand; Gina C. Gordon; Christopher R. Mehrer; Brian F. Pfleger
      Pages: 78 - 83
      Abstract: Publication date: December 2017
      Source:Metabolic Engineering Communications, Volume 5
      Author(s): Jacqueline M. Rand, Gina C. Gordon, Christopher R. Mehrer, Brian F. Pfleger
      Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts.

      PubDate: 2017-11-27T08:51:57Z
      DOI: 10.1016/j.meteno.2017.10.001
      Issue No: Vol. 5 (2017)
  • Metabolic engineering of Ustilago trichophora TZ1 for improved malic acid

    • Authors: Thiemo Zambanini; Hamed Hosseinpour Tehrani; Elena Geiser; Christiane K. Sonntag; Joerg M. Buescher; Guido Meurer; Nick Wierckx; Lars M. Blank
      Pages: 12 - 21
      Abstract: Publication date: June 2017
      Source:Metabolic Engineering Communications, Volume 4
      Author(s): Thiemo Zambanini, Hamed Hosseinpour Tehrani, Elena Geiser, Christiane K. Sonntag, Joerg M. Buescher, Guido Meurer, Nick Wierckx, Lars M. Blank
      Ustilago trichophora RK089 has been found recently as a good natural malic acid producer from glycerol. This strain has previously undergone adaptive laboratory evolution for enhanced substrate uptake rate resulting in the strain U. trichophora TZ1. Medium optimization and investigation of process parameters enabled titers and rates that are able to compete with those of organisms overexpressing major parts of the underlying metabolic pathways. Metabolic engineering can likely further increase the efficiency of malate production by this organism, provided that basic genetic tools and methods can be established for this rarely used and relatively obscure species. Here we investigate and adapt existing molecular tools from U. maydis for use in U. trichophora. Selection markers from U. maydis that confer carboxin, hygromycin, nourseothricin, and phleomycin resistance are applicable in U. trichophora. A plasmid was constructed containing the ip-locus of U. trichophora RK089, resulting in site-specific integration into the genome. Using this plasmid, overexpression of pyruvate carboxylase, two malate dehydrogenases (mdh1, mdh2), and two malate transporters (ssu1, ssu2) was possible in U. trichophora TZ1 under control of the strong P etef promoter. Overexpression of mdh1, mdh2, ssu1, and ssu2 increased the product (malate) to substrate (glycerol) yield by up to 54% in shake flasks reaching a titer of up to 120gL−1. In bioreactor cultivations of U. trichophora TZ1 P etef ssu2 and U. trichophora TZ1 P etef mdh2 a drastically lowered biomass formation and glycerol uptake rate resulted in 29% (Ssu1) and 38% (Mdh2) higher specific production rates and 38% (Ssu1) and 46% (Mdh2) increased yields compared to the reference strain U. trichophora TZ1. Investigation of the product spectrum resulted in an 87% closed carbon balance with 134gL−1 malate and biomass (73gL−1), succinate (20gL−1), CO2 (17gL−1), and α-ketoglutarate (8gL−1) as main by-products. These results open up a wide range of possibilities for further optimization, especially combinatorial metabolic engineering to increase the flux from pyruvate to malic acid and to reduce by-product formation.

      PubDate: 2017-01-22T21:58:04Z
      DOI: 10.1016/j.meteno.2017.01.002
      Issue No: Vol. 4 (2017)
  • Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased

    • Authors: Feiyan Liang; Peter Lindblad
      Pages: 29 - 36
      Abstract: Publication date: Available online 20 February 2017
      Source:Metabolic Engineering Communications
      Author(s): Feiyan Liang, Peter Lindblad
      The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium Synechocystis PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and in vitro RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the in vitro RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium Synechocystis PCC 6803 under certain growth conditions.

      PubDate: 2017-02-23T21:19:34Z
      DOI: 10.1016/j.meteno.2017.02.002
      Issue No: Vol. 4 (2017)
  • Metabolic engineering of Escherichia coli for the biosynthesis of

    • Authors: Jingwei Zhang; Emily Kao; George Wang; Edward E.K. Baidoo; Matthew Chen; Jay. D. Keasling
      Pages: 1 - 7
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Jingwei Zhang, Emily Kao, George Wang, Edward E.K. Baidoo, Matthew Chen, Jay. D. Keasling
      2-Pyrrolidone is a valuable bulk chemical with myriad applications as a solvent, polymer precursor and active pharmaceutical intermediate. A novel 2-pyrrolidone synthase, ORF27, from Streptomyces aizunensis was identified to catalyze the ring closing dehydration of γ-aminobutyrate. ORF27's tendency to aggregate was resolved by expression at low temperature and fusion to the maltose binding protein (MBP). Recombinant Escherichia coli was metabolically engineered for the production of 2-pyrrolidone from glutamate by expressing both the genes encoding GadB, a glutamate decarboxylase, and ORF27. Incorporation of a GadB mutant lacking H465 and T466, GadB_ΔHT, improved the efficiency of one-pot 2-pyrrolidone biosynthesis in vivo. When the recombinant E. coli strain expressing the E. coli GadB_ΔHT mutant and the ORF27-MBP fusion was cultured in ZYM-5052 medium containing 9g/L of l-glutamate, 7.7g/L of l-glutamate was converted to 1.1g/L of 2-pyrrolidone within 31h, achieving 25% molar yield from the consumed substrate.
      Graphical abstract image

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2015.11.001
      Issue No: Vol. 3 (2017)
  • Liposomes modified with cardiolipin can act as a platform to regulate the
           potential flux of NADP+-dependent isocitrate dehydrogenase

    • Authors: Keishi Suga; Akari Hamasaki; Junpei Chinzaka; Hiroshi Umakoshi
      Pages: 8 - 14
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Keishi Suga, Akari Hamasaki, Junpei Chinzaka, Hiroshi Umakoshi
      Cardiolipin (CL) is a phospholipid found in the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM) in animal cells. Isocitrate dehydrogenase (ICDH) is an important catalytic enzyme that is localized at the cytosol and mitochondria; the metabolic pathway catalyzed by ICDH differs between the OMM and IMM. To estimate the possible role of lipid membrane in the enzymatic activity of NADP+-dependent ICDH, CL-modified liposomes were prepared using CL/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol (Ch), and their characteristics were analyzed based on the fluorescent probe method. The relative enzymatic activity of ICDH decreased in the presence of CL/DPPC/Ch=(30/50/20) liposome, whereas activity increased in the presence of CL/DPPC/Ch=(5/75/20) liposome. NADP+ had the greatest substrate affinity and was dominant in the regulation of ICDH activity. Analysis of membrane properties indicated that membranes in CL-modified liposomes were dehydrated by ICDH binding. Using circular dichroism analysis, CL/DPPC/Ch=(30/50/20) liposome induced a conformational change in ICDH, indicating that CL-rich membrane domains could inhibit ICDH activity. These results suggest that lipid membranes, including CL molecules, could act as a platform to regulate ICDH-related metabolic pathways such as the tricarboxylic acid cycle and lipid synthesis.
      Graphical abstract image

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2015.11.002
      Issue No: Vol. 3 (2017)
  • Hyaluronan production and molecular weight is enhanced in
           pathway-engineered strains of lactate dehydrogenase-deficient Lactococcus

    • Authors: Mandeep Kaur; Guhan Jayaraman
      Pages: 15 - 23
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Mandeep Kaur, Guhan Jayaraman
      The potential advantages of hyaluronic acid (HA) production by metabolically-engineered Lactococcus lactis is constrained by the lower molecular weight and yield of HA obtained in these strains, compared to natural producers. Earlier studies have correlated lower HA yield with excessive lactate production in L. lactis cultures (Chauhan et al., 2014). In the present study, a three-fold increase was observed in the amount as well as molecular weight of HA produced by recombinant ldh-mutant L. lactis strains. The diversion from lactate production in the ldh-mutant strains resulted in excess ethanol and acetoin production and higher NAD+/NADH ratio in these cultures. The initial NAD+/NADH ratio showed a positive correlation with HA molecular weight as well as with the HA-precursor ratio (UDP-GlcUA/UDP-GlcNAc). The influence of NAD+/NADH ratio on regulation of the concerned metabolic pathways was assessed by transcriptional analysis of key genes having putative binding sites of the NADH-binding transcriptional factor, Rex.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.01.003
      Issue No: Vol. 3 (2017)
  • Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

    • Authors: Jeffrey G. Linger; Sarah E. Hobdey; Mary Ann Franden; Emily M. Fulk; Gregg T. Beckham
      Pages: 24 - 29
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Jeffrey G. Linger, Sarah E. Hobdey, Mary Ann Franden, Emily M. Fulk, Gregg T. Beckham
      Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.01.005
      Issue No: Vol. 3 (2017)
  • Promiscuous plasmid replication in thermophiles: Use of a novel
           hyperthermophilic replicon for genetic manipulation of Clostridium
           thermocellum at its optimum growth temperature

    • Authors: Joseph Groom; Daehwan Chung; Daniel G. Olson; Lee R. Lynd; Adam M. Guss; Janet Westpheling
      Pages: 30 - 38
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Joseph Groom, Daehwan Chung, Daniel G. Olson, Lee R. Lynd, Adam M. Guss, Janet Westpheling
      Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60°C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.01.004
      Issue No: Vol. 3 (2017)
  • Excessive by-product formation: A key contributor to low isobutanol yields
           of engineered Saccharomyces cerevisiae strains

    • Authors: N. Milne; S.A. Wahl; A.J.A. van Maris; J.T. Pronk; J.M. Daran
      Pages: 39 - 51
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): N. Milne, S.A. Wahl, A.J.A. van Maris, J.T. Pronk, J.M. Daran
      It is theoretically possible to engineer Saccharomyces cerevisiae strains in which isobutanol is the predominant catabolic product and high-yielding isobutanol-producing strains are already reported by industry. Conversely, isobutanol yields of engineered S. cerevisiae strains reported in the scientific literature typically remain far below 10% of the theoretical maximum. This study explores possible reasons for these suboptimal yields by a mass-balancing approach. A cytosolically located, cofactor-balanced isobutanol pathway, consisting of a mosaic of bacterial enzymes whose in vivo functionality was confirmed by complementation of null mutations in branched-chain amino acid metabolism, was expressed in S. cerevisiae. Product formation by the engineered strain was analysed in shake flasks and bioreactors. In aerobic cultures, the pathway intermediate isobutyraldehyde was oxidized to isobutyrate rather than reduced to isobutanol. Moreover, significant concentrations of the pathway intermediates 2,3-dihydroxyisovalerate and α-ketoisovalerate, as well as diacetyl and acetoin, accumulated extracellularly. While the engineered strain could not grow anaerobically, micro-aerobic cultivation resulted in isobutanol formation at a yield of 0.018±0.003 mol/mol glucose. Simultaneously, 2,3-butanediol was produced at a yield of 0.649±0.067mol/mol glucose. These results identify massive accumulation of pathway intermediates, as well as overflow metabolites derived from acetolactate, as an important, previously underestimated contributor to the suboptimal yields of ‘academic’ isobutanol strains. The observed patterns of by-product formation is consistent with the notion that in vivo activity of the iron–sulphur-cluster-requiring enzyme dihydroxyacid dehydratase is a key bottleneck in the present and previously described ‘academic’ isobutanol-producing yeast strains.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.01.002
      Issue No: Vol. 3 (2017)
  • Interaction of storage carbohydrates and other cyclic fluxes with central
           metabolism: A quantitative approach by non-stationary 13C metabolic flux

    • Authors: C.A. Suarez-Mendez; M. Hanemaaijer; Angela ten Pierick; J.C. Wolters; J.J. Heijnen; S.A. Wahl
      Pages: 52 - 63
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): C.A. Suarez-Mendez, M. Hanemaaijer, Angela ten Pierick, J.C. Wolters, J.J. Heijnen, S.A. Wahl
      13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307h−1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold) and higher fluxes relative to the glucose uptake rate (up to 16%). Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.01.001
      Issue No: Vol. 3 (2017)
  • Tet-On lentiviral transductants lose inducibility when silenced for
           extended intervals in mammary epithelial cells

    • Authors: Yang Yu; Michelle Montag Lowy; Randolph C. Elble
      Pages: 64 - 67
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Yang Yu, Michelle Montag Lowy, Randolph C. Elble
      Silencing of virally transduced genes by promoter methylation and histone deacetylation has been a chronic problem both experimentally and therapeutically. We observed frequent silencing of the tetracycline-inducible Tet-On promoter borne by the Tripz lentivirus in mammary epithelial cell lines. We found that silencing could be prevented by continuous induction, but uninduced Tet-On gradually became uninducible, suggesting promoter modification. Accordingly, silencing was reversible by a common inhibitor of histone deacetylases, sodium butyrate. The effect was cell-line dependent, as HEK293 cells exhibited only moderate silencing that could be partly reversed by extended induction. These results indicate the need to test individual cell lines prior to using this system for studies that require induction after long periods of repression such as in animal models or RNA interference screens.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.03.001
      Issue No: Vol. 3 (2017)
  • Mitochondrial targeting increases specific activity of a heterologous
           valine assimilation pathway in Saccharomyces cerevisiae

    • Authors: Kevin V. Solomon; Elisa Ovadia; Fujio Yu; Wataru Mizunashi; Michelle A. O’Malley
      Pages: 68 - 75
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Kevin V. Solomon, Elisa Ovadia, Fujio Yu, Wataru Mizunashi, Michelle A. O’Malley
      Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.03.004
      Issue No: Vol. 3 (2017)
  • An optimized method for accurate quantification of cell migration using
           human small intestine cells

    • Authors: Steffen Nyegaard; Brian Christensen; Jan Trige Rasmussen
      Pages: 76 - 83
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Steffen Nyegaard, Brian Christensen, Jan Trige Rasmussen
      Quantifying the ability of a compound to modulate cell migration rate is a crucial part of many studies including those on chemotaxis, wound healing and cancer metastasis. Existing migration assays all have their strengths and weaknesses. The “scratch” assay is the most widely used because it seems appealingly simple and inexpensive. However, the scratch assay has some important limitations, as the tool introducing the “wound” might injure/stress the boundary cells and/or harm underlying matrix coatings, which in both cases will affect cell migration. This described method is a Cell Exclusion Zone Assay, in which cell-free areas are created by growing cells around removable silicone stoppers. Upon appropriate staining with fluorescent dyes and microscopically visualizing the monolayers, the migration rate is then quantified by counting the cells (nuclei) intruding the void area left by the silicone insert. In the current study human small intestine epithelial cells were seeded on a physiological substrate matrix to produce collectively migrating monolayers. Different substrates were tested to determine the optimal surface for enterocyte adherence and migration and morphological changes monitored. Recombinant human epidermal growth factor and osteopontin purified from urine were tested to see if the established migration assay produces accurate and reliable migration data with human small intestine cells. The obtained data accurately confirmed that the two bioactive proteins modulate cellular migration in a dose-dependent manner. The presented assay can likely be converted for use with other adherent cell lines or substrate matrices and allows for high throughput, while cost is kept low and versatility high. Co-staining can be applied in order to assay for cell death, different cell types, cell stress and others allowing intricate analysis of migration rate of mixed populations and correction for cell viability.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.03.002
      Issue No: Vol. 3 (2017)
  • Acidithiobacillus ferrooxidans's comprehensive model driven analysis of
           the electron transfer metabolism and synthetic strain design for biomining

    • Authors: Miguel A. Campodonico; Daniela Vaisman; Jean F. Castro; Valeria Razmilic; Francesca Mercado; Barbara A. Andrews; Adam M. Feist; Juan A. Asenjo
      Pages: 84 - 96
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Miguel A. Campodonico, Daniela Vaisman, Jean F. Castro, Valeria Razmilic, Francesca Mercado, Barbara A. Andrews, Adam M. Feist, Juan A. Asenjo
      Acidithiobacillus ferrooxidans is a gram-negative chemolithoautotrophic γ-proteobacterium. It typically grows at an external pH of 2 using the oxidation of ferrous ions by oxygen, producing ferric ions and water, while fixing carbon dioxide from the environment. A. ferrooxidans is of great interest for biomining and environmental applications, as it can process mineral ores and alleviate the negative environmental consequences derived from the mining processes. In this study, the first genome-scale metabolic reconstruction of A. ferrooxidans ATCC 23270 was generated (iMC507). A total of 587 metabolic and transport/exchange reactions, 507 genes and 573 metabolites organized in over 42 subsystems were incorporated into the model. Based on a new genetic algorithm approach, that integrates flux balance analysis, chemiosmotic theory, and physiological data, the proton translocation stoichiometry for a number of enzymes and maintenance parameters under aerobic chemolithoautotrophic conditions using three different electron donors were estimated. Furthermore, a detailed electron transfer and carbon flux distributions during chemolithoautotrophic growth using ferrous ion, tetrathionate and thiosulfate were determined and reported. Finally, 134 growth-coupled designs were calculated that enables Extracellular Polysaccharide production. iMC507 serves as a knowledgebase for summarizing and categorizing the information currently available for A. ferrooxidans and enables the understanding and engineering of Acidithiobacillus and similar species from a comprehensive model-driven perspective for biomining applications.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.03.003
      Issue No: Vol. 3 (2017)
  • Dynamics of benzoate metabolism in Pseudomonas putida KT2440

    • Authors: Suresh Sudarsan; Lars M. Blank; Alexander Dietrich; Oliver Vielhauer; Ralf Takors; Andreas Schmid; Matthias Reuss
      Pages: 97 - 110
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Suresh Sudarsan, Lars M. Blank, Alexander Dietrich, Oliver Vielhauer, Ralf Takors, Andreas Schmid, Matthias Reuss
      Soil microorganisms mineralize lignin-derived aromatic carbon sources using oxidative catabolic pathways, such as the β-ketoadipate pathway. Although this aromatic pathway is one of the best-studied pathways in biochemistry, the complete pathway, including its regulation by aromatic carbon sources, has not been integrated into the metabolic network. In particular, information about the in vivo operation (e.g., kinetics and flux capacity) of the pathway is lacking. In this contribution, we use kinetic modeling and thermodynamic analysis to evaluate the in vivo operation of this key aromatic multi-step pathway. The resulting ab initio deterministic model of benzoate degradation via the β-ketoadipate (ortho-cleavage) pathway in Pseudomonas putida KT2440 is presented. The kinetic model includes mechanistic rate expressions for the enzymes and transport processes. The design and experimental validation of the model are driven by data generated from short-term perturbation experiments in a benzoate-limited continuous culture. The results of rigorous modeling of the in vivo dynamics provide strong support for flux regulation by the benzoate transporter and the enzymes forming and cleaving catechol. Revisiting the β-ketoadipate pathway might be valuable for applications in different fields, such as biochemistry and metabolic engineering, that use lignin monomers as a carbon source.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.03.005
      Issue No: Vol. 3 (2017)
  • Enhancing muconic acid production from glucose and lignin-derived aromatic
           compounds via increased protocatechuate decarboxylase activity

    • Authors: Christopher W. Johnson; Davinia Salvachúa; Payal Khanna; Holly Smith; Darren J. Peterson; Gregg T. Beckham
      Pages: 111 - 119
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Christopher W. Johnson, Davinia Salvachúa, Payal Khanna, Holly Smith, Darren J. Peterson, Gregg T. Beckham
      The conversion of biomass-derived sugars and aromatic molecules to cis,cis-muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p-coumarate by 50% and resulted in a titer of >15g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92±0.48g/L muconate. This study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.04.002
      Issue No: Vol. 3 (2017)
  • Development of a plasmid-based expression system in Clostridium
           thermocellum and its use to screen heterologous expression of bifunctional
           alcohol dehydrogenases (adhEs)

    • Authors: Shuen Hon; Anthony A. Lanahan; Liang Tian; Richard J. Giannone; Robert L. Hettich; Daniel G. Olson; Lee R. Lynd
      Pages: 120 - 129
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Shuen Hon, Anthony A. Lanahan, Liang Tian, Richard J. Giannone, Robert L. Hettich, Daniel G. Olson, Lee R. Lynd
      Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.04.001
      Issue No: Vol. 3 (2017)
  • Improved sugar-free succinate production by Synechocystis sp. PCC 6803
           following identification of the limiting steps in glycogen catabolism

    • Authors: Tomohisa Hasunuma; Mami Matsuda; Akihiko Kondo
      Pages: 130 - 141
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Tomohisa Hasunuma, Mami Matsuda, Akihiko Kondo
      Succinate produced by microorganisms can replace currently used petroleum-based succinate but typically requires mono- or poly-saccharides as a feedstock. The cyanobacterium Synechocystis sp. PCC6803 can produce organic acids such as succinate from CO2 not supplemented with sugars under dark anoxic conditions using an unknown metabolic pathway. The TCA cycle in cyanobacteria branches into oxidative and reductive routes. Time-course analyses of the metabolome, transcriptome and metabolic turnover described here revealed dynamic changes in the metabolism of Synechocystis sp. PCC6803 cultivated under dark anoxic conditions, allowing identification of the carbon flow and rate-limiting steps in glycogen catabolism. Glycogen biosynthesized from CO2 assimilated during periods of light exposure is catabolized to succinate via glycolysis, the anaplerotic pathway, and the reductive TCA cycle under dark anoxic conditions. Expression of the phosphoenolpyruvate (PEP) carboxylase gene (ppc) was identified as a rate-limiting step in succinate biosynthesis and this rate limitation was alleviated by ppc overexpression, resulting in improved succinate excretion. The sugar-free succinate production was further enhanced by the addition of bicarbonate. In vivo labeling with NaH13CO3 clearly showed carbon incorporation into succinate via the anaplerotic pathway. Bicarbonate is in equilibrium with CO2. Succinate production by Synechocystis sp. PCC6803 therefore holds significant promise for CO2 capture and utilization.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.04.003
      Issue No: Vol. 3 (2017)
  • The Saccharomyces cerevisiae pheromone-response is a metabolically active
           stationary phase for bio-production

    • Authors: Thomas C. Williams; Bingyin Peng; Claudia E. Vickers; Lars K. Nielsen
      Pages: 142 - 152
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Thomas C. Williams, Bingyin Peng, Claudia E. Vickers, Lars K. Nielsen
      The growth characteristics and underlying metabolism of microbial production hosts are critical to the productivity of metabolically engineered pathways. Production in parallel with growth often leads to biomass/bio-product competition for carbon. The growth arrest phenotype associated with the Saccharomyces cerevisiae pheromone-response is potentially an attractive production phase because it offers the possibility of decoupling production from population growth. However, little is known about the metabolic phenotype associated with the pheromone-response, which has not been tested for suitability as a production phase. Analysis of extracellular metabolite fluxes, available transcriptomic data, and heterologous compound production (para-hydroxybenzoic acid) demonstrate that a highly active and distinct metabolism underlies the pheromone-response. These results indicate that the pheromone-response is a suitable production phase, and that it may be useful for informing synthetic biology design principles for engineering productive stationary phase phenotypes.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.05.001
      Issue No: Vol. 3 (2017)
  • Improving the flux distributions simulated with genome-scale metabolic
           models of Saccharomyces cerevisiae

    • Authors: Rui Pereira; Jens Nielsen; Isabel Rocha
      Pages: 153 - 163
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Rui Pereira, Jens Nielsen, Isabel Rocha
      Genome-scale metabolic models (GEMs) can be used to evaluate genotype-phenotype relationships and their application to microbial strain engineering is increasing in popularity. Some of the algorithms used to simulate the phenotypes of mutant strains require the determination of a wild-type flux distribution. However, the accuracy of this reference, when calculated with flux balance analysis, has not been studied in detail before. Here, the wild-type simulations of selected GEMs for Saccharomyces cerevisiae have been analysed and most of the models tested predicted erroneous fluxes in central pathways, especially in the pentose phosphate pathway. Since the problematic fluxes were mostly related to areas of the metabolism consuming or producing NADPH/NADH, we have manually curated all reactions including these cofactors by forcing the use of NADPH/NADP+ in anabolic reactions and NADH/NAD+ for catabolic reactions. The curated models predicted more accurate flux distributions and performed better in the simulation of mutant phenotypes.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.05.002
      Issue No: Vol. 3 (2017)
  • A genetic screen for increasing metabolic flux in the isoprenoid pathway
           of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen

    • Authors: M. Wadhwa; A.K. Bachhawat
      Pages: 164 - 172
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): M. Wadhwa, A.K. Bachhawat
      A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.
      Graphical abstract image

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.05.004
      Issue No: Vol. 3 (2017)
  • Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning
           and genetic engineering design, simulation and management software for
           complex synthetic biology and metabolic engineering projects

    • Authors: Zhenyu Shi; Claudia E. Vickers
      Pages: 173 - 186
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Zhenyu Shi, Claudia E. Vickers
      Molecular Cloning Designer Simulator (MCDS) is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1) it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2) it can perform a user-defined workflow of cloning steps in a single execution of the software; (3) it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4) it includes experimental information to conveniently guide wet lab work; and (5) it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.05.003
      Issue No: Vol. 3 (2017)
  • Investigation of useful carbon tracers for 13C-metabolic flux analysis of
           Escherichia coli by considering five experimentally determined flux

    • Authors: Kousuke Maeda; Nobuyuki Okahashi; Yoshihiro Toya; Fumio Matsuda; Hiroshi Shimizu
      Pages: 187 - 195
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Kousuke Maeda, Nobuyuki Okahashi, Yoshihiro Toya, Fumio Matsuda, Hiroshi Shimizu
      The 13C-MFA experiments require an optimal design since the precision or confidence intervals of the estimated flux levels depends on factors such as the composition of 13C-labeled carbon sources, as well as the metabolic flux distribution of interest. In this study, useful compositions of 13C-labeled glucose for 13C-metabolic flux analysis (13C-MFA) of Escherichia coli are investigated using a computer simulation of the stable isotope labeling experiment. Following the generation of artificial mass spectra datasets of amino acid fragments using five literature-reported flux distributions of E. coli, the best fitted flux distribution and the 95% confidence interval were estimated by the 13C-MFA procedure. A comparison of the precision scores showed that [1, 2-13C]glucose and a mixture of [1-13C] and [U-13C]glucose at 8:2 are one of the best carbon sources for a precise estimation of flux levels of the pentose phosphate pathway, glycolysis and the TCA cycle. Although the precision scores of the anaplerotic and glyoxylate pathway reactions were affected by both the carbon source and flux distribution, it was also shown that the mixture of non-labeled, [1-13C], and [U-13C]glucose at 4:1:5 was specifically effective for the flux estimation of the glyoxylate pathway reaction. These findings were confirmed by wet 13C-MFA experiments.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.06.001
      Issue No: Vol. 3 (2017)
  • System wide cofactor turnovers can propagate metabolic stability between

    • Authors: Y. Yang; Y.H. Guan; J. Villadsen
      Pages: 196 - 204
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Y. Yang, Y.H. Guan, J. Villadsen
      Metabolic homeostasis, or low-level metabolic steady state, has long been taken for granted in metabolic engineering, and research priority has always been given to understand metabolic flux control and regulation of the reaction network. In the past, this has not caused concerns because the metabolic networks studied were invariably associated with living cells. Nowadays, there are needs to reconstruct metabolic networks, and so metabolic homeostasis cannot be taken for granted. For metabolic steady state, enzyme feedback control has been known to explain why metabolites in metabolic pathways can avoid accumulation. However, we reasoned that there are further contributing mechanisms. As a new methodology developed, we separated cofactor intermediates (CIs) from non-cofactor intermediates, and identified an appropriate type of open systems for operating putative reaction topologies. Furthermore, we elaborated the criteria to tell if a multi-enzyme over-all reaction path is of in vivo nature or not at the metabolic level. As new findings, we discovered that there are interactions between the enzyme feedback inhibition and the CI turnover, and such interactions may well lead to metabolic homeostasis, an emergent property of the system. To conclude, this work offers a new perspective for understanding the role of CIs and the presence of metabolic homeostasis in the living cell. In perspective, this work might provide clues for constructing non-natural metabolic networks using multi-enzyme reactions or by degenerating metabolic reaction networks from the living cell.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.06.002
      Issue No: Vol. 3 (2017)
  • The impact of respiration and oxidative stress response on recombinant
           α-amylase production by Saccharomyces cerevisiae

    • Authors: José L. Martínez; Eugenio Meza; Dina Petranovic; Jens Nielsen
      Pages: 205 - 210
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): José L. Martínez, Eugenio Meza, Dina Petranovic, Jens Nielsen
      Studying protein production is important for fundamental research on cell biology and applied research for biotechnology. Yeast Saccharomyces cerevisiae is an attractive workhorse for production of recombinant proteins as it does not secrete many endogenous proteins and it is therefore easy to purify a secreted product. However, recombinant production at high rates represents a significant metabolic burden for the yeast cells, which results in oxidative stress and ultimately affects the protein production capacity. Here we describe a method to reduce the overall oxidative stress by overexpressing the endogenous HAP1 gene in a S. cerevisiae strain overproducing recombinant α-amylase. We demonstrate how Hap1p can activate a set of oxidative stress response genes and meanwhile contribute to increase the metabolic rate of the yeast strains, therefore mitigating the negative effect of the ROS accumulation associated to protein folding and hence increasing the production capacity during batch fermentations.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.06.003
      Issue No: Vol. 3 (2017)
  • Enhanced fatty acid production in engineered chemolithoautotrophic
           bacteria using reduced sulfur compounds as energy sources

    • Authors: Harry R. Beller; Peng Zhou; Talia N.M. Jewell; Ee-Been Goh; Jay D. Keasling
      Pages: 211 - 215
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Harry R. Beller, Peng Zhou, Talia N.M. Jewell, Ee-Been Goh, Jay D. Keasling
      Chemolithoautotrophic bacteria that oxidize reduced sulfur compounds, such as H2S, while fixing CO2 are an untapped source of renewable bioproducts from sulfide-laden waste, such as municipal wastewater. In this study, we report engineering of the chemolithoautotrophic bacterium Thiobacillus denitrificans to produce up to 52-fold more fatty acids than the wild-type strain when grown with thiosulfate and CO2. A modified thioesterase gene from E. coli (‘tesA) was integrated into the T. denitrificans chromosome under the control of Pkan or one of two native T. denitrificans promoters. The relative strength of the two native promoters as assessed by fatty acid production in engineered strains was very similar to that assessed by expression of the cognate genes in the wild-type strain. This proof-of-principle study suggests that engineering sulfide-oxidizing chemolithoautotrophic bacteria to overproduce fatty acid-derived products merits consideration as a technology that could simultaneously produce renewable fuels/chemicals as well as cost-effectively remediate sulfide-contaminated wastewater.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.07.001
      Issue No: Vol. 3 (2017)
  • Computational metabolic engineering strategies for growth-coupled biofuel
           production by Synechocystis

    • Authors: Kiyan Shabestary; Elton P. Hudson
      Pages: 216 - 226
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Kiyan Shabestary, Elton P. Hudson
      Chemical and fuel production by photosynthetic cyanobacteria is a promising technology but to date has not reached competitive rates and titers. Genome-scale metabolic modeling can reveal limitations in cyanobacteria metabolism and guide genetic engineering strategies to increase chemical production. Here, we used constraint-based modeling and optimization algorithms on a genome-scale model of Synechocystis PCC6803 to find ways to improve productivity of fermentative, fatty-acid, and terpene-derived fuels. OptGene and MOMA were used to find heuristics for knockout strategies that could increase biofuel productivity. OptKnock was used to find a set of knockouts that led to coupling between biofuel and growth. Our results show that high productivity of fermentation or reversed beta-oxidation derived alcohols such as 1-butanol requires elimination of NADH sinks, while terpenes and fatty-acid based fuels require creating imbalances in intracellular ATP and NADPH production and consumption. The FBA-predicted productivities of these fuels are at least 10-fold higher than those reported so far in the literature. We also discuss the physiological and practical feasibility of implementing these knockouts. This work gives insight into how cyanobacteria could be engineered to reach competitive biofuel productivities.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.07.003
      Issue No: Vol. 3 (2017)
  • Quantifying complexity in metabolic engineering using the LASER database

    • Authors: James D. Winkler; Andrea L. Halweg-Edwards; Ryan T. Gill
      Pages: 227 - 233
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): James D. Winkler, Andrea L. Halweg-Edwards, Ryan T. Gill
      We previously introduced the LASER database (Learning Assisted Strain EngineeRing, (Winkler et al. 2015) to serve as a platform for understanding past and present metabolic engineering practices. Over the past year, LASER has been expanded by 50% to include over 600 engineered strains from 450 papers, including their growth conditions, genetic modifications, and other information in an easily searchable format. Here, we present the results of our efforts to use LASER as a means for defining the complexity of a metabolic engineering “design”. We evaluate two complexity metrics based on the concepts of construction difficulty and novelty. No correlation is observed between expected product yield and complexity, allowing minimization of complexity without a performance trade-off. We envision the use of such complexity metrics to filter and prioritize designs prior to implementation of metabolic engineering efforts, thereby potentially reducing the time, labor, and expenses of large-scale projects. Possible future developments based on an expanding LASER database are then discussed.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.07.002
      Issue No: Vol. 3 (2017)
  • Creating metabolic demand as an engineering strategy in Pseudomonas putida
           – Rhamnolipid synthesis as an example

    • Authors: Till Tiso; Petra Sabelhaus; Beate Behrens; Andreas Wittgens; Frank Rosenau; Heiko Hayen; Lars Mathias Blank
      Pages: 234 - 244
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Till Tiso, Petra Sabelhaus, Beate Behrens, Andreas Wittgens, Frank Rosenau, Heiko Hayen, Lars Mathias Blank
      Metabolic engineering of microbial cell factories for the production of heterologous secondary metabolites implicitly relies on the intensification of intracellular flux directed toward the product of choice. Apart from reactions following peripheral pathways, enzymes of the central carbon metabolism are usually targeted for the enhancement of precursor supply. In Pseudomonas putida, a Gram-negative soil bacterium, central carbon metabolism, i.e., the reactions required for the synthesis of all 12 biomass precursors, was shown to be regulated at the metabolic level and not at the transcriptional level. The bacterium's central carbon metabolism appears to be driven by demand to react rapidly to ever-changing environmental conditions. In contrast, peripheral pathways that are only required for growth under certain conditions are regulated transcriptionally. In this work, we show that this regulation regime can be exploited for metabolic engineering. We tested this driven-by-demand metabolic engineering strategy using rhamnolipid production as an example. Rhamnolipid synthesis relies on two pathways, i.e., fatty acid de novo synthesis and the rhamnose pathway, providing the required precursors hydroxyalkanoyloxy-alkanoic acid (HAA) and activated (dTDP-)rhamnose, respectively. In contrast to single-pathway molecules, rhamnolipid synthesis causes demand for two central carbon metabolism intermediates, i.e., acetyl-CoA for HAA and glucose-6-phosphate for rhamnose synthesis. Following the above-outlined strategy of driven by demand, a synthetic promoter library was developed to identify the optimal expression of the two essential genes (rhlAB) for rhamnolipid synthesis. The best rhamnolipid-synthesizing strain had a yield of 40% rhamnolipids on sugar [CmolRL/CmolGlc], which is approximately 55% of the theoretical yield. The rate of rhamnolipid synthesis of this strain was also high. Compared to an exponentially growing wild type, the rhamnose pathway increased its flux by 300%, whereas the flux through de novo fatty acid synthesis increased by 50%. We show that the central carbon metabolism of P. putida is capable of meeting the metabolic demand generated by engineering transcription in peripheral pathways, thereby enabling a significant rerouting of carbon flux toward the product of interest, in this case, rhamnolipids of industrial interest.
      Graphical abstract image

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.08.002
      Issue No: Vol. 3 (2017)
  • Microbial synthesis of a branched-chain ester platform from organic waste

    • Authors: Donovan S. Layton; Cong T. Trinh
      Pages: 245 - 251
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Donovan S. Layton, Cong T. Trinh
      Processing of lignocellulosic biomass or organic wastes produces a plethora of chemicals such as short, linear carboxylic acids, known as carboxylates, derived from anaerobic digestion. While these carboxylates have low values and are inhibitory to microbes during fermentation, they can be biologically upgraded to high-value products. In this study, we expanded our general framework for biological upgrading of carboxylates to branched-chain esters by using three highly active alcohol acyltransferases (AATs) for alcohol and acyl CoA condensation and modulating the alcohol moiety from ethanol to isobutanol in the modular chassis cell. With this framework, we demonstrated the production of an ester library comprised of 16 out of all 18 potential esters, including acetate, propionate, butanoate, pentanoate, and hexanoate esters, from the 5 linear, saturated C2-C6 carboxylic acids. Among these esters, 5 new branched-chain esters, including isobutyl acetate, isobutyl propionate, isobutyl butyrate, isobutyl pentanoate, and isobutyl hexanoate were synthesized in vivo. During 24h in situ fermentation and extraction, one of the engineered strains, EcDL208 harnessing the SAAT of Fragaria ananassa produced ~63mg/L of a mixture of butyl and isobutyl butyrates from glucose and butyrate co-fermentation and ~127mg/L of a mixture of isobutyl and pentyl pentanoates from glucose and pentanoate co-fermentation, with high specificity. These butyrate and pentanoate esters are potential drop-in liquid fuels. This study provides better understanding of functional roles of AATs for microbial biosynthesis of branched-chain esters and expands the potential use of these esters as drop-in biofuels beyond their conventional flavor, fragrance, and solvent applications.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.08.001
      Issue No: Vol. 3 (2017)
  • The expression of glycerol facilitators from various yeast species
           improves growth on glycerol of Saccharomyces cerevisiae

    • Authors: Mathias Klein; Zia-ul Islam; Peter Boldsen Knudsen; Martina Carrillo; Steve Swinnen; Mhairi Workman; Elke Nevoigt
      Pages: 252 - 257
      Abstract: Publication date: December 2016
      Source:Metabolic Engineering Communications, Volume 3
      Author(s): Mathias Klein, Zia-ul Islam, Peter Boldsen Knudsen, Martina Carrillo, Steve Swinnen, Mhairi Workman, Elke Nevoigt
      Glycerol is an abundant by-product during biodiesel production and additionally has several assets compared to sugars when used as a carbon source for growing microorganisms in the context of biotechnological applications. However, most strains of the platform production organism Saccharomyces cerevisiae grow poorly in synthetic glycerol medium. It has been hypothesized that the uptake of glycerol could be a major bottleneck for the utilization of glycerol in S. cerevisiae. This species exclusively relies on an active transport system for glycerol uptake. This work demonstrates that the expression of predicted glycerol facilitators (Fps1 homologues) from superior glycerol-utilizing yeast species such as Pachysolen tannophilus, Komagataella pastoris, Yarrowia lipolytica and Cyberlindnera jadinii significantly improves the growth performance on glycerol of the previously selected glycerol-consuming S. cerevisiae wild-type strain (CBS 6412-13A). The maximum specific growth rate increased from 0.13 up to 0.18h−1 and a biomass yield coefficient of 0.56gDW/gglycerol was observed. These results pave the way for exploiting the assets of glycerol in the production of fuels, chemicals and pharmaceuticals based on baker's yeast.
      Graphical abstract image

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2016.09.001
      Issue No: Vol. 3 (2017)
  • Microbial production of 1-octanol: A naturally excreted biofuel with
           diesel-like properties

    • Authors: M. Kalim Akhtar; Hariharan Dandapani; Kati Thiel; Patrik R. Jones
      Pages: 1 - 5
      Abstract: Publication date: December 2015
      Source:Metabolic Engineering Communications, Volume 2
      Author(s): M. Kalim Akhtar, Hariharan Dandapani, Kati Thiel, Patrik R. Jones
      The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4mg 1-octanolL−1 h−1 which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB–TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2014.11.001
      Issue No: Vol. 2 (2017)
  • Development of a plasmid addicted system that is independent of
           co-inducers, antibiotics and specific carbon source additions for
           bioproduct (1-butanol) synthesis in Escherichia coli

    • Authors: Rick Laguna; Sarah J. Young; Chih-Chin Chen; Natividad Ruiz; Shang-Tian Yang; F. Robert Tabita
      Pages: 6 - 12
      Abstract: Publication date: December 2015
      Source:Metabolic Engineering Communications, Volume 2
      Author(s): Rick Laguna, Sarah J. Young, Chih-Chin Chen, Natividad Ruiz, Shang-Tian Yang, F. Robert Tabita
      Synthetic biology approaches for the synthesis of value-based products provide interesting and potentially fruitful possibilities for generating a wide variety of useful compounds and biofuels. However, industrial production is hampered by the costs associated with the need to supplement large microbial cultures with expensive but necessary co-inducer compounds and antibiotics that are required for up-regulating synthetic gene expression and maintaining plasmid-borne synthetic genes, respectively. To address these issues, a metabolism-based plasmid addiction system, which relies on lipopolysaccharide biosynthesis and maintenance of cellular redox balance for 1-butanol production; and utilizes an active constitutive promoter, was developed in Escherichia coli. Expression of the plasmid is absolutely required for cell viability and 1-butanol production. This system abrogates the need for expensive antibiotics and co-inducer molecules so that plasmid-borne synthetic genes may be expressed at high levels in a cost-effective manner. To illustrate these principles, high level and sustained production of 1-butanol by E. coli was demonstrated under different growth conditions and in semi-continuous batch cultures, in the absence of antibiotics and co-inducer molecules.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2014.12.001
      Issue No: Vol. 2 (2017)
  • CRISPR–Cas system enables fast and simple genome editing of industrial
           Saccharomyces cerevisiae strains

    • Authors: Vratislav Stovicek; Irina Borodina; Jochen Forster
      Pages: 13 - 22
      Abstract: Publication date: December 2015
      Source:Metabolic Engineering Communications, Volume 2
      Author(s): Vratislav Stovicek, Irina Borodina, Jochen Forster
      There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of Saccharomyces cerevisiae will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR–Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.

      PubDate: 2017-12-07T09:35:13Z
      DOI: 10.1016/j.meteno.2015.03.001
      Issue No: Vol. 2 (2017)
  • Genome-scale model guided design of Propionibacterium for enhanced
           propionic acid production

    • Authors: Laura Navone; Tim McCubbin; Axayacatl R. Gonzalez-Garcia; Lars K. Nielsen; Esteban Marcellin
      Abstract: Publication date: Available online 24 November 2017
      Source:Metabolic Engineering Communications
      Author(s): Laura Navone, Tim McCubbin, Axayacatl R. Gonzalez-Garcia, Lars K. Nielsen, Esteban Marcellin
      Production of propionic acid by fermentation of propionibacteria has gained increasing attention in the past few years. However, biomanufacturing of propionic acid cannot compete with the current oxo-petrochemical synthesis process due to its well-established infrastructure, low oil prices and the high downstream purification costs of microbial production. Strain improvement to increase propionic acid yield is the best alternative to reduce downstream purification costs. The recent generation of genome-scale models for a number of Propionibacterium species facilitates the rational design of metabolic engineering strategies and provides a new opportunity to explore the metabolic potential of the Wood-Werkman cycle. Previous strategies for strain improvement have individually targeted acid tolerance, rate of propionate production or minimisation of by-products. Here we used the P. freudenreichii subsp. shermanii and the pan-Propionibacterium GEMs to simultaneously target these combined issues. This was achieved by focussing on strategies which yield higher energies and directly suppress acetate formation. Using P. freudenreichii subsp. shermanii, two strategies were assessed. The first tested the ability to manipulate the redox balance to favour propionate production by over-expressing the first two enzymes of the pentose-phosphate pathway (PPP), Zwf (glucose-6-phosphate 1-dehydrogenase) and Pgl (6-phosphogluconolactonase). Results showed a 4-fold increase in propionate to acetate ratio during the exponential growth phase. Secondly, the ability to enhance the energy yield from propionate production by over-expressing an ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) and sodium-pumping methylmalonyl-CoA decarboxylase (MMD) was tested, which extended the exponential growth phase. Together, these strategies demonstrate that in silico design strategies are predictive and can be used to reduce by-product formation in Propionibacterium. We also describe the benefit of carbon dioxide to propionibacteria growth, substrate conversion and propionate yield.

      PubDate: 2017-11-27T08:51:57Z
      DOI: 10.1016/j.meteno.2017.11.001
  • YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway
           engineering in Yarrowia lipolytica

    • Authors: Lynn Wong; Jake Engel; Erqing Jin; Benjamin Holdridge; Peng Xu
      Abstract: Publication date: Available online 1 October 2017
      Source:Metabolic Engineering Communications
      Author(s): Lynn Wong, Jake Engel, Erqing Jin, Benjamin Holdridge, Peng Xu
      Effective metabolic engineering of microorganisms relies on balanced expression of both heterologous and endogenous genes to channel metabolic flux towards products of interest while achieving reasonable biomass buildup. To facilitate combinatorial pathway engineering and facile genetic operation, we engineered a set of modular cloning vectors compatible with BioBrick standards, called YaliBricks, to allow for rapid assembly of multigene pathways with customized genetic control elements (promoters, intronic sequences and terminators) in the oleaginous yeast Yarrowia lipolytica. We established a sensitive luciferase reporter and characterized a set of 12 native promoters to expand the oleaginous yeast genetic toolbox for transcriptional fine-tuning. We harnessed the intron alternative splicing mechanism and explored three unique gene configurations that allow us to encode genetic structural variations into metabolic function. We elucidated the role of how these genetic structural variations affect gene expression. To demonstrate the simplicity and effectiveness of streamlined genetic operations, we assembled the 12kb five-gene violacein biosynthetic pathway in one week. We also expanded this set of vectors to accommodate self-cleavage ribozymes and efficiently deliver guide RNA (gRNA) for targeted genome-editing with a codon-optimized CRISPR-Cas9 nuclease. Taken together, the tools built in this study provide a standard procedure to streamline and accelerate metabolic pathway engineering and genetic circuits construction in Yarrowia lipolytica.

      PubDate: 2017-10-03T22:49:42Z
      DOI: 10.1016/j.meteno.2017.09.001
  • Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome
           editing for lactic acid production from glucose and cellobiose

    • Authors: Aiko Ozaki; Rie Konishi; Chisako Otomo; Mayumi Kishida; Seiya Takayama; Takuya Matsumoto; Tsutomu Tanaka; Akihiko Kondo
      Abstract: Publication date: Available online 24 August 2017
      Source:Metabolic Engineering Communications
      Author(s): Aiko Ozaki, Rie Konishi, Chisako Otomo, Mayumi Kishida, Seiya Takayama, Takuya Matsumoto, Tsutomu Tanaka, Akihiko Kondo
      Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2g/L from 35.5g/L of consumed glucose with a yield of 0.71g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4g/L from 30g/L of cellobiose in minimal medium, with a yield of 0.68g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

      PubDate: 2017-08-25T12:49:32Z
      DOI: 10.1016/j.meteno.2017.08.002
  • UP Finder: A COBRA toolbox extension for identifying gene overexpression
           strategies for targeted overproduction

    • Authors: Xi Wang; Liang Yu; Shulin Chen
      Abstract: Publication date: Available online 16 August 2017
      Source:Metabolic Engineering Communications
      Author(s): Xi Wang, Liang Yu, Shulin Chen
      Overexpression of key genes is a basic strategy for overproducing target products via metabolic engineering. Traditionally, identifying those key genes/pathways largely relies on the knowledge of biochemistry and bioinformatics. In this study, a modeling tool named UP Finder was developed to facilitate the rapid identification of gene overexpression strategies. It was based on the COBRA toolbox under MATLAB environment. All the key gene/pathway targets are identified in one click after simply loading a Systems Biology Markup Language model and specifying a metabolite as the targeted product. The outputs are also quantitatively ranked to show the preference for determining overexpression strategies in pathway design. Analysis examples for overproducing lycopene precursor in Escherichia coli and fatty acyl-ACP in the cyanobacterium Synechocystis sp. PCC 6803 by the UP Finder showed high degree of agreement with the reported key genes in the literatures.
      Graphical abstract image

      PubDate: 2017-08-25T12:49:32Z
      DOI: 10.1016/j.meteno.2017.08.001
  • Flux Balance Analysis Indicates that Methane Is the Lowest Cost Feedstock
           for Microbial Cell Factories

    • Authors: Austin D. Comer; Matthew R. Long; Jennifer L. Reed; Brian F. Pfleger
      Abstract: Publication date: Available online 10 July 2017
      Source:Metabolic Engineering Communications
      Author(s): Austin D. Comer, Matthew R. Long, Jennifer L. Reed, Brian F. Pfleger
      The low cost of natural gas has driven significant interest in using C1 carbon sources (e.g. methane, methanol, CO, syngas) as feedstocks for producing liquid transportation fuels and commodity chemicals. Given the large contribution of sugar and lignocellulosic feedstocks to biorefinery operating costs, natural gas and other C1 sources may provide an economic advantage. To assess the relative costs of these feedstocks, we performed flux balance analysis on genome-scale metabolic models to calculate the maximum theoretical yields of chemical products from methane, methanol, acetate, and glucose. Yield calculations were performed for every metabolite (as a proxy for desired products) in the genome-scale metabolic models of three organisms: Escherichia coli (bacterium), Saccharomyces cerevisiae (yeast), and Synechococcus sp. PCC 7002 (cyanobacterium). The calculated theoretical yields and current feedstock prices provided inputs to create comparative feedstock cost surfaces. Our analysis shows that, at current market prices, methane feedstock costs are consistently lower than glucose when used as a carbon and energy source for microbial chemical production. Conversely, methanol is costlier than glucose under almost all price scenarios. Acetate feedstock costs could be less than glucose given efficient acetate production from low-cost syngas using nascent biological gas to liquids (BIO-GTL) technologies. Our analysis suggests that research should focus on overcoming the technical challenges of methane assimilation and/or yield of acetate via BIO-GTL to take advantage of low-cost natural gas rather than using methanol as a feedstock.

      PubDate: 2017-07-10T07:14:54Z
      DOI: 10.1016/j.meteno.2017.07.002
  • Eliminating a global regulator of carbon catabolite repression enhances
           the conversion of aromatic lignin monomers to muconate in Pseudomonas
           putida KT2440

    • Authors: Christopher W. Johnson; Paul E. Abraham; Jeffrey G. Linger; Payal Khanna; Robert L. Hettich; Gregg T. Beckham
      Abstract: Publication date: Available online 31 May 2017
      Source:Metabolic Engineering Communications
      Author(s): Christopher W. Johnson, Paul E. Abraham, Jeffrey G. Linger, Payal Khanna, Robert L. Hettich, Gregg T. Beckham
      Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce the muconate yield and productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36hours was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72hours was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24hours (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72hours (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering of pseudomonads for conversion of renewable feedstocks to value-added chemicals.

      PubDate: 2017-06-02T17:30:21Z
      DOI: 10.1016/j.meteno.2017.05.002
  • Comparison of orthologous cyanobacterial aldehyde deformylating oxygenases
           in the production of volatile C3-C7 alkanes in engineered E. coli

    • Authors: Pekka Patrikainen; Veronica Carbonell; Kati Thiel; Eva-Mari Aro; Pauli Kallio
      Abstract: Publication date: Available online 8 May 2017
      Source:Metabolic Engineering Communications
      Author(s): Pekka Patrikainen, Veronica Carbonell, Kati Thiel, Eva-Mari Aro, Pauli Kallio
      Aldehyde deformylating oxygenase (ADO) is a unique enzyme found exclusively in photosynthetic cyanobacteria, which natively converts acyl aldehyde precursors into hydrocarbon products embedded in cellular lipid bilayers. This capacity has opened doors for potential biotechnological applications aiming at biological production of diesel-range alkanes and alkenes, which are compatible with the nonrenewable petroleum-derived end-products in current use. The development of production platforms, however, has been limited by the relative inefficiency of ADO enzyme, promoting research towards finding new strategies and information to be used for rational design of enhanced pathways for hydrocarbon over-expression. In this work we present an optimized approach to study different ADO orthologs derived from different cyanobacterial species in an in vivo set-up in Escherichia coli. The system enabled comparison of alternative ADOs for the production efficiency of short-chain volatile C3-C7 alkanes, propane, pentane and heptane, and provided insight on the differences in substrate preference, catalytic efficiency and limitations associated with the enzymes. The work concentrated on five ADO orthologs which represent the most extensively studied cyanobacterial species in the field, and revealed distinct differences between the enzymes. In most cases the ADO from Nostoc punctiforme PCC 73102 performed the best in respect to yields and initial rates for the production of the volatile hydrocarbons. At the other extreme, the system harboring the ADO form Synechococcus sp. RS9917 produced very low amounts of the short-chain alkanes, primarily due to poor accumulation of the enzyme in E. coli. The ADOs from Synechocystis sp. PCC 6803 and Prochlorococcus marinus MIT9313, and the corresponding variant A134F displayed less divergence, although variation between chain-length preferences could be observed. The results confirmed the general trend of ADOs having decreasing catalytic efficiency towards precursors of decreasing chain-length, while expanding the knowledge on the species-specific traits, which may aid future pathway design and structure-based engineering of ADO for more efficient hydrocarbon production systems.
      Graphical abstract image

      PubDate: 2017-05-13T16:11:16Z
      DOI: 10.1016/j.meteno.2017.05.001
  • Development of a high efficiency integration system and promoter library
           for rapid modification of Pseudomonas putida KT2440

    • Authors: Joshua R Elmore; Anna Furches; Gara N. Wolff; Kent Gorday; Adam M Guss
      Abstract: Publication date: Available online 15 April 2017
      Source:Metabolic Engineering Communications
      Author(s): Joshua R Elmore, Anna Furches, Gara N. Wolff, Kent Gorday, Adam M Guss
      Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Further, most heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. To improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, −35 sequences, and −10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. An additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.

      PubDate: 2017-04-18T20:13:03Z
      DOI: 10.1016/j.meteno.2017.04.001
  • Selection Finder (SelFi): A Computational Metabolic Engineering Tool to
           Enable Directed Evolution of Enzymes

    • Authors: Neda Hassanpour; Ehsan Ullah; Mona Yousofshahi; Nikhil U. Nair; Soha Hassoun
      Abstract: Publication date: Available online 1 March 2017
      Source:Metabolic Engineering Communications
      Author(s): Neda Hassanpour, Ehsan Ullah, Mona Yousofshahi, Nikhil U. Nair, Soha Hassoun
      Directed evolution of enzymes consists of an iterative process of creating mutant libraries and choosing desired phenotypes through screening or selection until the enzymatic activity reaches a desired goal. The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s) with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi), to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. Importantly, SelFi advances directed evolution of enzymes as there is currently no known generalized strategies or computational techniques for identifying high-throughput selections for engineering enzymes.

      PubDate: 2017-03-03T22:26:12Z
      DOI: 10.1016/j.meteno.2017.02.003
  • Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by
           Pseudomonas putida KT2440

    • Authors: Michael T. Guarnieri; Mary Ann Franden; Christopher W. Johnson; Gregg T. Beckham
      Abstract: Publication date: Available online 8 February 2017
      Source:Metabolic Engineering Communications
      Author(s): Michael T. Guarnieri, Mary Ann Franden, Christopher W. Johnson, Gregg T. Beckham
      The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

      PubDate: 2017-02-10T19:03:04Z
      DOI: 10.1016/j.meteno.2017.02.001
  • Overexpression of the primary sigma factor gene sigA improved carotenoid
           production by Corynebacterium glutamicum: application to production of
           β-carotene and the non-native linear C50 carotenoid

    • Authors: Hironori Taniguchi; Nadja A. Henke; Sabine A.E. Heider; Volker F. Wendisch
      Abstract: Publication date: Available online 13 January 2017
      Source:Metabolic Engineering Communications
      Author(s): Hironori Taniguchi, Nadja A. Henke, Sabine A.E. Heider, Volker F. Wendisch
      Corynebacterium glutamicum shows yellow pigmentation due to biosynthesis of the C50 carotenoid decaprenoxanthin and its glycosides. This bacterium has been engineered for production of various non-native cyclic C40 and C50 carotenoids such as β-carotene, astaxanthin or sarcinaxanthin. In this study, the effect of modulating gene expression more broadly by overexpression of sigma factor genes on carotenoid production by C. glutamicum was characterized. Overexpression of the primary sigma factor gene sigA improved lycopene production by recombinant C. glutamicum up to 8-fold. In C. glutamicum wild type, overexpression of sigA led to 2-fold increased accumulation of the native carotenoid decaprenoxanthin in the stationary growth phase. Under these conditions, genes related to thiamine synthesis and aromatic compound degradation showed increased RNA levels and addition of thiamine and the aromatic iron chelator protocatechuic acid to the culture medium enhanced carotenoid production when sigA was overexpressed. Deletion of the gene for the alternative sigma factor SigB, which is expected to replace SigA in RNA polymerase holoenzymes during transition to the stationary growth phase, also increased carotenoid production. The strategy of sigA overexpression could be successfully transferred to production of the non-native carotenoids β-carotene and bisanhydrobacterioruberin (BABR). Production of the latter is the first demonstration that C. glutamicum may accumulate a non-native linear C50 carotenoid instead of the native cyclic C50 carotenoid decaprenoxanthin.
      Graphical abstract image

      PubDate: 2017-01-15T21:27:08Z
      DOI: 10.1016/j.meteno.2017.01.001
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