Disruption of poly (3-hydroxyalkanoate) depolymerase gene and overexpression of three poly (3-hydroxybutyrate) biosynthetic genes improve poly (3-hydroxybutyrate) production from nitrogen ... View Full Text


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Article Info

DATE

2019-12

AUTHORS

Jyumpei Kobayashi, Akihiko Kondo

ABSTRACT

BACKGROUND: Due to various environmental problems, biodegradable polymers such as poly (3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production. RESULTS: We constructed a poly (3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJΔphaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5 days of culture. The HJΔphaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJΔphaZ (phaA3), HJΔphaZ (phaB2), and HJΔphaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJΔphaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJΔphaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88 ± 0.08 g l-1 and with 100 mM ammonium sulfate; 0.99 ± 0.05 g l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively. CONCLUSION: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly (3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria. More... »

PAGES

40

References to SciGraph publications

  • 1995-04. Accumulation of poly-(hydroxybutyrate) by a non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides RV at different pH in BIOTECHNOLOGY LETTERS
  • 2016-12. Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator in MICROBIAL CELL FACTORIES
  • 2016-09. Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by Halomonas boliviensis in BIOPROCESS AND BIOSYSTEMS ENGINEERING
  • 2014-12. Improved production of poly(lactic acid)-like polyester based on metabolite analysis to address the rate-limiting step in AMB EXPRESS
  • 2015-12. Metabolic engineering of Escherichia coli for poly(3-hydroxybutyrate) production via threonine bypass in MICROBIAL CELL FACTORIES
  • 2017-10. Polyhydroxyalkanoate production from sucrose by Cupriavidus necator strains harboring csc genes from Escherichia coli W in APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
  • 1991-03. The accumulation of poly(3-hydroxyalkanoates) in Rhodobacter sphaeroides in ARCHIVES OF MICROBIOLOGY
  • 2009-07. Expression of four pha genes involved in poly-β-hydroxybutyrate production and accumulation in Rhodobacter sphaeroides FJ1 in MOLECULAR GENETICS AND GENOMICS
  • 2014-12. Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions in BMC BIOTECHNOLOGY
  • 1999-03. Expression of luciferase gene under control of the puf promoter from Rhodobacter sphaeroides in APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
  • 2008-10. Poly-β-hydroxyalkanoate production by halotolerant Rhodobacter sphaeroides U7 in WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
  • Journal

    TITLE

    Microbial Cell Factories

    ISSUE

    1

    VOLUME

    18

    Author Affiliations

    From Grant

  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1186/s12934-019-1088-y

    DOI

    http://dx.doi.org/10.1186/s12934-019-1088-y

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/30808422


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        "description": "BACKGROUND: Due to various environmental problems, biodegradable polymers such as poly\u00a0(3-hydroxybutyrate) (PHB) have gained much attention in recent years. Purple non-sulfur (PNS) bacteria have various attractive characteristics useful for environmentally harmless PHB production. However, production of PHB by PNS bacteria using genetic engineering has never been reported. This study is the first report of a genetically engineered PNS bacterial strain with a high PHB production.\nRESULTS: We constructed a poly\u00a0(3-hydroxyalkanoate) depolymerase (phaZ) gene-disrupted Rhodobacter sphaeroides HJ strain. This R. sphaeroides HJ\u0394phaZ (pLP-1.2) strain showed about 2.9-fold higher volumetric PHB production than that of the parent HJ (pLP-1.2) strain after 5\u00a0days of culture. The HJ\u0394phaZ strain was further improved for PHB production by constructing strains overexpressing each of the eight genes including those newly found and annotated as PHB biosynthesis genes in the KEGG GENES Database. Among these constructed strains, all of gene products exhibited annotated enzyme activities in the recombinant strain cells, and HJ\u0394phaZ (phaA3), HJ\u0394phaZ (phaB2), and HJ\u0394phaZ (phaC1) showed about 1.1-, 1.1-, and 1.2-fold higher volumetric PHB production than that of the parent HJ\u0394phaZ (pLP-1.2) strain. Furthermore, we constructed a strain that simultaneously overexpresses all three phaA3, phaB2, and phaC1 genes; this HJ\u0394phaZ (phaA3/phaB2/phaC1) strain showed about 1.7- to 3.9-fold higher volumetric PHB production (without ammonium sulfate; 1.88\u2009\u00b1\u20090.08\u00a0g\u00a0l-1 and with 100\u00a0mM ammonium sulfate; 0.99\u2009\u00b1\u20090.05\u00a0g\u00a0l-1) than those of the parent HJ (pLP-1.2) strain grown under nitrogen limited and rich conditions, respectively.\nCONCLUSION: In this study, we identified eight different genes involved in PHB biosynthesis in the genome of R. sphaeroides 2.4.1, and revealed that their overexpression increased PHB accumulation in an R. sphaeroides HJ strain. In addition, we demonstrated the effectiveness of a phaZ disruption for high PHB accumulation, especially under nitrogen rich conditions. Furthermore, we showed that PNS bacteria may have some unidentified genes involved in poly\u00a0(3-hydroxyalkanoates) (PHA) biosynthesis. Our findings could lead to further improvement of environmentally harmless PHA production techniques using PNS bacteria.", 
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