Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production (original) (raw)
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Many bacterial species, such as the chemolytotrophs Cupriavidus necator (Ralstonia eutropha), Cupriavidus metallidurans and Alcaligenes latus [4], Pseudomonas putida, P. aeruginosa , P. pseudoflava and other Pseudomonas spp [16], Azotobacter vinelandii [17], Halomonas campisalis [18], Enterobacter spp. [19], Thermus thermophilus [20]; Bacillus subtilis [21,22] and Bacillus cereus [23] among others, can synthesise and accumulate and store polyhydroxyalkanoates (PHAs) in intracellular organelles [24], co-polymers composed of hydroxypropionate, hydroxybutyrate and hydroxyvalerate, depending on the availability of precursors (such as octanoic acid) [11,25-27] and intermediate compounds regulating the
PRODUCTION OF PHA BY RECOMBINANT ORGANISMS
Polyhydroxyalkanoates (PHA) are biodegradable, thermoplastic polyesters produced from renewable carbon sources by a number of bacteria. However, their application is limited by high production costs. One of the strategies aimed to reduce their costs is the development of recombinant strains able to utilize different carbon sources. Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano/ microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. Natural PHA producers have become accustomed to accumulating PHA during evolution; they often have a long generation time, relatively low optimal growth temperature, are often hard to lysis and contain pathways for PHA degradation. This led to the development of recombinant PHA producers, capable of high PHA accumulation and/or free of PHA degradative pathways. This review summarizes the production of PHA by recombinant bacteria as well as some higher organisms in brief.
Starch based polyhydroxybutyrate production in engineered Escherichia coli
Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2 % (w/v) starch in M9 media containing 0.15 % (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4 % of cell dry mass.
We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escher- ichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudo- monas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2- hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1Ps6-19 and PctCp was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concen- trations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2- ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co- LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.
Applied and Environmental Microbiology, 2012
Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production. Volume 78 Number 9 aem.asm.org 3179 on April 9, 2012 by guest http://aem.asm.org/ Downloaded from FIG 3 Molecular weight distributions of P(3HB) synthesized by recombinant E. coli DH5␣ harboring pGET109-pha series plasmids. The cultivation time was 12 h (dashed lines) or 72 h (solid lines). Determination of molecular weight was carried out in triplicate, and typical distributions for each polymer are shown.
Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review
International Journal of Biological Macromolecules, 1999
Poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoates (PHAs) have been drawing much attention as biodegradable substitutes for conventional nondegradable plastics. For the economical production of P(3HB), various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed for the production of P(3HB) with high concentration and productivity. To reduce the cost of carbon substrate, several processes for P(3HB) production from cheap carbon sources were also developed. P(3HB) can now be produced to a content of 80% of cell dry weight with the productivity greater than 4 g/l per h. Fermentation strategy was also developed for the efficient production of medium chain length PHA by high cell density culture. With all these advances, P(3HB) and PHAs can be produced by bacterial fermentation at a cost (ca. $2/kg) similar to that of other biodegradable polymers under development.
Recent developments in bioreactor scale production of bacterial polyhydroxyalkanoates
Bioprocess and Biosystems Engineering, 2019
Polyhydroxyalkanoates (PHAs) are biological plastics that are sustainable alternative to synthetic ones. Numerous microorganisms have been identified as PHAs producers they store PHAs as cellular inclusions to use as an energy source backup. They can be produced in shake flasks and in bioreactors under defined fermentation and physiological culture conditions using suitable nutrients. Their production at bioreactor scale depends on various factors such as carbon source, nutrients supply, temperature, dissolved oxygen level, pH and processes. Once produced, PHAs find diverse applications in multiple fields of science and technology particularly in the medical sector. The present review covers some recent developments in sustainable bioreactor scale production of PHAs and identifies some areas in which future research in this field might be focused.
Antonie van Leeuwenhoek, 2008
Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated b-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and 1 H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of b-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.