Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose (original) (raw)
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Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli
Microbial Cell Factories, 2012
Background Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). Results Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (...
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.
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.
2013
Various isolates from activated sludge and soil samples were investigated for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HBco-3HV), from structurally unrelated single carbon substrates. Isolated Gram-negative bacteria, Agrobacterium sp. SH-1 and GW-014, could synthesize PQHB-co3HV) with 3HV monomer mole percent in the range from 1.3 to 11.4 from single carbon substrate, such as glucose, xylose or sucrose. Terpolyesters of 3HB, 3HV and 4-hydroxybutyrate (4HB) were also accumulated by Agrobucterium sp. SH-1 and GW-014 in a nitrogen-free production stage with various hydroxyafhanoate substrates. The structure of P(3HB-co-3HV) synthesized from glucose was confirmed by gas chromatographicmass spectrometric (GC-MS) analysis, and its molecular weight, polydispersity index and melting temperature were determined to be 628000,1.2 and 178"C, respectively. Total polymer content up to 78% (w/w) of dry cell weight and 3HV mole percent up to 50% could be achieved by fed-batch production from glucose and propionate.
Microbial cell factories, 2014
The most successful polyhydroxyalkanoate (PHA) in medical applications is poly(4-hydroxybutyrate) (P4HB), which is due to its biodegradability, biocompatibility and mechanical properties. One of the major obstacles for wider applications of P4HB is the cost of production and purification. It is highly desired to obtain P4HB in large scale at a competitive cost. In this work, we studied the possibility to increase P4HB productivity by using high cell density culture. To do so, we investigated for the first time some of the most relevant factors influencing P4HB biosynthesis in recombinant Escherichia coli. We observed that P4HB biosynthesis correlated more with limitations of amino acids and less with nitrogen depletion, contrary to the synthesis of many other types of PHAs. Furthermore, it was found that using glycerol as the primary carbon source, addition of acetic acid at the beginning of a batch culture stimulated P4HB accumulation in E. coli. Fed-batch high cell density culture...
Scientific reports, 2016
While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of β-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) f...
FEMS Microbiology Letters, 2003
Acyl-CoA dehydrogenase gene (yafH) of Escherichia coli was expressed together with polyhydroxyalkanoate synthase gene (phaC Ac ) and (R)-enoyl-CoA hydratase gene (phaJ Ac ) from Aeromonas caviae. The expression plasmids were introduced into E. coli JM109, DH5K and XL1-blue, respectively. Compared with the strains harboring only phaC Ac and phaJ Ac , all recombinant E. coli strains harboring yafH, phaC Ac and phaJ Ac accumulated at least four times more poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). Cell dry weights produced by all recombinants containing yafH were also considerably higher than that without yafH. The addition of acrylic acid which serves as inhibitor for L-oxidation and may lead to more precursor supply for PHA synthesis did not result in improved PHBHHx production compared with that of the overexpression of yafH. It appeared that the overexpression of acyl-CoA dehydrogenase gene (yafH) enhanced the supply of enoyl-CoA which is the substrate of (R)-enoyl-CoA hydratase. With the enhanced precursor supply, the recombinants accumulated more PHBHHx.