Metabolic engineering of poly(3‐hydroxybutyrate‐<i>co</i>‐3‐hydroxyvalerate) composition in recombinant <i>Salmonella enterica</i> serovar typhimurium

100

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon (phaBCA) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2R)-methylmalonyl-CoA mutase and a (2R)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B 12 ) concentration in the medium.

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) in Recombinant Salmonella enterica Serovar Typhimurium

Aldor

Kim

Prather

et al. 2002

100

“…Two samples were subjected to acid extraction before they were fixed and examined by electron microscopy (F and G). Bars ϭ 1 M. (4,6,20) because then the nitrogen for CGP would also be missing. The only conditions under which significantly larger amounts of CGP were accumulated was addition of arginine as a carbon source at relatively high concentrations (75 mM).…”

Section: Fig 3 Microscopic Inspection Of Cells Of a Calcoaceticusmentioning

confidence: 99%

Physiological Conditions Conducive to High Cyanophycin Content in Biomass ofAcinetobacter calcoaceticusStrain ADP1

Elbahloul

Krehenbrink

Reichelt

et al. 2005

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30°C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 g of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::⍀Km mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.

“…1). One leads to the formation of a C 4 monomer (3-hydroxybutyrate) in the copolymer, and the other leads to the formation of a C 5 monomer (3-hydroxyvalerate) (1,36). The C 5 monomer is formed through the condensation of acetyl-coenzyme A (acetyl-CoA) and propionyl-CoA by the action of ␤-ketothiolase.…”

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confidence: 99%

“…In addition, many bacteria showed poor 3HV yields from substrate propionate (Y 3HV/Prop ) (32). The overexpression of propionyl-CoA synthetase (encoded by prpE) in recombinant Salmonella enterica or Escherichia coli improved the 3HV fraction in the copolymer up to 25 mol% (1,40). Besides the uptake limitation of propionate, the endogenous catabolism of propionyl-CoA is also a limiting factor for producing PHBV with a high 3HV fraction.…”

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confidence: 99%

Production in Escherichia coli of Poly(3-Hydroxybutyrate- co -3-Hydroxyvalerate) with Differing Monomer Compositions from Unrelated Carbon Sources

Chen

Wang

Wei

et al. 2011

The industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis in Escherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing the thrABC operon in E. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.