Dataset Information

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

ABSTRACT: Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1 _Ps6-19) was commonly utilized in the de novo biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC _Cs ) from Chromobacterium sp. USM2 in engineered Escherichia coli for the de novo biosynthesis of PLA from glucose. The results indicated that PhaC _Cs had better performance in PLA production than that of class II synthase PhaC1 _Ps6-19. In addition, the sulA gene was engineered in PLA-producing strains for morphological engineering. The morphologically engineered strains present increased PLA production. This study also tested fused propionyl-CoA transferase and lactate dehydrogenase A (fused Pct _Cp /LdhA) in engineered E. coli and found that fused Pct _Cp /LdhA did not apparently improve the PLA production. After systematic engineering, the highest PLA production was achieved by E. coli MS6 (with PhaC _Cs and sulA), which could produce up to 955.0 mg/L of PLA in fed-batch fermentation with the cell dry weights of 2.23%, and the average molecular weight of produced PLA could reach 21,000 Da.

SUBMITTER: Shi M

PROVIDER: S-EPMC9261260 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Shi Mengxun M Li Mengdi M Yang Anran A Miao Xue X Yang Liu L Pandhal Jagroop J Zou Huibin H

Frontiers in bioengineering and biotechnology 20220623

Polylactic acid (PLA), a homopolymer of lactic acid (LA), is a bio-derived, biocompatible, and biodegradable polyester. The evolved class II PHA synthase (PhaC1 Ps6-19) was commonly utilized in the de novo biosynthesis of PLA from biomass. This study tested alternative class I PHA synthase (PhaC Cs ) from Chromobacterium sp. USM2 in engineered Escherichia coli for the de novo biosynthesis of PLA from glucose. The results indicated t ...[more]

PMID: 35814019

Similar Datasets

Project description:Mevalonate is a biochemical precursor to a wide range of isoprenoids. The mevalonate pathway uses three moles of acetyl-CoA, and therefore native pathways which metabolize acetyl-CoA compete with mevalonate synthesis. Moreover, the final step in mevalonate formation, mediated by hydroxymethylglutaryl-CoA reductase, requires NADPH as a co-substrate. This study focuses on chromosomal modification of citrate synthase (GltA) involved in acetyl-CoA utilization and phosphofructokinase (PfkA) involved in NADPH formation to increase the yield and productivity of mevalonate in Escherichia coli overexpressing the three genes of the heterologous mevalonate pathway. Nine GltA variants were compared for mevalonate production with the ΔgltA knockout and the wild-type GltA strain in shake flasks in the absence and presence of casamino acids. In the presence of casamino acids, all variants generated mevalonate at a greater yield than the wild-type control, but less than the GltA knockout. In the absence of casamino acids, the strain expressing wild-type GltA generated the greatest yield of mevalonate, while most variants instead accumulated primarily acetate. Using the wild-type strain and two citrate synthase variants, four phosphofructokinase variants were also compared with the ΔpfkA knockout and the wild-type strain, but PfkA variants generated less mevalonate than the corresponding wild-type PfkA strain. Controlled processes at the 1-liter scale comparing five strains demonstrated the inverse relationship between yield and productivity, with the GltA[K167A] variant showing the best balance for the yield (0.20 g/g) and productivity (0.87 g/L h). A nitrogen-limited process using the GltA[K167A] variant generated 36.9 g/L mevalonate in 31 h at a yield of 0.31 g/g. This study demonstrates that GltA variants offer a means to affect intracellular acetyl-CoA pools for the generation of acetyl-CoA derived products and that the acetyl-CoA pool rather than NADPH availability is the important limiting factor for mevalonate production.

Project description:Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE(Dm) and phaC(Dm) genes. PhaC(Dm) and PhaE(Dm) were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC(Dm) alone (pBBRMCS-2::phaC(Dm)) and of phaE(Dm)C(Dm) (pBBRMCS-2::phaE(Dm)C(Dm)) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB(-)4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE(Dm)C(Dm) small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC(Dm) and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.

Dataset Information

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Publications

Class I Polyhydroxyalkanoate (PHA) Synthase Increased Polylactic Acid Production in Engineered Escherichia Coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets