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. 2022 Jun 23:10:919969.
doi: 10.3389/fbioe.2022.919969. eCollection 2022.

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

Mengxun Shi  1   2 , Mengdi Li  1 , Anran Yang  1 , Xue Miao  1 , Liu Yang  1 , Jagroop Pandhal  2 , Huibin Zou  1   3
Affiliations

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

Mengxun Shi et al. Front Bioeng Biotechnol. .

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.

Keywords: biopolyester; class I polyhydroxyalkanoate synthase; degradable polymer; engineered E. coli; polylactic acid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Metabolic and morphologic engineering of E. coli strains for polylactic acid production. Exogenous vectors expressed the genes of key enzymes which are involved in the biosynthesis pathway of polylactic acid. The gene of ackA was deleted in the engineered strains to reduce the competitive by-product of acetic acid. The gene of sulA was expressed in engineered stains for larger cell space. Full names of abbreviations are as follows: PLA, polylactic acid; PhaC, PHA synthase; PCT, propionyl-CoA transferase; LDH, lactate dehydrogenase; Glu, glucose; PEP, phosphoenolpyruvate; PYR, pyruvate; Ac-CoA, acetyl co-enzyme A; AA, acetic acid; LA, lactic acid.
FIGURE 2
FIGURE 2
Comparison of the PLA production by five different engineered strains. The PLA production was recorded by wt% (weight of PLA/dry cell weight). The data shown are expressed as means of three repeated experiments, and the error bars present their standard deviation. Statistical difference (p value) between different groups is also present. (A) Strains which express the gene of PhaC1 Cs (class I PHA synthase) and sulA (for morphological engineering) have higher PLA production. (B) Strain which expresses the fused enzyme of Pct Cp /LdhA has similar PLA production with the control strain.
FIGURE 3
FIGURE 3
Morphological comparison of strains with or without morphological engineering using SEM and TEM assays. (A) SEM assay of cells from the morphologically engineered strain (with the expression of sulA). (B) SEM assay of cells from the control strain without expressing sulA. (C) TEM assay of cells from the morphologically engineered strain (with the expression of sulA). (D) TEM assay of cells from the control strain without expressing sulA.
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