Engineering of fast-growing Vibrio natriegens for biosynthesis of poly(3-hydroxybutyrate-co-lactate).

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is a highly promising valuable biodegradable material with good biocompatibility and degradability. Vibrio natriegens, owing to its fast-growth, wide substrate spectrum characteristics, was selected to produce P(3HB-co-LA). Herein, the crucial role of acetyltransferase PN96-18060 for PHB synthesis in V. natriegens was identified. Heterologous pathway of P(3HB-co-LA) was introduced into V. natriegens successfully, in addition, overexpression of the dldh gene led to 1.84 fold enhancement of the lactate content in P(3HB-co-LA). Finally, the production of P(3HB-co-LA) was characterized under different carbon sources. The lactate fraction in P(3HB-co-LA) was increased to 28.3 mol% by the modification, about 1.84 times of that of the control. This is the first successful case of producing the P(3HB-co-LA) in V. natriegens. Collectively, this study showed that V. natriegens is an attractive host organism for producing P(3HB-co-LA) and has great potential to produce other co-polymers.

Enhancement of lactate fraction in poly(lactate-co-3-hydroxybutyrate) biosynthesized by metabolically engineered E. coli.

Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] is a high-molecular-weight biomaterial with excellent biocompatibility and biodegradability. In this study, the properties of P(LA-co-3HB) were examined and found to be affected by its lactate fraction. The efficiency of lactyl-CoA biosynthesis from intracellular lactate significantly affected the microbial synthesis of P(LA-co-3HB). Two CoA transferases from Anaerotignum lactatifermentans and Bacillota bacterium were selected for use in copolymer biosynthesis from 11 candidates. We found that cotAl enhanced the lactate fraction by 31.56% compared to that of the frequently used modified form of propionyl-CoA transferase from Anaerotignum propionicum. In addition, utilizing xylose as a favorable carbon source and blocking the lactate degradation pathway further enhanced the lactate fraction to 30.42 mol% and 52.84 mol%, respectively. Furthermore, when a 5 L bioreactor was used for fermentation utilizing xylose as a carbon source, the engineered strain produced 60.60 wt% P(46.40 mol% LA-co-3HB), which was similar to the results of our flask experiments. Our results indicate that the application of new CoA transferases has great potential for the biosynthesis of other lactate-based copolymers.

Efficient poly(3-hydroxybutyrate-co-lactate) production from corn stover hydrolysate by metabolically engineered Escherichia coli.

Poly(3-hydroxybutyrate-co-lactate)[P(3HB-co-LA)], is a biodegradable and biocompatible bioplastic, and the monomeric composition of the copolymer plays an important role in affecting its mechanical properties. Corn stover hydrolysate (CSH), the waste by-product in agriculture, has been considered as an important carbon source for value-added biochemical production. Therefore, the effect of CSH on P(3HB-co-LA) biosynthesis was investigated in this study. Taking CSH as the carbon source, the lactate (LA) fraction in the copolymer reached 7.1 mol% by the engineered stain. The results of shake flask fermentation demonstrated that reducing the activity of electron transport system resulted in a higher LA fraction. Furthermore, we replaced the promoter of the key gene pctth with ldhA gene promoter, so that the expression of pctth gene could be dynamically modulated as well as the lactic acid content changed. This study suggests that CSH is a promising carbon source for the production of biodegradable P(3HB-co-LA).

[Effect of short-chain thioesterase deficiency on P(3HB-co-LA) biosynthesis in Escherichia coli].

Polyhydroxyalkanoates (PHAs) have obtained much attention in biomaterial fields due to their similar physicochemical properties to those of the petroleum-derived plastics. Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is one member of the PHAs family, and has better toughness and transparency compared to existing polylactic acid (PLA) and poly[(R)-3-hydroxybutyrate] [P(3HB)]. First, we confirmed the one-step biosynthesis of P(LA-co-3HB) with the lactate fraction of 23.8 mol% by introducing P(3HB-co-LA) production module into Escherichia coli MG1655. Then, the lactate fraction was increased to 37.2 mol% in the dld deficient strain WXJ01-03. The genes encoding the thioesterases, ydiI and yciA, were further knocked out, and the lactate fraction in the P(3HB-co-LA) was improved to 42.3 mol% and 41.1 mol% respectively. Strain WXJ03-03 with dld, ydiI and yciA deficient was used for the production of the LA-enriched polymer, and the lactate fraction was improved to 46.1 mol%. Notably, the lactate fraction in P(3HB-co-LA) from xylose was remarkably higher than from glucose, indicating xylose as a potent carbon source for P(3HB-co-LA) production. Therefore, the deficiency of thioesterase may be considered as an effective strategy to improve the lactate fraction in P(3HB-co-LA) in xylose fermentation.

Recent advances in the microbial synthesis of lactate-based copolymer.

Due to the increasing environmental pollution of un-degradable plastics and the consumption of non-renewable resources, more attention has been attracted by new bio-degradable/based polymers produced from renewable resources. Polylactic acid (PLA) is one of the most representative bio-based materials, with obvious advantages and disadvantages, and has a wide range of applications in industry, medicine, and research. By copolymerizing to make up for its deficiencies, the obtained copolymers have more excellent properties. The development of a one-step microbial metabolism production process of the lactate (LA)-based copolymers overcomes the inherent shortcomings in the traditional chemical synthesis process. The most common lactate-based copolymer is poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], within which the difference of LA monomer fraction will cause the change in the material properties. It is necessary to regulate LA monomer fraction by appropriate methods. Based on synthetic biology and systems metabolic engineering, this review mainly focus on how did the different production strategies (such as enzyme engineering, fermentation engineering, etc.) of P(LA-co-3HB) optimize the chassis cells to efficiently produce it. In addition, the metabolic engineering strategies of some other lactate-based copolymers are also introduced in this article. These studies would facilitate to expand the application fields of the corresponding materials.