Cell-growth phase-dependent promoter replacement approach for improved poly(lactate-co-3-hydroxybutyrate) production in Escherichia coli

• Published in: Microbial cell factories Vol. 22; no. 1; p. 131
• Main Authors: Nagao, Yuki, Koh, Sangho, Taguchi, Seiichi, Shimada, Tomohiro
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 19.07.2023; BioMed Central; BMC
• Subjects: Analysis; Bacteria; Carbon; Carbon sources; Cell growth; Chromosomes; Cloning; E coli; Enzymes; Escherichia coli; Gene expression; Genes; Glucose; Lactate fraction; Lactic acid; Methods; P(LA-co-3HB); Plasmids; Polymerization; Production processes; Promoter; Promoters; Stationary phase; Japan; Promoter; P(LA-co-3HB); Gene expression; Lactate fraction; Escherichia coli
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-023-02143-w

Escherichia coli is a useful platform for producing valuable materials through the implementation of synthetic gene(s) derived from other organisms. The production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] was carried out in E. coli using a set of five other species-derived genes: Pseudomonas sp. 61-3-derived phaC1STQK (for polymerization), Cupriavidus necator-derived phaAB (for 3HB-CoA generation), and Megasphaera elsdenii-derived pct (for LA-CoA generation) cloned into pTV118NpctphaC1p (ST/QK)AB. Here, we aimed to optimize the expression level and timing of these genes to improve the production of P(LA-co-3HB) and to manipulate the LA fraction by replacing the promoters with various promoters in E. coli. Evaluation of the effects of 21 promoter replacement plasmids revealed that the phaC1STQK-AB operon is critical for the stationary phase for P(LA-co-3HB) production. Interestingly, the effects of the promoters depended on the composition of the medium. In glucose-supplemented LB medium, the dps promoter replacement plasmid resulted in the greatest effect, increasing the accumulation to 8.8 g/L and an LA fraction of 14.1 mol% of P(LA-co-3HB), compared to 2.7 g/L and 8.1 mol% with the original plasmid. In xylose-supplemented LB medium, the yliH promoter replacement plasmid resulted in the greatest effect, with production of 5.6 g/L and an LA fraction of 40.2 mol% compared to 3.6 g/L and 22.6 mol% with the original plasmid. These results suggest that the selection of an appropriate promoter for expression of the phaC1STQK-AB operon could improve the production and LA fraction of P(LA-co-3HB). Here, we propose that the selection of cell-growth phase-dependent promoters is a versatile biotechnological strategy for effective intracellular production of polymeric materials such as P(LA-co-3HB), in combination with the selection of sugar-based carbon sources.