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

Abstract

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_s(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.

Keywords: Escherichia coli; Gene expression; Lactate fraction; P(LA-co-3HB); Promoter.

Fig. 1

Biosynthetic pathway scheme of P(LA-co-3HB) production in E. coli used in this study. Two sugars, glucose and xylose, derived from biomass were used as carbon sources for production of P(LA-co-3HB) in Escherichia coli. LDH: lactate dehydrogenase; PCT: propionyl-CoA transferase; LPE: lactate-polymerizing enzyme [29] engineered from PHA synthase of Pseudomonas sp. 61-3; PhaA: β-ketothiolase; PhaB: NADPH-dependent acetoacetyl-CoA reductase

Fig. 2

P(LA-co-3HB) production and expression levels of phaC1STQK-AB operon over the course of cultivation time by E. coli harboring pTV118NpctphaC1p_s(ST/QK)AB in glucose-supplemented LB medium. A The bars indicate the amount of 3HB units in the polymer (black), the amount of LA units in the polymer (white), and the fluorescent intensities of Nile-red-stained cells (gray). B Diagonal stripe bars indicate the mRNA levels of phaC. The line graph shows cell growth

Fig. 3

P(LA-co-3HB) production by replacement of the promoter fused to the phaC1STQK-AB operon. Each P(LA-co-3HB) production was measured by the fluorescent intensities of Nile-red staining. pTV indicates the conventional plasmid pTV118NpctphaC1p_s(ST/QK)AB. The gene name indicates the promoter replaced with the phaC promoter

Fig. 4

Correlation between P(LA-co-3HB) production and the mRNA levels of phaC. The x-axis shows the expression ratio of phaC mRNA to the ribosomal RNA, rrsA, quantified by RT-qPCR after 16 h of incubation. The y-axis shows the production of P(LA-co-3HB) as measured by Nile-red staining of cells after 48 h of incubation. The gene name of the promoter used for expression of the phaC1STQK-AB operon is indicated next to each dot

Fig. 5

P(LA-co-3HB) production by replacement of the promoter fused to pct. The bars indicate the amount of 3HB units in the polymer (black), the amount of LA units in the polymer (white), and the LA fraction (vertical stripe). pTV indicates the conventional plasmid pTV118NpctphaC1p_s(ST/QK)AB. The gene name indicates the promoter replaced with the lac promoter located upstream of pct

Fig. 6

P(LA-co-3HB) production and expression level of the phaC1STQK-AB operon by replacement of the promoter fused to the phaC1STQK-AB operon in xylose-supplemented LB medium. A The bars indicate the amount of 3HB units in the polymer (black), the amount of LA units in the polymer (white), and the LA fraction (gray). B, C The x-axis shows the expression ratio of phaC mRNA to the ribosomal RNA, rrsA, quantified by RT-qPCR after 24 h of incubation. The y-axis shows the production of P(LA-co-3HB) after 48 h of incubation (B), and the LA fraction (C). pTV indicates the conventional plasmid pTV118NpctphaC1p_s(ST/QK)AB. The gene name indicates the promoter replaced with the phaC promoter