School of Agriculture, Meiji University, 1-1-1 Kawasaki-Shi, Kanagawa, 214-8571, Japan.
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-Cho, Nada, Kobe, 657-8501, Japan.
Microb Cell Fact. 2023 Jul 19;22(1):131. 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.
大肠杆菌是一种有用的平台，可以通过实施来自其他生物体的合成基因来生产有价值的材料。通过在大肠杆菌中使用一组来自其他五种物种的基因，生产了基于乳酸（LA）的聚酯聚[LA-共-3-羟基丁酸（3HB）]：来自假单胞菌 sp. 61-3 的 phaC1STQK（用于聚合）、来自铜绿假单胞菌的 phaAB（用于 3HB-CoA 生成）和来自巨大芽孢杆菌的 pct（用于 LA-CoA 生成）克隆到 pTV118NpctphaC1p（ST/QK）AB 中。在这里，我们旨在优化这些基因的表达水平和时间，以提高 P（LA-co-3HB）的产量，并通过用不同的启动子替换大肠杆菌中的启动子来操纵 LA 分数。评估 21 个启动子替换质粒的效果表明，phaC1STQK-AB 操纵子对 P（LA-co-3HB）生产的静止期至关重要。有趣的是，启动子的效果取决于培养基的组成。在补充有葡萄糖的 LB 培养基中，dps 启动子替换质粒的效果最大，与原始质粒相比，积累量增加到 8.8 g/L，LA 分数为 14.1 mol%的 P（LA-co-3HB），而在补充有葡萄糖的 LB 培养基中，与原始质粒相比，积累量增加到 8.8 g/L，LA 分数为 14.1 mol%的 P（LA-co-3HB）。在补充有木糖的 LB 培养基中，yliH 启动子替换质粒的效果最大，与原始质粒相比，产量为 5.6 g/L，LA 分数为 40.2 mol%的 P（LA-co-3HB）。这些结果表明，选择合适的启动子来表达 phaC1STQK-AB 操纵子可以提高 P（LA-co-3HB）的产量和 LA 分数。在这里，我们提出选择细胞生长阶段依赖性启动子是一种有效的生物技术策略，可与选择糖基碳源相结合，有效在细胞内生产聚合材料，如 P（LA-co-3HB）。
