Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida.
Biotechnol Bioeng. 2018 Feb;115(2):453-463. doi: 10.1002/bit.26472. Epub 2017 Oct 30.
Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h g cells ) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.
聚乳酸（PLA）基塑料是可再生的、基于生物的和可生物降解的。尽管目前的 PLA 主要由 L-乳酸组成，但预计 L-和 D-乳酸共聚物将提高 PLA 的质量并扩大其用途。为了增加产生 D-乳酸的耐热微生物生物催化剂的数量，对在 50°C 下生长的枯草芽孢杆菌 168 菌株进行了代谢工程改造。由于枯草芽孢杆菌缺乏编码 D-乳酸脱氢酶（ldhA）的基因，因此评估了五个异源 ldhA 基因（凝结芽孢杆菌 ldhA 和 gldA101，以及来自三种德氏乳杆菌的 ldhA）。相应的 D-LDH 被纯化并进行了生化特性分析。在这些基因中，来自德氏乳杆菌亚种保加利亚的 D-LDH 在 37°C 下（枯草芽孢杆菌菌株 DA12）支持最高的 D-LA 产量（约 1M）和生产力（2 g h g 细胞）。在 48°C 时，D-LA 的产量约为 0.6M，产率为 0.99（g D-LA g 消耗的葡萄糖）。菌株 DA12 还在无机盐培养基中发酵葡萄糖生成乳酸，产率为 0.89 g g 葡萄糖，D-乳酸的浓度为 180±4.5 mM。这些结果表明，枯草芽孢杆菌作为一种平台生物具有在 48°C 下生产化学品的代谢工程潜力，可以最大限度地降低工艺成本。
