Non-sterilized fermentation of high optically pure D-lactic acid by a genetically modified thermophilic Bacillus coagulans strain

• Published in: Microbial cell factories 2017-11, Vol.16 (1), p.213-213, Article 213
• Main Authors: Zhang, Caili, Zhou, Cheng, Assavasirijinda, Nilnate, Yu, Bo, Wang, Limin, Ma, Yanhe
• Format: Article
• Language: English
• Subjects: Acid production; Analysis; Bacillus (Bacteria); Bacillus coagulans; Cloning; Comparative analysis; D-Lactate dehydrogenase; d-Lactic acid; Dehydrogenase; Dehydrogenases; E coli; Evolution & development; Fermentation; Genes; Genetic engineering; Genetic modification; Glycerol; High temperature; Insertion; L-Lactate dehydrogenase; Laboratories; Lactate dehydrogenase; Lactic acid; Lactobacillus bulgaricus; Lactobacillus delbrueckii; Leucine dehydrogenase; Metabolism; Non-sterilized fermentation; Optically pure; Plasmids; Polylactic acid; Protein expression; Proteins; Sterilization; Studies; Thermophilic bacteria; Yeast
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-017-0827-1

Optically pure D-lactic acid (≥ 99%) is an important precursor of polylactic acid. However, there are relatively few studies on D-lactic acid fermentation compared with the extensive investigation of L-lactic acid production. Most lactic acid producers are mesophilic organisms. Optically pure D-lactic acid produced at high temperature not only could reduce the costs of sterilization but also could inhibit the growth of other bacteria, such as L-lactic acid producers. Thermophilic Bacillus coagulans is an excellent producer of L-lactic acid with capable of growing at 50 °C. In our previous study, the roles of two L-lactic acid dehydrogenases have been demonstrated in B. coagulans DSM1. In this study, the function of another annotated possible L-lactate dehydrogenase gene (ldhL3) was verified to be leucine dehydrogenase with an activity of 0.16 units (μmol/min) per mg protein. Furthermore, the activity of native D-lactate dehydrogenase was too low to support efficient D-lactic acid production, even under the control of strong promoter. Finally, an engineered B. coagulans D-DSM1 strain with the capacity for efficient production of D-lactic acid was constructed by deletion of two L-lactate dehydrogenases genes (ldhL1 and ldhL2) and insertion of the D-lactate dehydrogenase gene (LdldhD) from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 at the position of ldhL1. This genetically engineered strain produced only D-lactic acid under non-sterilized condition, and finally 145 g/L of D-lactic acid was produced with an optical purity of 99.9% and a high yield of 0.98 g/g. This is the highest optically pure D-lactic acid titer produced by a thermophilic strain.