Department of Microbiology and Cell Science, University of Florida, Box 110700, Gainesville, FL 32611, USA.
J Ind Microbiol Biotechnol. 2011 Mar;38(3):441-50. doi: 10.1007/s10295-010-0788-4. Epub 2010 Jul 31.
Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50-55 °C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55 °C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35 °C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55 °C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.
凝结芽孢杆菌是一种产芽孢的乳酸细菌，最适生长温度为 50-55°C，能从己糖和戊糖产生乳酸作为主要发酵产物。与最佳生长温度为 35°C 的酿酒酵母的同步糖化发酵（SSF）相比，在 55°C 下进行 SSF 所需的真菌纤维素酶的量以前报道要低三到四倍。产乙醇凝结芽孢杆菌有望通过 55°C 的 SSF 降低纤维素乙醇的纤维素酶负荷和生产成本。作为将凝结芽孢杆菌开发为产乙醇微生物生物催化剂的第一步，通过突变（菌株 Suy27）去除了主要发酵酶 L-乳酸脱氢酶的活性。在 pH7.0 下生长时（每发酵 1g 葡萄糖产生 0.33g 乙醇），菌株 Suy27 以乙醇作为主要发酵产物。在该突变体中，依次作用的丙酮酸脱氢酶（PDH）和醇脱氢酶（ADH）贡献了大约 55%的乙醇，而丙酮酸甲酸裂解酶和 ADH 则负责其余部分。由于在 pH5.0 下发酵生长时凝结芽孢杆菌中缺乏 PDH 活性，l-ldh 突变体无法在 pH5.0 下进行厌氧生长。菌株 Suy27-13 是 l-ldh 突变体菌株 Suy27 的衍生物，在 pH5.0 下进行厌氧生长时产生 PDH 活性，在该 pH 下生长并产生乙醇作为发酵产物（每发酵 1g 葡萄糖产生 0.39g 乙醇）。这些结果表明，构建产乙醇凝结芽孢杆菌除了需要去除 LDH 活性以支持生长和乙醇生产外，还需要 PDH 活性的最佳表达。
