丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

The role of thioredoxin system in solvent production process of Clostridium acetobutylicum

摘要:

【目的】探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能。【方法】使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因（trxB）进行插入失活，得到突变株，通过Southern杂交方法验证插入内含子的拷贝数；在基本培养基中进行分批发酵，比较并分析突变株的生长特点；通过pH控制，利用限磷的连续发酵方法使丙酮丁醇梭菌稳定地在产酸期和产溶剂期生长，分析野生型菌株和突变株在稳定的产酸期和产醇期的生长和产物合成情况；通过添加不同浓度的过氧化氢检测野生型和突变株的抗氧化压力。【结果】抗性筛选和基因测序结果表明，成功构建了硫氧还蛋白还原酶失活的突变株，命名为Clostridium acetobutylicum trxB：：int（29）。在分批发酵中，突变株和野生型菌株的最大生长量相近，细胞在600 nm处的光吸收值（OD₆₀₀）达到6.5，但是突变株在36 h的OD₆₀₀达到最大，较野生型推迟12 h；在连续发酵的产酸期，野生型菌株与突变株生长变化不大，OD₆₀₀分别稳定在4.6和4.4，且葡萄糖的消耗和酸产量相差不大；在产溶剂期，突变株的OD₆₀₀稳定在3.5，低于野生型的4.0，同时，丙酮产量达到25.5 mmol/L，高于野生型（18 mmol/L），丁醇产量达到36.1 mmol/L，低于野生型（48 mmol/L）；添加低浓度的过氧化氢对突变株的影响不大，高浓度的过氧化氢对突变株造成的损伤大于野生型。【结论】硫氧还蛋白还原酶基因的失活不是致死突变，突变株在产酸期的生长和产酸能力与野生型相近，表明该基因的失活没有影响产酸期的代谢过程；trxB基因的失活主要影响了产溶剂期的氧化还原平衡，造成丙酮/丁醇的比率升高。

关键词:丙酮丁醇梭菌;硫氧还蛋白;限磷连续发酵;ClosTron系统

Abstract:

[Objective] This study aimed at investigating the roles of thioredoxin dependent reduction pathway in growth and metabolism of Clostridium acetobutylicum.[Methods] We used ClosTron technology to inactivate the thioredoxin reductase genes (trxB) of Trx-dependent reduction system in C. acetobutylicum to obtain the mutant, which was further verified by Southern blotting to test the copy number of the intron. Batch fermentation was implemented in minimum medium to analyze the growth and metabolic products of the wild type and mutant. The wild type and mutant were maintained at acidogenesis and solventogenesis phase to investigate the growth and production in the phosphorus-limited continuous fermentation. Antioxidant capacity was also studied by adding different concentrations of hydrogen peroxide.[Results] The results of resistance screening and gene sequencing showed that a thioredoxin reductase inactivated mutant named C. acetobutylicum trxB::int(29) was successfully constructed. In batch fermentation, the maximum optical density (OD₆₀₀) value of mutant and wild type was similar, reaching 6.5; but the maximum OD₆₀₀ of mutant was obtained at 36 h, 12 h later than that of wild type. Nevertheless, in the acidogenic phase of continuous fermentation, the growth of wild type and mutant had little change. OD₆₀₀ was stable at 4.6 and 4.4, respectively; glucose consumption and acid production were also similar. At the solventogenenic phase, OD₆₀₀ of the mutant was 3.5 and the butanol titer decreased to 36.1 mmol/L, lower than those of the wild type (4.0 and 48 mmol/L); but the acetone titer increased to 25.5 mmol/L compared to the wild type of 18 mmol/L. Furthermore, low concentration of hydrogen peroxide had little effect on the mutant, while high concentration caused more damage to the mutant than the wild type.[Conclusion] The inactivation of thioredoxin reductase gene was not a lethal mutation. The growth and acid production ability of the mutant were similar to those of the wild type, indicating that the inactivation of thioredoxin reductase gene did not affect the metabolic process of acetogenesis. The inactivation of trxB gene mainly affected the redox balance during solventogenic phase, resulting in the increase of acetone/butanol ratio.

Key words:Clostridium acetobutylicum;thioredoxin;phosphorus-limited continuous fermentation;ClosTron system