丙酮丁醇梭菌半胱氨酸合成途径中铁氧还蛋白和胱硫醚-γ-裂解酶基因功能

Role of ferredoxin and cystathionine γ-lyase in cysteine biosynthesis of Clostridium acetobutylicum

摘要:

[目的] 探究丙酮丁醇梭菌半胱氨酸合成代谢途径上铁氧还蛋白和胱硫醚-γ-裂解酶基因的功能。[方法] 使用ClosTron系统对半胱氨酸合成途径上的铁氧还蛋白基因（fer）和胱硫醚-γ-裂解酶基因（mccB）进行失活，得到突变株；在不同硫源的培养基中进行分批发酵，分析突变株的生长特点；通过pH控制，使用限磷的连续发酵方法将丙酮丁醇梭菌维持在产酸期和产溶剂期，分析野生型菌株和突变株在连续发酵中的生长情况。[结果] 成功构建Δfer和ΔmccB突变株。在分批发酵中，敲除fer基因的突变株无法利用硫酸盐作为硫源，但添加亚硫酸盐或半胱氨酸可以使其恢复生长；在以半胱氨酸为唯一硫源进行分批发酵时，其终浓度1 mmol/L时不会影响野生型与Δfer突变株的生长，但高于1 mmol/L时生长均会受到抑制。在连续发酵中，Δfer突变株不能在产溶剂阶段生长，添加过量的半胱氨酸也不能恢复生长；敲除mccB基因的突变株仍能在添加甲硫氨酸的培养基中生长，但最大OD仅为野生型的57%；相较于野生型，ΔmccB突变株在产酸期和产溶剂期的生长均受到抑制。[结论] fer基因为半胱氨酸合成途径中硫酸盐还原为亚硫酸盐的关键基因，其控制合成的半胱氨酸不能完全由外源的半胱氨酸替代，敲除后对生长的抑制主要表现在连续发酵中的产溶剂阶段。mccB基因参与调控甲硫氨酸转化为半胱氨酸的过程，其敲除会影响甲硫氨酸到半胱氨酸的转化，但不会阻断该生物反应过程。

Abstract:

[Objective] This study aims to investigate the roles of ferredoxin and cystathionine γ-lyase genes located in cysteine metabolic pathway of Clostridium acetobutylicum.[Methods] ClosTron technology is used to inactivate target genes to obtain mutants. The mutants, cultured in the phosphate-limited medium, are maintained at acidogenesis and solventogenesis phase at controlled pH under continuous fermentation condition. The OD and glucose uptake of wild type and mutants are determined.[Results] The Δfer and ΔmccB mutants are obtained by using ClosTron system. The Δfer mutant cannot use sulfate as the sole sulfur source, but it will recover growth by adding sulfite or cysteine in batch fermentation. Moreover, cysteine is harmful to the growth of C. acetobutylicum and the Δfer mutant as the sole sulfur source, and it mainly influences the growth in solventogenic phase. The ΔmccB mutant also grows poorly during acidogenic and solventogenic phases with methionine or cysteine as the sole sulfur source.[Conclusion] Cysteine is a key sulfur-containing compound in the metabolic pathway of C. acetobutylicum and its metabolism is tightly controlled. Ferredoxin participates in its biosynthesis through reduction reactions of sulfate to sulfite; while the cystathionine γ-lyase participates in another cysteine biosynthesis pathway from methionine to cysteine, which is not essential.