秸秆水解液发酵产丁二酸高产菌株的选育及其代谢调控

采用酸解与酶解处理秸秆并对菌株代谢途径进行分析，以软X射线诱变选育乙酸及乳酸代谢阻断突变株，并围绕HMP与EMP流量比调控H还原力以及围绕木酮糖激酶实施五、六碳糖共代谢调控。结果表明200 g秸秆水解得57 g葡萄糖和11 g木糖，出发株代谢流量显示丁二酸、乳酸及乙酸流量分别为2.547、0.726、0.611 mmol•g^-1•h^-1。乳酸脱氢酶突变株HF-1代谢分析显示，其乳酸流量降至0.0296 mmol•g^-1•h^-1，氟乙酸抗性突变株HF-2的代谢分析显示，其乙酸流量降至0.100 mmol•g^-1•h^-1，丁二酸流量有所下降。H还原力代谢平衡调控显示，少量柠檬酸盐的添加使HMP与EMP流量比从1.389∶0.389增至1.684∶0.330时，间接促进丁二酸代谢流量从3.005 mmol•g^-1•h^-1增至3.468 mmol•g^-1•h^-1。酶活调控显示，碳酸镁取代碳酸钙作为中和剂促使木酮糖激酶酶活从76 U•mg^-1增至560 U•mg^-1，进而使木糖代谢流量从0.0444 mmol•g^-1•h^-1增至0.453 mmol•g^-1•h^-1，最终五、六碳糖共代谢产丁二酸达68.7 g•L^-1。

Directed breeding and metabolic regulation for Actinobacillus succinogenes producing succinic acid from crop straw hydrolyte

Pentose and hexose hydrolyzed from crop straw with acid and enzyme were determined by HPLC.Based on the discussion of corresponding product and enzyme, the metabolic pathway was analyzed.For interdicting lactic acid metabolic pathway and acetic acid metabolic pathway, the parent strain was mutated by soft X-ray of synchronous radiation.In order to improve the balance of H reducing power, the flux ratio of HMP to EMP was modulated by adding citrate.Then the xylulokinase activity was regulated for the co-fermentation of pentose and hexose.The results showed that about 57 g glucose and 11 g xylose were hydrolyzed from 200 g crop straw.Metabolic flux analysis of the parent strain indicated that the flux of succinic acid,lactic acid and acetic acid were 2.547 mmol•g^-1•h^-1, 0.726 mmol•g^-1•h^-1 and 0.611 mmol•g^-1•h^-1, respectively.Lactic acid flux of Ldh mutant strain HF-1 decreased markedly to 0.0296 mmol•g^-1•h^-1.Acetic acid flux of anti-fluoroacetic mutant strain HF-2 decreased distinctly to 0.100 mmol•g^-1•h^-1.However, the decrease of acetic acid resulted in not strengthening but weakening the metabolism of succinic acid.By adding citrate the flux ratio of HMP to EMP increased from 1.389∶0.389 to 1.684∶0.330 and thus the H reducing power was better balanced, then the succinic acid flux increased from 3.005 mmol•g^-1•h^-1 to 3.468 mmol•g^-1•h^-1 accordingly.Substituting MgCO₃ for CaCO₃ as neutralizer increased xylulokinase specific activity to 560 U•mg^-1 from 76 U•mg^-1, and thus the consumed xylose flux increased from 0.0444 mmol•g^-1•h^-1 to 0.453 mmol•g^-1•h^-1 accordingly, then pentose and hexose were co-fermented and the succinic acid yield reached 68.7 g•L^-1.