基于丙酰辅酶A通量优化的安丝菌素P-2(AP-2)高产

Optimization of the Metabolic Flux of Propionyl-Co A towards Ansamitocin P-2(AP-2)Yield Improvement

微生物来源的安丝菌素是植物来源的美登素的结构类似物,在传统工业生产中常用来替代美登素作为抗体药物偶联物(ADC)的毒素分子部分,因此获得安丝菌素高产菌株及高产培养基尤为重要。本研究首先在初始培养基中依次添加异亮氨酸、正丙醇和丙酸钠等三碳单元前体,对发酵培养基进行优化。发酵结果表明,添加10 mmol/L异亮氨酸的培养基(FI)可使AP-2产量提高1.23倍,达到(22.12±1.5)mg/L;在FI培养基中添加1.5%正丙醇(FIP),可使AP-2产量再增加74.5%,达到(36.85±3.62)mg/L;在FIP培养基中添加25 mmol/L丙酸钠(FIPP),可使AP-2产量再增加近31%,达到(48.31±0.67)mg/L。在高产培养基基础上,为了进一步提高AP-2产量,使用同源重组双交换方法缺失NXJ24菌株中的丙酰辅酶A羧化酶基因APASM6339;发酵结果表明,使用FIP培养基进行发酵,丙酰辅酶A羧化酶基因APASM6339缺失突变株的AP-2产量比出发菌株NXJ24提高了9.4倍,达到(98.93±0.19)mg/L,获得AP-2高产菌株ZY-1。该研究表明,提高珍贵束丝放线菌中胞内丙酰辅酶A的代谢通量是提高AP-2产量的重要策略,且通过改变安丝菌素C3位不同酰基侧链在珍贵束丝放线菌胞内的比例,可定向改变安丝菌素不同组分的生产比例。

Ansamitocins are analogues of maytansine produced by bacterium Actinosynnema pretiosum,which replace maytansine as the toxic molecule part of ADCs in industry.So it’s very important to improve the yield of ansamitocins by optimizing the medium and engineering the high-yield strain.In this study,three carbon precursors such as isoleucine,N-propanol and sodium propionate were added successively to the culture medium to optimize the fermentation medium.Compared with the starting fermentation medium(F),supplementation of10 mmol/L isoleucine(FI)resulted in an AP-2 titer of(22.12±1.5)mg/L(increased by 1.23 folds);further addition of 1.5%n-propanol(FIP)resulted in an AP-2 titer of(36.85±3.62)mg/L(additional increase of 74.5%),and additional supplementation of 25 mmol/L sodium propionate(FIPP)resulted in an AP-2 titer of(48.31±0.67)mg/L(further increase of 31%).Then,we constructed a mutant ZY-1 to further improve the yield of AP-2 based on the optimized medium,in which the propionyl-CoA carboxylase gene APASM6339 was deleted.Compared with the starting strain NXJ24,the deletion of propionyl-CoA carboxylase gene increased the AP-2 titer to(98.93±0.19)mg/L(net increase of 9.46 folds)in FIP medium.The results suggested that it’s an important strategy for increasing the yield of AP-2,through increasing the metabolic flux of intracellular propionyl-CoA.And the ratio of different components of ansamitocins can be changed by adjusting the ratio of the acyl side chain precursors of C3 in ansamitocins.