代谢工程改造肉桂地链霉菌还原力供给系统提升莫能菌素产量

Enhancement of monensin production by metabolic engineering of the redox cofactor supply in Streptomyces cinnamonensis

摘要:

莫能菌素是由肉桂地链霉菌(Streptomyces cinnamonensis)合成的一种聚醚类离子载体抗生素，在畜牧领域应用广泛。为解决其工业发酵中胞内还原力(NADPH/NADH)供给不足导致的产量瓶颈，本研究采用多途径协同代谢工程策略进行理性改造。通过将磷酸戊糖途径基因zwf (编码葡萄糖-6-磷酸脱氢酶)和gnd (编码6-磷酸葡萄糖酸脱氢酶)，与脂肪酸β-氧化途径基因fadB (编码3-羟酰基-ACP脱水酶)串联构建融合表达盒，克隆至含ermE强启动子和fd终止子的整合型载体pSET152中，并通过属间接合转移导入肉桂地链霉菌SDSL6002 (WT)，获得重组菌株S-zwf-gnd-fadB。摇瓶发酵结果显示，工程菌胞内NADPH与NADH总量约为野生型的1.8倍，莫能菌素效价达22.6 g/L，产量提高51.7%，在无抗性传代5代后产量保持稳定。进一步在50 L和5 m³生产规模发酵罐中进行放大验证，工程菌株效价达37.2 g/L和40.4 g/L，较对照菌株分别提升16.3%和18.8%，展现出优异的工艺放大适应性与规模化生产潜力。本研究通过多基因协同表达系统强化了还原力供给网络，为莫能菌素的工业生物制造提供了具有应用潜力的高产工程菌株和可行的代谢工程策略。

Abstract:

Monensin, a polyether ionophore antibiotic produced by Streptomyces cinnamonensis, is widely used in the livestock industry. To address the yield bottleneck caused by insufficient intracellular reducing power (NADPH/NADH) during industrial fermentation, we employed a multi-pathway collaborative metabolic engineering strategy. We fused the genes zwf (encoding glucose-6-phosphate dehydrogenase) and gnd (encoding 6-phosphogluconate dehydrogenase) from the pentose phosphate pathway with fadB (encoding 3-hydroxyacyl-ACP dehydratase) from the fatty acid β-oxidation pathway to construct a synthetic expression cassette. This cassette was cloned into the integrative vector pSET152 under the control of the strong ermE promoter and fd terminator and then introduced into S. cinnamonensis SDSL6002 (WT) via intergeneric conjugation, yielding the recombinant strain S-zwf-gnd-fadB. Shake-flask fermentation demonstrated that the engineered strain achieved the intracellular NADPH/NADH level 1.8 folds of that in the wild type, with a monensin titer of 22.6 g/L (a 51.7% increase). The yield remained stable after five generations of antibiotic-free subculturing. Further scale-up validation in 50 L and 5 m³ industrial fermenters demonstrated that the engineered strain achieved monensin titers of 37.2 g/L and 40.4 g/L, which represented 16.3% and 18.8% improvements, respectively, over that of the control strain. These results highlight the exceptional scalability and industrial production potential of the engineered strain. This study establishes a multi-gene co-expression system to reinforce the reducing power network, providing a high yield engineered strain and a feasible metabolic engineering approach for industrial biosynthesis of monensin.