State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Microb Cell Fact. 2018 Jun 7;17(1):87. doi: 10.1186/s12934-018-0936-5.
Cephalosporin C (CPC) produced by Acremonium chrysogenum is one of the most important drugs for treatment of bacterial infectious diseases. As the major stimulant, methionine is widely used in the industrial production of CPC. In this study, we found methionine stimulated CPC production through enhancing the accumulation of endogenous S-adenosylmethionine (SAM). To overcome the methionine dependent stimulation of CPC production, the methionine cycle of A. chrysogenum was reconstructed by metabolic engineering.
Three engineered strains were obtained by overexpressing the SAM synthetase gene AcsamS and the cystathionine-γ-lyase gene mecB, and disrupting a SAM dependent methyltransferase gene Acppm1, respectively. Overexpression of AcsamS resulted in fourfold increase of CPC production which reached to 129.7 µg/mL. Disruption of Acppm1 also increased CPC production (up to 135.5 µg/mL) through enhancing the accumulation of intracellular SAM. Finally, an optimum recombinant strain (Acppm1DM-mecBOE) was constructed through overexpressing mecB in the Acppm1 disruption mutant. In this strain, CPC production reached to the maximum value (142.7 µg/mL) which was 5.5-fold of the wild-type level and its improvement was totally independent of methionine stimulation.
In this study, we constructed a recombinant strain in which the improvement of CPC production was totally independent of methionine stimulation. This work provides an economic route for improving CPC production in A. chrysogenum through metabolic engineering.
产黄青霉产生的头孢菌素 C（CPC）是治疗细菌感染性疾病的最重要药物之一。作为主要的刺激物，蛋氨酸广泛应用于 CPC 的工业生产。在这项研究中，我们发现蛋氨酸通过增强内源性 S-腺苷甲硫氨酸（SAM）的积累来刺激 CPC 的产生。为了克服蛋氨酸对 CPC 生产的依赖性刺激，通过代谢工程重建了产黄青霉的蛋氨酸循环。
通过过表达 SAM 合酶基因 AcsamS 和半胱氨酸-γ-裂解酶基因 mecB，以及敲除一个依赖 SAM 的甲基转移酶基因 Acppm1，分别获得了三株工程菌。AcsamS 的过表达使 CPC 的产量增加了四倍，达到 129.7µg/mL。Acppm1 的敲除也通过增强细胞内 SAM 的积累增加了 CPC 的产量（达到 135.5µg/mL）。最后，通过在 Acppm1 敲除突变株中过表达 mecB，构建了一个最佳的重组菌株（Acppm1DM-mecBOE）。在该菌株中，CPC 的产量达到了最大值（142.7µg/mL），是野生型水平的 5.5 倍，其提高完全不依赖于蛋氨酸的刺激。
在这项研究中，我们构建了一个重组菌株，其中 CPC 产量的提高完全不依赖于蛋氨酸的刺激。这项工作通过代谢工程为提高产黄青霉中的 CPC 生产提供了一种经济的途径。
