Shanghai Institute of Pharmaceutical Industry, China.
Mol Biotechnol. 2010 Feb;44(2):101-9. doi: 10.1007/s12033-009-9214-4.
Cephalosporins are widely used as anti-infectious beta-lactam antibiotics in clinic. For the purpose of increasing the yield of cephalosporin C (CPC) fermentation, especially in an industrial strain, A. chrysogenum genes cefEF and cefG, which encode the ultimate and penultimate steps in CPC biosynthesis, cefT, which encodes a CPC efflux pump, and vgb, which encodes a bacterial hemoglobin gene were transformed in various combinations into an industrial strain of A. chrysogenum. Both PCR and Southern blotting indicated that the introduced genes were integrated into the chromosome of A. chrysogenum. Carbon monoxide difference spectrum absorbance assay was performed and the result showed that Vitreoscilla hemoglobin was successfully expressed in A. chrysogenum and had biological activity. HPLC analysis of fermentation broth of recombinant A. chrysogenum showed that most transformants had a higher CPC production level than the parental strain. Multiple transformants containing an additional copy of cefG showed a significant increase in CPC production. However, cefT showed little effect on CPC production in this high producer. The highest improvement of CPC titer was observed in the mutant with an extra copy of cefG + cefEF + vgb whose CPC production was increased by 116.3%. This was the first report that three or more genes were introduced simultaneously into A. chrysogenum. Our results also demonstrated that the combination of these genes had a synergy effect in a CPC high producer.
头孢菌素作为抗感染的β-内酰胺类抗生素在临床上被广泛应用。为了提高头孢菌素 C (CPC)发酵的产量，特别是在工业菌株中，我们将编码 CPC 生物合成最后和倒数第二步的 A. chrysogenum 基因 cefEF 和 cefG、编码 CPC 外排泵的 cefT 和编码细菌血红蛋白基因 vgb 分别或组合转化到一株工业生产用的 A. chrysogenum 菌株中。PCR 和 Southern blot 分析表明，这些基因都已整合到 A. chrysogenum 的染色体上。一氧化碳差光谱吸收检测表明，Vitreoscilla 血红蛋白在 A. chrysogenum 中成功表达并具有生物活性。HPLC 分析重组 A. chrysogenum 的发酵液表明，大多数转化子的 CPC 产量水平均高于亲本菌株。含有额外拷贝 cefG 的多个转化子的 CPC 产量显著增加。然而，在这个高产菌株中，cefT 对 CPC 的产量影响较小。带有额外拷贝 cefG + cefEF + vgb 的突变体的 CPC 效价提高最高，达到 116.3%。这是首次报道同时向 A. chrysogenum 中导入三个或更多基因。我们的结果还表明，这些基因的组合在 CPC 高产菌株中具有协同作用。
