Comparison Of Primary Metabolism In High- And Low-yield Acremonium Chrysogenum And Study On CPC Methylation

Cephalosporin C(CPC) is a major precursor of β-lactam antibiotics for the treatment of bacterial infections. The filamentous fungus Acremonium chrysogenum is an important CPC producer. During classical strain improvement procedures, recursive rounds of mutagenesis and selection resulted in a significantly higher CPC producing strain when compared to the wild-type. However, the exact genetic changes underlying the high-yield production are still unclear. Primary metabolism is intrinsically linked to secondary metabolism via the supply of precursors and cofactors, which are limiting factors for antibiotic production in high-yield strains. In this study, we used RNA sequencing data and metabolite profiling to compare primary metabolism in a high-yield and low-yield strain.Firstly, the transcriptional levels of genes involved in central carbon metabolism of Acremonium chrysogenum were analyzed. Genes involved in glycolysis were upregulated at the early stage of fermentation to consume glucose rapidly and begin CPC biosynthesis in the high-yield strain. At the late stage of fermentation, the genes located upstream of the gluconeogenesis pathway were upregulated, while genes located downstream of 3-phosphoglycerate(3PG) were downregulated. We speculated that the carbon flux through gluconeogenesis was channeled to 3PG for serine synthesis in the high-yield strain. In the tricarboxylic acid(TCA) cycle, citrate synthase gene was lower expressed in the high-yield strain, suggesting less pyruvate entered the TCA cycle thus favoring valine synthesis. Moreover, the succinate dehydrogenase gene was upregulated, indicating that malate levels increase, which was oxidized to oxaloacetate entering gluconeogenesis pathway for 3PG synthesis. Transcript levels of isocitrate lyase and malate synthase genes were higher in the high-yield strain, suggesting the glyoxylate cycle in the high-yield strain was more active so that more succinate and was produced. Upregulation of the pentose phosphate pathway genes coding glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase at the early stage of fermentation, and the γ-amino butyric acid shunt genes coding glutamate dehydrogenase and succinate semialdehyde dehydrogenase, especially at the late stage of fermentation, led to more NADPH supply to enhance CPC production in the high-yield strain. Intracellular concentrations of some primary metabolism intermediates were compared between the high-yield strain and low-yield strain. The results showed comparatively great differences in intracellular concentrations of glucose, 3PG, malate and succinate. The variation trend of those primary metabolism intermediates concentrations was consistent with the transcriptional levels of relevant genes.Secondly, the transcriptional levels of genes involved in biosynthesis pathways of CPC amino acid precursors were analyzed. The transcriptions of cysteine biosynthetic gene coding Cystathionine-γ-lyase, Cystathionine-β-synthase and S-adenosylmethionine synthase increased in the high-yield strain. The transcriptional levels of the serine and valine biosynthetic genes in the high-yield strain were higher than that in low-yield strain. The transcriptional analysis of the lysine biosynthesis pathway showed that the first enzyme in the pathway was upregulated and the last enzyme in the pathway was downregulated in the HY strain. Moreover, the concentrations of serine, cysteine, valine and α-aminoadipic acid in the high-yield strain were higher compared to the low-yield strain in agreement with the transcriptional levels of relevant genes.Finally, a study on the methylation of CPC was carried out in vitro and vivo. Gene cmc I and cmc J from Streptomyces clavuligerus was cloned respectively into E.coli expression vector p ET-28a(+) in vitro. Recombinant Cmc I and Cmc J were successful expressed and purified. The purified Cmc I and Cmc J were added into lysates of Streptomyces lividans and Acremonium chrysogenum to convert CPC. The genes cmc I and cmc J were applied to construct two different double expression plasmids in vivo. The two plasmids were transferred respectively to Acremonium chrysogenum and then the fermentation broth of correct transformants was analyzed by LC-MS.