Fermentation Optimization For S-adenosyl-L-methionine Prodution And Transcriptome Analysis In Recombinant Pichia Pastoris Strains

As the second most widely used enzyme substrate after ATP, S-adenosyl-L-methionine (SAM) has wide applications in clinic and health care. The SAM producing strain DS16 had been constructed in our lab by integration of a recombinant methionine adenosyltransferase gene(dsl6) in Pichia pastoris strain GS115, and recombinant strains G12-CBS, G/Dspe, G/Dmsm and G/Dsah had been obtained by further knocking out or downregulation of genes involved in SAM conversion pathways. In this paper, the fermentation process of SAM production in G12-CBS was optimized, and comparative transcriptome analysis was performed using these recombinant strains.Genetic manipulation did not affect cell growth in these four recombinant strains in the shake flasks and fermentors. In comparison with the original strain DS16, SAM production was increased by 73.7%,38.9%,46.1% and 86.6% in G/Dspe, G/Dmsm, G/Dsah and G12-CBS, respctively, in the fermentors. In order to enhance SAM production in G12-CBS, L-methionine feeding strategy was optimized. The maximum SAM production and cell growth were reached with the L-Met addition amount of 0.2%(w/v)/24 h in the shake flasks. In 15 L fermentors,the optimal L-Met feeding rate was 0.4 g/L/h,at which speed the maximum SAM production (13.01 g/L) was obtained. The analyses of key metabolites and enzyme activity indicated that the bottleneck for SAM production with the low L-Met feeding rate was the insufficient L-Met supply, but both the tricarboxylic acid cycle and nitrogen uptake were reduced with the high L-Met feeding rate, which was probably the reason for the low SAM accumulation and cell growth inhibition.The effect of dsl6 overexpression, cys4 downregulation and L-Met addition on recombinant strains was investigated by comparative transcriptome analysis in the recombinant strain. The results showed that dsl6 was transcribed intensively in DS16, and the genes related to methanol energy metabolism, TCA cycle and nitrogen uptake were remarkably upregulated, which might be the reason for improved cell growth and SAM production in DS16. The additional downregulation of cysA in G12-CBS resulted in the further upregulation of ds16, the alteration of transcription level of genes involved in SAM conversion leading to the low SAM transformation in G12-CBS, and downregulation of the genes involved in TCA cycle and nitrogen uptake. In response to L-Met addition in G12-CBS, although the genes related to methanol energy metabolism were upregulated, but the genes involved in AMP synthesis, TCA cycle and nitrogen uptake were significantly downregulated, which might affect ATP synthesis, cell growth and SAM synthesis.