Metabolic Engineering Of Pichia Pastoris For S-adenosyl-L-methionine Production

S-adenosyl-L-methionine(SAM)is an important biological sulfonium compound that is involved in many essential biochemical processes,and can be used in pharmaceutical and healthcare area.So,it has broad application prospects.Several SAM producer strains have been constructed in our lab from the wild type Pichia pastoris GS115:DS16(overproducing a recombinant SAM synthetase),G12(down regulating the expression of cystathionine β-synthase gene cys4 in DS16),G/Dspe(knocking-out SAM decarboxylase gene spe2 in DS16),DS16/DsvA and DS16/DsvP(knocking-in Vitreoscilla hemoglobin gene vgb at the spe2 locus in DS16,driven by PAOX and PsADH2 respectively).To further enhance SAM synthesis,we carried out metabolic engineering by analyzing the physiological properties of these recombinant strains.Under oxygen non-limiting conditions,DS16/DsvA and DS16/DsvP showed higher maximum specific growth rate and viability,and exhibited an increase of 14.3%and 4.6%in SAM production,respectively,in comparison to G/Dspe.Under oxygen limiting conditions,DS16/DsvA and DS16/DsvP exhibited a higher viability and showed an increase of 19.1%and 9.6%in SAM production,respectively,in comparison to G/Dspe.In a 5-L fermenter,the effect of VHb on the growth and production performance in recombinant strains was investigated at the dissolved oxygen level of 10-15%.The SOUR values of DS16/DsvA and DS16/DsvP were significantly higher than those of DS16 and G/Dspe at 30 h after induction.At the end of fermentation,it was found that inserting vgb gene at the spe2 locus could increase SAM production compared with deletion of the spe2 gene.The highest production was achieved in DS16/DsvP,where the SAM production was 56.8%and 17.0%higher than in DS16 and G/Dspe,respectively.Meanwhile,the methanol conversion rate was increased by 100%and 33.3%,in comparison with DS16 and G/Dspe.Based on the above results,it was concluded that expression of vgb gene could not only promot cell growth,but also improve SAM production under oxygen limiting conditions.The comparative transcriptome analysis was performed to identify genes that responded to the accumulationof SAM using engineered strains DS16 and G12.Differentially expressed genes associated with TCA cycle,pentose phosphate pathway,purine metabolism,and nitrogen pathways were analyzed to screen for candidate genes for further improving SAM production through targeted knocking-in or knocking-out.Overexpression of mdh and idh did not result in significant change in production,the insertion of gnd caused a decrease of 14.7%in SAM production,and the overexpression of gin led to a 13.5%increase.The Rps28b knocked-out strain was unable to grow,while the deletion of gdh2 led to a 20.9%decrease in SAM production.These results will provide useful guidance for fermentation process optimization and metabolic engineering in different recombinant strains.