Effects Of PAS＿chr1-3＿0225 Gene Deletion On S-adenosyl Methionine Synthesis In Pichia Pastoris

Pichia pastoris is a platform strain widely used for recombinant protein expression and biocatalysis.It can not only carry out post-translational modification but also achieve high cell density fermentation.In recent decades,many proteins of P.pastoris have been successfully expressed,but few studies have been carried out to modify the structure of it.Some cell wall structures are not necessary for P.pastoris growth,resulting in waste of carbon source and energy.Based on this,this study attempted to reasonably simplify the redundant structure of cell wall to reduce carbon source consumption,promote growth and product synthesis,so as to obtain excellent chassis cells.Our team has conducted many years of research on the synthesis of S-adenosine methionine（SAM）catalyzed by P.pastoris（A methyl donor for the treatment of depression and osteoarthritis）.Therefore,in this paper,we investigated the expression of SAM synthase and the efficiency of SAM synthesis in the modified P.pastoris.Firstly,P.pastoris GS115 was used as the starting strain for rapid and unlabeled genome engineering.In this study,we investigated the effects of deletion of some genes related to the cell wall of P.pastoris on the growth and expression of target proteins,and used the modified deletion strains for the fermentation expression of SAM synthase and the effects of glycerol to methanol carbon source conversion on lipidomics of P.pastoris were investigated.The lipid changes in the process of glycerol to methanol carbon source conversion were analyzed and compared,and the lipid oxidation caused by the addition of methanol in P.pastoris fermentation was investigated.The methods and results are as follows:（1）Using CRISPR/Cas9 technology,β-1,3-glucan,β-1,6-glucan andα-1,2-mannose-related genes of P.pastoris GS115 cell wall were knocked out.Four single gene deletion strains were successfully constructed:ΔPAS＿chr3＿0370、ΔPAS＿chr1-3＿0225、ΔPAS＿chr1-3＿0114 andΔPAS＿chr3＿0960.One double gene deletion strain:ΔPAS＿chr3＿0370ΔPAS＿chr1-3＿0225.（2）Growth test results show that:The growth rate ofΔPAS＿chr3＿0370、ΔPAS＿chr1-3＿0225 andΔPAS＿chr3＿0370ΔPAS＿chr1-3＿0225 was significantly higher than GS115.When glucose was used as carbon source,ΔPAS＿chr3＿0370ΔPAS＿chr1-3＿0225 had the highest carbon source yield of 0.72 g·g^-1,which was 0.07 g·g^-1 higher than GS115.When glycerol was used as carbon source,ΔPAS＿chr3＿0370 had the highest carbon source yield of 0.51g·g^-1,which was 0.10 g·g^-1 higher than GS115.Cell wall polysaccharides showed that the structure of the cell wall was flexible and complex.The results show thatΔPAS＿chr1-3＿0225 andΔPAS＿chr3＿0370ΔPAS＿chr1-3＿0225 achieve the goal of cell wall reduction.Comparing the expression of green fluorescent protein,the results showed thatΔPAS＿chr1-3＿0225 had the best expression of green fluorescent protein,and the fluorescence intensity ofΔPAS＿chr1-3＿0225was 3594,which was 1.6 times of GS115.（3）SAM synthase expression was compared in shaker and fermenter levels.ΔPAS＿chr1-3＿0225 produced the highest SAM yield at shaker level,which was 0.94 g·L^-1,17.8%higher than GS115.The biomass of SAM produced byΔPAS＿chr1-3＿0225 and maximum yield were204 g·L^-1 and 2.13 g·L^-1,respectively,in a 2 L fermenter after methanol induction for 96 h,which were 13 g·L^-1 and 0.54 g·L^-1 higher than GS115,respectively.The yield of SAM per unit wet weight was 10.44 mg·g^-1,which was 2.12 mg·g^-1 higher than GS115.（4）Due to the bacterial decline and oxidative stress of induced P.pastoris,the mechanism of GS115 was explored and the foundation was laid for further optimization of GS115.The changes of lipidome in induced P.pastoris during carbon source transformation were studied.The result showed:253 lipids were identified during the conversion of carbon source from glycerol to methanol,including glycerol,glycerophospholipids and sphinolipids.It was found that the changes of lipids containing unsaturated double bonds were closely related to oxidative stress induced by methanol addition,and the relative content of sphinolipids increased suddenly at the late stage of methanol induction,which may be related to apoptosis induced by oxidative stress.Studies have shown that deletion of PAS＿chr1-3＿0225 can not only improve the growth rate and maximum biomass,but also is beneficial to the expression of SAM synthase and SAM synthesis.Therefore,GS115ΔPAS＿chr1-3＿0225 is superior to GS115 platform strain.This study will provide support for the transformation of platform strain of P.pastoris.