The Construction And Metabolic Transformation Of Alkylresorcinol Producing Engineering Saccharomyces Cerevisiae

ARs are 1,3-dihydroxybenzene derivatives with an odd-numbered alkyl chain at position 5 of the benzene ring.They are important active constituents and markers for the cortex of cereals.The food industry standard(LS/T3244-2015)judges important biomarkers of whole wheat flour,and has physiological activities such as anti-oxidation,anti-parasitic,anti-tumor,antibacterial,hypoglycemic and lipid-lowering.At present,most of the ARs are extracted from plants,and the presence of low ARs cannot meet the market demand in plants.This paper uses biosynthesis methods to mass produce ARs.Saccharomyces cerevisiae(S.cerevisiae)serves as a widley used biotechnological production organism as well as a eukaryotic model system and known genome.It has the advantages of non-toxicity,acid resistance,simple genetic manipulation and easy cultivation.In this study,S.cerevisiae was used as the expression host,and the optimized Secale cereale,sorghum bicolor ARSs gene and pRS42 H vector were used to construct the recombinant plasmid,and the recombinant plasmid was transformed into S.cerevisiae by LiAc/PEG chemical transformation method.The ARs genetic engineered strains SN001,SN002 and SN003 were cultured and fermented in 30 °C and YPD medium.The ARs produced were 44.5±3.1 ?g/mL,23.5±2.3 ?g/mL and 28.3 ± 2.9 ?g/mL by SN001,SN002 and SN003 recombinant yeast,respectively.Acetyl-CoA is an important active intermediate in vivo.In order to study its effect on the synthesis and accumulation of ARs in yeast,knockout the gene encoding the branch of acetyl-CoA in yeast and overexpress the gene synthesized by acetyl-CoA.In this paper knock out MLS1(Malate synthase)and CIT2(Citrate synthase)genes,and investigate the synthesis ability of each mutant strain ARs.It was found that the deletion of MLS1 and CIT2 genes alone is beneficial for improve to synthesis and accumulation of ARs by recombinant yeast strains.The deletion of MLS1 gene resulted in the highest yields of ARs for SN009,SN011 and SN013 strains of 60.9±2.2 ?g/mL,54.6±3.7 ?g/mL and 50.5±4.3 ?g/mL,respectively.which were 0.4-,1.3-and 0.65-fold higher than recombinant yeast strains.The CIT2 gene deletion resulted in the highest yields of ARs for SN010,SN012 and SN014 strains was 58.7±3.2 ?g/mL,50.3±2.6 ?g/mL and 63.1±2.1 ?g/mL,respectively,which was 0.56-,1.19-,and 1.22-fold higher than that of the recombinant yeast strain.in order to further increase the yield of ARs,and knock out the MLS1 and CIT2 genes in S.cerevisiae,the highest yields of ARs for SN016,SN017 and SN018 strains was 75.4±1.8 ?g/mL,60.9±1.2 ?g/mL and 68.6±2.6?g/mL,respectively.which were 0.7-,1.67-and 1.41-fold higher than recombinant yeast,respectively.S.cerevisiae genome was used as a template to amplify the ACS1(Acetyl-CoA synthetase,ACS1)and DGA1(DiacylGlycerol Acyltransferase,DGA1)genes,and the transformed S.cerevisiae expression vector pESC-trp was ligated with ACS1 and DGA1 genes and the target gene.Overexpression of the plasmid,and then overexpression was transformed plasmid into yeast to construct an overexpressing yeast strain.The results showed that the highest yields of ARs for SN020,SN022 and SN024 strains were 72.6±3.3 ?g/mL,51.6±3.2 ?g/mL and 59.3±3.5 ?g/mL,respectively,which were improved 0.6-,1.2-and 1.1-fold compared with recombinant yeast strains.The highest yields of ARs for SN021,SN023 and SN025 strains reached 54.6±2.9 ?g/mL,32.8±2.5 ?g/mL and 39.6±3.1 ?g/mL,respectively,which were 0.2-,0.4-and 0.4-fold higher than that of recombinant yeast strains.In this paper,the optimized strains of ARSs(Alkylresorcinol synthase,ARSs)was used to construct the ARSs of S.cerevisiae engineering strains,and the ability of different engineering bacteria to biosynthesize alkylresorcinol was studied.Metabolic transformation lays the foundation for the preparation of ARs by biosynthesis.