Construction Of Genetically Engineered Strain Of Saccharomyces Cerevisiae With High Yield Of Methylparaben

Methylparaben(MP)is an aromatic compound,which has great demand in the industrial market as an antibacterial agent,preservative and feed additive,and also has potential application value in the preparation of bio-based polyetherester materials.It is of great significance to develop an environmentally friendly MP synthesis method by means of synthetic biology.Saccharomyces cerevisiae,as a typical eukaryotic model bacteria,provides a broad platform for the synthesis of aromatic compounds and their derivatives.In this paper,using Saccharomyces cerevisiae BY4741 as the chassis cell,the gene encoding pyruvate lyase Ubic derived from Escherichia coli and the gene Bsmt-1 encoding benzoate carboxymethyltransferase derived from Australian tobacco were introduced into Saccharomyces cerevisiae cells,biosynthetic pathways to achieve MP.To further enhance the biosynthetic yield of MP,various metabolic engineering strategies were employed in this study.First,the central carbon metabolism pathway of the host cell was modified to enhance the central metabolic flux:(1)The carbon flux of the shikimate pathway was enhanced by overexpression of key enzyme genes of the shikimate pathway and knockout of the competing pathway;(2)Increase the flux of phosphoenolpyruvate(PEP)into shikimate by replacing the pyruvate kinase gene with a weaker promoter and knocking out the genes encoding pyruvate decarboxylase(PDC1,PDC5,PDC6);(3)The introduction of phosphoketolase derived from Bifidobacterium breve enables the rapid and efficient synthesis of 4-phospho-erythrose(E4P)from fructose-6-phosphate,thereby increasing the content of E4 P.The MP content of the optimized strain was 41.91 mg/L.Secondly,on the basis of enhancing the level of precursor supply,various strategies and regulatory mechanisms were explored to improve the activity of benzoate carboxymethyltransferase:(1)The promoter of pyruvate lyase and benzoate carboxymethyltransferase were combined.They were replaced with constitutive promoters of different strengths to obtain the most efficient promoter-driven combination;(2)The chorismate pyruvate lyase and the benzoate carboxymethyltransferase were designed as fusion proteins to increase the benzoate carboxylate Methyltransferase expression;(3)Overexpression of the MYB transcription factor-EOBI that regulates the shikimate pathway.Comparing the three strategies,the recombinant strains can synthesize MP up to 51.78 mg/L.Finally,a single-factor variation experiment was carried out on the composition and content of the fermentation medium,and an orthogonal experiment was designed to optimize the carbon source,nitrogen source,and carbon-nitrogen ratio,and the optimal strain obtained by combining a variety of metabolic engineering strategies produced MP up to 68.59 mg/ L.This is also the highest yield of MP biosynthesis by Saccharomyces cerevisiae,and the modified optimal strain also provides a production platform for other aromatic compounds.