Synthesis Of β-amyrin By Enhancing The Acetyl-CoA Pathway In Saccharomyces Cerevisiae

β-amyrin is a pentacyclic triterpene compound,which is also a biosynthetic framework of other oleanane-type triterpenes,such as glycyrrhetinic acid and soybean saponin,β-amyrin has good pharmacological properties such as anti-inflammatory,antibacterial and anti-virus.The synthesis ofβ-amyrin has been successfully achieved in Saccharomyces cerevisiae by metabolic engineering and synthetic biology.However,the yield ofβ-amyrin is relatively low due to insufficient supply of precursors,accumulation of toxic intermediates and feedback regulation,greatly limiting its large-scale production and wide application.Aiming at the supply of acetyl-Co A,an important precursor in the synthesis ofβ-amyrin,metabolic engineering strategies were used to enhance the synthesis of cytoplasmic acetyl-Co A and to overexpress t HMG1,ERG genes involved in the synthesis ofβ-amyrin to enhance the synthesis of squalene to achieve efficient synthesis ofβ-amyrin in S.cerevisiae.The results are as follows:(1)For increasing the content of cytoplasmic acetyl-Co A in S.cerevisiae and increasing the yield ofβ-amyrin,we focused on enhancing the endogenous acetyl-Co A pathway and constructing the exogenous acetyl-Co A pathway in S.cerevisiae.Strengthening of the endogenous acetyl-Co A pathway includes:(1)Through overexpression of S.cerevisiae alcohol dehydrogenase ADH2 in plasmid,which catalyses ethanol to acetyl-Co A synthetic pathway,β-amyrin content reached 3.97mg/g,1.52 times relative to the base yeast strain;(2)Overexpression of aldehyde dehydrogenase and acetyl-Co A synthase genes ACS1 and ACS2 at S.cerevisiae HO site,the yield ofβ-amyrin reached 30.4and 31.24 mg/L,which were 1.3 times and 1.34 times relative to the base yeast strain.However,using 1 molecule of glucose as substrate,2 molecules of acetyl-Co A were synthesized by the pyruvate dehydrogenase bypass pathway,and the net consumption of 2molecules of ATP affected the production efficiency ofβ-amyrin.Pyruvate dehydrogenase bypass pathway in S.cerevisiae consumes more ATP,leading to the low efficiency of acetyl-Co A synthesis,in order to reduce or eliminate the ATP consumption of cytoplasmic acetyl-Co A synthesis,researchers introduced exogenous acetyl-Co A synthetic pathways into S.cerevisiae.The construction of the exogenous acetyl-Co A pathway for promoting the synthesis ofβ-amyrin in S.cerevisiae genome mainly includes:(1)The pyruvate dehydrogenase pathway was constructed in S.cerevisiae by introducing three heterologous genes:pyruvate dehydrogenase gene(ace E),dihydrolipoyl transacetylase gene(ace F)and dihydrofolious lipase gene(lpdm).By knocking out glucose-6-phosphate dehydrogenase gene,the pyruvate dehydrogenase complex can use more NADP~+,which effectively increasedβ-amyrin production to 33.2mg/L,1.49 times relative to the base yeast strain;(2)By introducing the codon-optimized citrate lyase from Aspergillus nidulans,the citrate lyase pathway was successfully constructed in S.cerevisiae,directly catalyzing cytoplasmic citrate to acetyl-Co A.By adding citric acid in vitro,theβ-amyrin content reach 31.05mg/L,1.53 times relative to the base yeast strain;(3)By introducing acetoacetaldehyde dehydrogenase,xylulose-specific phosphoketolase and phosphotransacetylase,two heterologous acetyl-Co A synthetic pathways were successfully constructed in Saccharomyces cerevisiae,and the NADH balance of the pathway was enhanced by expressing NADH-HMGR from Silicibacter pomeroyi.As a result,the yield ofβ-amyrin reached 39.49 mg/L,1.95 times relative to the base yeast strain.Knocking out citrate synthase to prevent the loss of cytoplasmic acetyl-Co A,further increasedβ-amyrin yield to 69.64 mg/L,3.34 times relative to the base yeast strain;(2)To strengthen the squalene synthesis,the rate-limiting enzyme t HMG1 of mevalonate pathway was overexpressed to enhance squalene synthesis,which increasedβ-amyrin yield to 4.19mg/g,1.7 times relative to the base yeast strain;Finally,ADH2 and t HMG1 were overexpressed in Saccharomyces cerevisiae,effectively increasedβ-amyrin yield to 32.01 mg/L,1.37 times relative to the base yeast strain;(3)In terms of fermentation optimization,the strain BA40 with high yield ofβ-amyrin was performed by fed-batch fermentation.β-amyrin yield reached 272.42mg/L,3.91 times of shake flask fermentation,the highest yield reported in S.cerevisiae to our knowledge.