Biosynthesis Of Squalene From Recombinant Saccharomyces Cerevisiae

Squalene（C30H50）is a polyunsaturated triterpenoid compound with an all-trans linear structure.Squalene has natural antioxidant and anti-cancer activities and is widely used in food additives,drug delivery and skin care.The traditional production method of squalene is animal and plant extraction,but this method has some problems,such as complicated extraction steps,long production cycle,large amount of raw materials,and high cost.The chemical production of squalene has complex synthesis routes,demanding catalyst requirements and environmental pollution.Compared with the above two production methods,the biosynthetic production of squalene has the advantages of shorter production cycle,environmental protection,simple extraction and mild production conditions.In this study,the recombinant S.cerevisiae strain was constructed by metabolic engineering to efficiently synthesize squalene,which laid the foundation for squalene biosynthesis.The main results were as follows:（1）Enhancing the expression of key enzymes in squalene biosynthesis pathway to enhance squalene accumulation.First,the key enzyme genes in the squalene biosynthesis pathway affecting squalene concentration were identified by individually enhancing the expression of the enzyme genes in the squalene biosynthesis pathway.Then,the key enzyme genes were combined to enhance expression,and six recombinant S.cerevisiae strains were constructed.The effects of different combinations of key enzyme genes on the concentrations of squalene and ergosterol were investigated.The regulation of squalene epoxidase（ERG1）expression level by promoter modification and point mutation was studied.The results showed that t HMG1,MVD1,ERG20,IDI1,ERG9 and ERG8 were the key genes affecting squalene concentration in the squalene synthesis pathway.After fermentation with recombinant strains IL,ID,IH,ILD,ILH and ILDH,strain IL showed the highest concentration of squalene at 34.0 mg/L,while strain ILDH showed the highest concentration of ergosterol at 12.2 mg/L.After the expression level of ERG1 in IL,ID,ILD,ILH and ILDH was down-regulated by promoter modification,the squalene concentration of recombinant ILp reached 46.2 mg/L,which was 35.9%higher than that before the modification.The concentration of squalene in strain ILDHpreached 38.6 mg/L,which was 294.3%higher than that before the transformation,and the highest concentration of squalene was 42.9 mg/L detected at 24 h.Strain ILp G30S was constructed by introducing ERG1 mutant on the basis of ILp.The ergosterol content of strain ILp G30S was slightly lower than that of strain ILp,but the concentration of squalene was not further increased.（2）Enhancing the supply of precursor substances for squalene synthesis promotes the further accumulation of squalene.The constructed recombinant ILp,ILp G30S and ILDHpwere used as starting strains to convert acetyl-Co A in mitochondria to MVA,the precursor of squalene synthesis,using mitochondrial localization signal peptides.The transcription level of mevalonate pathway（MVA pathway）genes in the constructed recombinant strain was analyzed,and the expression of low transcription level genes was enhanced by increasing the copy number of low transcription level genes.Enhancing the ethanol metabolic pathway to convert ethanol to acetyl-coa provides more precursors for squalene production.The results showed that after overexpression of ERG10,ERG13 and t HMG1 in mitochondria using the galactose promoter,the mitochondrial acetyl-coa was successfully introduced into squalene production,and the mitochondrial engineering recombinant strains ILp C,ILp G30SC and ILDHpC were constructed.The concentration of squalene in the recombinant strain ILDHpC reached 261.1 mg/L（47.1 mg/g DCW）,which was 576.4%higher than that before mitochondrial engineering.Furthermore,increasing the copy number of genes with low expression levels of MVA pathway in strain ILDHpC did not have any significant effect on squalene production.The recombinant strain R was constructed by enhancing ADA and ADH2 expression in the recombinant strain ILDHpC,which could effectively metabolze ethanol to acetyl-coa,the precursor of squalene production.The maximum concentration of squalene in strain R reached 316.1 mg/L（56.7 mg/g DCW）,which was 20.6%higher than that in ILDHpC.A squalene producing strain RD without galactose induction was obtained using recombinant strain R.（3）To optimize the fermentation process of high-yield engineering bacteria for squalene.By optimizing the type and concentration of carbon source,the type and addition ratio of nitrogen source,and the addition concentration of metal ions in shake flask,as well as the fed-batch fermentation process in a 5 L fermenter,the concentration of squalene synthesized by the engineered strain RD was effectively increased.The results showed that sucrose was more conducive to the accumulation of squalene than glucose,and the optimal addition concentration was 60 g/L.The optimal nitrogen source was the mixture of peptone and yeast powder,and the optimal total nitrogen concentration was 45 g/L.Addition of K₂HPO₄and MgSO4 favored the synthesis of squalene.On this basis,the RD nitrogen source addition ratio and metal ion addition concentration of engineering bacteria were optimized by response surface experimental design method,and the best nitrogen source addition ratio was peptone:The concentration of squalene reached 604.8 mg/L with yeast powder=4:1 and 0.1g/L K₂HPO₄and 0.35 g/L MgSO₄.The high yield engineered strain RD was used as the object to study the process of 5 L fermenter.When the inoculum amount was 2%,the dissolved oxygen was controlled at about 40%,and the pH was controlled at 5.8,A two-stage feeding process was adopted,in which medium A containing 360 g/L sucrose was added for12-48 h,and medium B containing 50%ethanol was added for 60-120 h.The maximum concentration of squalene reached 1929.7 mg/L.