Enhanced Biosynthesis Of Malonyl-CoA Derivatives In Saccharomyces Cerevisiae Based On β-Oxidation

Acetyl-CoA is a key metabolic intermediate of carbon metabolism,lipid metabolism and amino acid catabolism,which participates in many metabolic processes and biochemical reactions in cells and provides carbon source and energy for cell growth and metabolism.In Saccharomyces cerevisiae,acetyl-CoA is as the precursor of malonyl-CoA.The biosynthesis of acetyl-CoA is strictly regulated by phospholipid biosynthesis.Generally,the intracellular acetyl-CoA and malonyl-CoA are maintained at a low level.Regulating the metabolism of acetyl-CoA is the basis for regulating the metabolism of malonyl-CoA.Many high value-added compounds use malonyl-CoA as the biosynthetic precursor.However,the low level of malonyl-CoA in the cytoplasm limits the biosynthesis of compounds.Flavonoids such as naringenin is a kind of simple malonyl-CoA-derived compound,which requires three molecules of malonyl-CoA as the precursors.Anthraquinones such as carminic acid belongs to complex malonyl-CoA-derived compounds,which requires one molecule of acetyl-CoA and seven molecules of malonyl-CoA as precursors.In addition,the biosynthetic pathway of carminic acid in S.cerevisiae has not been resolved yet.In this paper,the complete biosynthetic pathway of carminic acid and its related enzymes were analyzed and de novo biosynthesis of carminic acid were reconstructed.Moreover,the key points of the biosynthetic pathway of carminic acid were analysed.Then,a lipid endocytosis mechanism of independent-transporters has been developed to further optimize the hydrophobic substrate absorption of fatty acid β-oxidation.Finally,the production of naringenin and carminic acid was optimized by regulating the fatty acid β-oxidation process.The main results of this paper are as follows:(1)The complete biosynthetic pathway and related enzymes of carminic acid in S.cerevisiae were identified.First,the enzymes related to the biosynthetic pathway of carminic acid in A.nidulans reported were expressed in S.cerevisiae: polyketone synthase OKS from Aloe arborescens,cyclase Zhu I and aromatase Zhu J from Streptomyces sp.R1128,and C-glucosyltransferase UGT2 from Dactylopius coccus.However,carminic acid was not detected.It was speculated that this was not a complete pathway for the biosynthesis of carminic acid in S.cerevisiae,and there may be enzymes involved in the biosynthesis of carminic acid in A.nidulans.By analyzing the genome of A.nidulans,the related enzymes involved in the biosynthesis of carminic acid in A.nidulans were discovered: 4’-phosphopantetheinyl transferases Npg A and monooxygenase Apt C(defined as flavokermesic acid anthrone oxidase in this paper).By integrating the heterologous genes of carminic acid biosynthetic pathway into the multiple copy site Ty2 of S.cerevisiae genome,a carminic acid-producing strain was constructed.The carminic acid production was 2664.6 μg/L in shake flask.(2)The key points of carminic acid biosynthetic pathway in S.cerevisiae were analysed.the hydroxylation process of carminic acid was analysed by knocking out the genes(SCS7,SUR2,TPA1 and CAT5)of hydroxylase in S.cerevisiae,when the knock out of CAT5 led to the loss of carminic acid biosynthesis,and the complementary expression of CAT5 restored the biosynthesis of carminic acid,showing that the endogenous hydroxylase Cat5 of S.cerevisiae(defined as flavokermesic acid hydroxylase in this paper)was involved in the biosynthesis of carminic acid.By overexpressing the genes of the biosynthetic pathway of carminic acid separately,it was concluded that the cyclization process was the key rate-limiting step of carminic acid biosynthesis.Through the fusion expression of polyketide synthase and its coenzyme,and the fusion expression of cyclase and aromatase,the effects on the biosynthesis of carminic acid were analysed.In addition,the effects of glycosylation on carminic acid biosynthesis were analysed by knocking out key endogenous hydrolases,enhancing UDP-glucose and expressing glycosyltransferases from different sources.In addition,the influence of oxidation level of intermediate products on the biosynthesis of carminic acid was analysed by adding different concentrations ascorbic acid.(3)Based on the expression of caveolin generated from human,a lipid endocytosis system of independent-transporters was constructed.To remove the dependence of transporters and the transport restriction of hydrophobic substrates,a new mechanism was required to strengthen the absorption and utilization of hydrophobic substrates by cells.First,caveolin was confirmed to form a microbubble structure in S.cerevisiae by the fusion expression of CAV1 and green fluorescent protein.Then,the endocytosis ability of the caveolin was verified by adding 5,6-carboxyfluorescein.In order to further verify the endocytosis ability of caveolin on hydrophobic substrate,adding soybean oil pre-stained with Nile Red and thin layer chromatography was used to identify the endocytosis ability of caveolin on oils.In addition,caveolin was applied to the chassis cells for biosynthesis of naringenin and carminic acid,and the role of oil endocytosis mechanism of caveolin in promoting naringenin and carminic acid was analysed.(4)The biosynthesis of malonyl-CoA derivatives was optimized by regulating the fatty acid β-oxidation process.First,the simple malonyl-CoA derivative(naringenin)-producing strain was as the chassis cell,and the effects of improvement of substrate level and the enhancement of fatty acid β-oxidation on the level of acetyl-CoA and malonyl-CoA and the production of naringenin was analysed.To further analyse the effect of fatty acid β-oxidation process on the biosynthesis of more complex malonyl-CoA derivative(carminic acid),the regulation strategy of fatty acid β-oxidation was applied to carminic acid-producing strain.To further strengthen the biosynthesis of carminic acid,the key genes of fatty acid β-oxidation and carminic acid biosynthetic pathway were combined overexpression,and the key combination expression frames were integrated into the genome multiple copy site Ty1cons1.Through the preliminary screening and re-screening,the carminic acid production was enhanced to 4092.0μg/L in the shake flask.Finally,the engineered naringenin-producing strain L07 and engineered carminic acid-producing strain CA133 were fermented in 5 L bioreactor,resulting in 1129.4 mg/L of naringenin production and 7983.8 μg/L of carminic acid production,respectively.