Metabolic Engineering Of Saccharomyces Cerevisiae To Produce Malonate

Malonate is an important dicarboxylic acid.As a high-value specialty chemical,it is widely used in the flavor and pharmaceutical industries.The current production of malonate mainly relies on chemical synthesis,but chemical synthesis has many by-products and difficulties in the disposal of wastes.Thus,microbial synthesis of malonate has attracted more and more attention in recent years as an environmental-friendly synthesis method.In this study,by taking Saccharomyces cerevisiae as a research object,malonate synthesis in Saccharomyces cerevisiae was investigated,including the construction of malonate synthesis pathway,genomic integration of the pathway genes and high-throughput screening of integrated strains,optimization of precursor synthesis pathway,inhibition of competing pathways and fermentation optimization.The main results in this study are highlighted below:（1）Construction and application of UAS（upstream activation sequence）libraries.The narrow scope and weak intensity of the current UAS in dynamic regulation limit to its applications.In order to obtain UAS with a wide dynamic range and enhanced intensity,a mutant library of UAS_TDH3 was primarily constructed by error-prone PCR.Hereafter,330validated mutant UAS were screened by Mi Seq high-throughput sequencing and a synthetic UAS library was constructed,in which the maximum intensity of UAS was 3.65 times that of UAS_TDH3,and the difference between the strongest and weakest UAS was approximately 37-fold.After analyzing the mutant UAS sequences,15 strength enhancing sites and their corresponding mutant bases were found,and the UAS library was rationally designed accordingly.To predict the intensity of rationally designed UAS,the relationship between UAS sequences and their intensity was established,and a prediction model was built based on the DNA curvature of UAS and its intensity.We demonstrated that the UAS library is a transcriptional regulation tool for regulation of the subsequent metabolic pathways.（2）Construction of theβ-alanine pathway for malonate synthesis.By genomic integration of exogenous genes Bc BAPAT and Tc PAND and overexpression of endogenous genes AAT2 and UGA2 from S.cerevisiae,theβ-alanine pathway for malonate synthesis was successfully constructed in S.cerevisiae,and malonate titer reached 7.21 mg/L.Through replacing the succinic acid semialdehyde dehydrogenase with yneI,the malonate titer was increased to 7.96 mg/L in the malonate producing strain BA-5.After optimization of the supplemental carbon source,the malonate titer of BA-5 strain reached 91.53 mg/L during the5-L fermenter fed-batch fermentation.（3）Construction of the malonyl-CoA pathway for malonate synthesis.To construct the malonyl-CoA pathway,the endogenous 3-hydroxyisobutyryl-CoA hydrolase encoding gene,EHD3 was mutated with the mitochondrial localization sequence,the active sites of F121 and E124 to obtain a EHD3***mutant,which was localized in the cytoplasm and had the malonyl-CoA hydrolase activity.Meanwhile,the acetyl-CoA carboxylase gene ACC1**with the mutated phosphorylation sites was overexpressed,and the final malonate producing strain LMA-S with episomal expressing EHD3***and ACC1**under the control of the mutant UAS promoters P_UAS1 and P_UAS2 was constructed,and its malonate titer reached 13.6 mg/L in shake flask fermentation,which was higher than that ofβ-alanine pathway after optimization.（4）Optimization the expression of key genes in malonyl-CoA pathway and the precursor acetyl-CoA supply.To stabilize gene expression and improve expression level,a delta sequence-based multi-copy genomic integration scheme was designed to integrate the target genes ACC1**and EHD3***along with the green fluorescent protein gene GFP as a copy number screening marker.And a high-throughput screening method based on fluorescence intensity was established.Finally,the high-copy integrated strain LMA-1 was screened by tube fermentation and shake flask fermentation with gradient expanding fermentation process.The malonate titer of this strain was 73.55 mg/L,which was about 5-fold higher compared with the episomal plasmid expressing strain LMA-S.To reduce the depletion of cytoplasmic acetyl-CoA to other competitive pathways and to increase the accumulation of the cytoplasmic acetyl-CoA,the expression of genes related to the acetyl-CoA metabolic pathway was optimized,the strain LMA-12,which overexpressed the PDC1 gene and deleted the MLS1 gene,could maximize the malonate titer,with a final malonate titer of 100.59 mg/L.（5）Fermentation optimization of malonyl-CoA pathway integrated strain.The malonate titer of LMA-12 strain was increased to 145 mg/L by supplying 5 g/L glucose at 12 h and 24h,which was 45%higher compared with that before optimization.The concentration and type of extra added metal ions were further optimized,and the extra addition of 200 mM MgCl₂improved the malonate titer most significantly.The presence of Mg²⁺could significantly increase the expression level of PDC1.By replacing the PDC1 promoter with the Mg²⁺-responsive promoter P_PCK1 to obtain the LMA-16 strain,and combining the extra addition of200 mM MgCl₂ and carbon source fermentation strategy,the final malonate titer of LMA-16was 187.25 mg/L,which was 23%higher than that of strain LMA-12.Finally,fermentation optimization with 5-L fermenter fed-batch fermentation using a strategy of slow flow addition of nitrogen source and glucose improved the malonate titer to 1.626 g/L.