| Saccharomyces cerevisiae,a promising eukaryotic microbial cell factory,is widely used in the synthesis of various chemical products,pharmaceuticals,and biofuels.Malonyl-CoA is a key precursor for the production a variety of value-added chemicals,such as polyketides,flavonoids,and fatty acids derived chemicals and biofuels.However,its intracellular availability is often limited in S.cerevisiae because of the tight regulation of the pathway and the competition with cellular metabolism.Therefore,it is highly desirable to enhance the malonyl-CoA availability to improve the synthesis of its downstream products.Although rational engineering approaches have been performed to increase the malonyl-CoA level,and thus it is still required to rapidly develop effective approaches for generating mutant strains with much more improved malonyl-CoA availability and identify new targets for further engineering.Malonyl-CoA is an intermediated of the metabolism,and its qutification is very difficult.Therefore,it is necessary to develop a simple and efficiecnt method to screening the mutanted strains and identify the key genes related to the malonyl-CoA availability for further improve the production of malonyl-CoA derived chemicals.Previously,we have constructed a malonyl-CoA activating biosensor and used it to control the expression of GFP to screening the mutants of ACC1 to improve its activity.In this study,we developed malonyl-CoA repressive biosensor for the first time.The biosensor was constructed and systematically engineered to optimize the dynamic range by comparing transcriptional activity of the activators,evaluating the numbers and positions of the DNA binding site in the promoter and comparing the effects of different promoters.Here,using malonyl-CoA biosensors,we created a growth-based screening system that linked the metabolite concentration to cell growth in S.cerevisiae.In addition,in vivo mutagenesis system in yeast was constructed by modifying the DNA replication and mismatch repair system.Using this growth-based screening system,we selected mutant strains with improved malonyl-CoA flux and identified the key targets related to malonyl-CoA availability by whole-genome sequencing,transcriptome analysis,and reverse engineering.The new process especially altered carbohydrate storage,and weakening lysine and arginine synthesis was found to improve malonyl-CoA flux.The details research of this study are as follows:(1)Construction and optimization of repressive biosensor in response to malonyl-CoA and other metabolitesMost of the biosensors constructed in S.cerevisiae have been designed to activate gene expression in response to an increase metabolite concentration,while few studies have been devoted to developing repressive regulation system.Here,we developed malonyl-CoA repressive biosensor for the first time.When the malonyl-CoA concentration is low,TF was fused with transcriptional activation domain(AD)of an activator to activate the transcription when it binds to the promoter.In the presence of malonyl-CoA,TF-AD is dissociated from the promoter to deactivate the transcription.thus down-regulating gene expression.To improve the sensitivity of biosensor,the activation efficiency of different activators(including yeast endogenous Gal4 and Med2;heterologous activators VP 16:hybrid Med2-Gal4 and triplet activator VPR)was compared.Among them.FapR-Med2 showed a much greater increase,and the fluorescence intensity was 46.4-fold higher than the fluorescence intensity of the control strain(without FapR-AD),and even better than that of FapR-VPR.With the graduated addition of cerulenin(20 μM).the fluorescence intensity decreased gradually and the fluorescence intensity of the strain expressing FapR-Med2 decreased by 72%.In promoter design,the strain with one fapO site inside the promoter(located 51 bp upstream of core element of promoter and the distance to the upstream TATA box was 57 bp)had relatively better activation efficiencies,and their fluorescence intensities were 53.5-fold than control.And,the fluorescence intensity decreased by 82%when 20μM cerulenin was added to the strain.Based on this design principle,another two biosensors,which sense acyl-CoA or xylose and downregulate gene expression,were also successfully constructed.Therefore,the design of this repressive sensor provides a design principle for the construction of other metabolite biosensors.(2)In vivo mutation system and metabolite coupling growth method were constructed to screen strains with improved malonyl-CoA flux.To screen for mutant strains that enhance malonyl-CoA synthesis,we introduced either a toxicity gene or an antibiotic resistance gene to the malonyl-CoA repressive biosensor and activated biosensor to construct a growth-coupled circuit.Among them,the activated circuit can link the metabolite concentration with the growth phenotype and therefore meets the requirements for screening.To further confirm the correlation of growth rate under resistant conditions with malonyl-CoA flux.ACL01 and ACL02,with different malonyl-CoA levels were constructed by expressing citrate lyase(ACL)and engineering citrate transports.We compared the fluorescence intensity,the maximum specific growth rate,intracellular malonylCoA concentration,and the production of 3-hydroxypropionic acid(3-HP),and verified that the growth rate was coupled to the intracellular malonyl-CoA level.To achieve in vivo evolution,we expressed the error-prone DNA polymerase δ(pol δ)and combined the modified mismatch repair system in S.cerevisiae,which resulting in the mutation rate reached 8.3 ×10-5,an increase of 2 orders of magnitude.Next,we created a mutagenesis library by subculturing the cells and screening for mutant strains using the growth-coupled circuit and screened four mutants with improved malonyl-CoA level.(3)Identification of causal genes by multi-omics analysisTo analyze the characteristics of the in vivo mutagenesis and investigate the possible genes that affect malonyl-CoA synthesis,we performed whole-genome sequencing and transcriptome analysis of these four mutant strains with improved 3-HP titer.From systems level analysis and we could identify common regulation patterns and thereby specified the general rules required for improved malonyl-CoA flux.In whole-genome sequencing,INO1,PGM3,UGP1 and GDB1 involved in the synthesis and metabolism of storage carbohydrates can increase the production of 3-HP.By measuring the glycogen content,it was further determined that the regulation of storage carbohydrates affected the carbon flux to central carbon metabolism and improved malonyl-CoA synthesis.In transcriptome analysis,the transcription level of the genes in 2-oxocarboxylic acid metabolism,arginine biosynthesis,and lysine biosynthesis were significantly downregulated in all four mutant strains.By regulating lysine and arginine metabolism,especially deleting LYS2 and ARG3 genes,the production of 3-HP increased by about 78%and 82%,respectively.Arginine and lysine synthesis requires acetyl-CoA;hence,downregulation of these pathways will save the carbon for the synthesis of desired products.Through the establishment of growth-coupled screening methods,the screening of highyielding strains and identification of key target genes,we discovered new pathway to enhance malonyl-CoA synthesis.In this study,we systematically optimized the malonyl-CoA biosensors for repressive regulation in yeast for the first time and provided useful information to develop such biosensors.Besides,the growth-based screening coupled with in vivo mutagenesis could circumvent the labor-intensive screening or the requirement of advanced equipment and obtain the desired phenotypes very quickly.Our work not only proposed the design principles of metabolite biosensors,but also offers a valuable complementary tool to other high-throughput screening approaches. |
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