| Malonyl-CoA is the key precursor of the biosynthesis of fatty-acid-derivatives,flavonoids and polyketides,these chemical compounds are important parts of pharmaceuticals,green energy and useful industry chemicals.Therefore,it has great potential to construct malonyl-CoA-platform strain.Saccharomyces cerevisiae is a commonly used industry strain for the beneficial qualities it possesses including clearly genetic background,good security and mature gene manipulation approach,so it can be used as potential chassis cell for malonyl-CoA biosynthesis.In saccharomyces cerevisiae,the metabolic intermediate malonyl-CoA is the basic precursor for the biosynthesis of acyl-CoAs,acyl-CoAs are responsible for the biosynthesis of phospholipids,triacylglycerol thus providing the essential nutrients for cell proliferation.Cytoplasmic malonyl-CoA is synthesized by acetyl-CoA carboxylase,which is the first step of acyl-CoAs biosynthesis and is also the rate-limiting step.The activity of acetyl-CoA carboxylase is regulated at both transcription level and post-translation level,the restrict feedback regulation limited the efficiency of malonyl-CoA generation.Besides,lipid compounds biosynthesis consumes a large amount of malonyl-CoA resulting in the low level of malonyl-CoA pool.Consequently,yeast cellular malonyl-CoA level is low,which limits the biosynthesis of malonyl-CoA-derived chemicals.Currently,research concerning improving cellular malonyl-CoA levels is carried out by enhancing the upstream metabolic flux,genome screening for new effective genes,reducing the consumption pathway.Whereas the most efficient way by combining multiple genes manipulation led to approximately 5-fold improvement of malonyl-CoA-derived chemical production,which is not enough to meet the industry need.Thereby,we need to explore more to improve malonyl-CoA levels.In this study,we reduced the synthetic pathway of phospholipids by modifying transcription factors,which effectively inhibited malonyl-CoA from consumption,thereby increasing the yield of its derivative product 3-hydroxypropionic acid.We constructed malonyl-CoA biosensors capable of responding to cellular malonyl-CoA concentrations,also optimized the sensitivity and signal output range of malonyl-CoA biosensors by combinating the biological components at multi-levels.Then the most efficient malonyl-CoA biosensor was used to screen phosphorylation sites mutations of Accl that can improve malonyl-CoA synthetic ability.The main research contents are summarized as fellows:(1)The phospholipids synthesis is the major consumption pathway of malonyl-CoA,but directly deletion the pathway is not advisable because phospholipids are components of biofilms.We reduced the synthesis of phospholipids by manipulating transcription regulators of phospholipid synthetic pathway through which malonyl-CoA was efficiently accumulated and the production of 3-hydroxypropionic acid was enhanced.We deleted transcription activators INO2,INO4,overexpressed transcription repressor OPI1.It showed that deletion of INO4 and overexpression of OPI1 did not improve the synthesis of malonyl-CoA-derived product 3-hydroxypropionic acid.Deletion of INO2 increased 3-hydroxypropionic acid titer by 1.8-fold even though cell growth was severely impaired,suggesting that deletion of INO2 can efficiently improve cellular malonyl-CoA availability.To further enhance the production,we intended to recover the cell growth.After supplying phospholipid precursors inositol and choline in the medium,the cell growth of ?INO2 recovered to 50%of the wild-type strain,and titer of 3-hydroxypropionic acid reached 477 mg/L,which was 9-fold improvement relative to the wild-type strain.This modification turns out to be the most efficient one for malonyl-CoA-derived product improvement so far.To reduce the cost of added nutrients,the lowest concentration of nutrients needed was explored.The results showed that only 0.02 mM choline was enough to increase the production of 3-hydroxypropionic acid to a satisfied level.To explore the possibilities of restoring cell growth and increasing the production without addition of nutrients,INO2 mutants were expressed in INO2-depleted strain,wherein the expression of the TAD1 mutant allowed the cell growth partially recovering and increased the titer of 3-hydroxypropionic acid by 1.8-fold.It suggested that expressing Ino2 mutant with partially activity can repair the cell growth caused by phospholipids biosynthesis limitation,therefore balance phospholipids requirement and malonyl-CoA accumulation,and make the metabolic flux more prone to malonyl-CoA accumulation.This study demonstrated the possibility of leading malonyl-CoA flux to product synthesis through transcription regulation.(2)Biosensors that respond to cellular intermetabolite are powerful tools for metabolic engineering development.Based on the mechanism that malonyl-CoA molecules regulate the transcription of fatty acid synthetic genes through specific binding to the transcription factor FapR in prokaryote,we construted two systems that can respond to malonyl-CoA concentrations in S.cerevisiae,which can repress or activate the transcription depending on malonyl-CoA concentrations.The first system was obtained by putting the FapR binding site,fapO,near the TATA box of the core promoter,and the transcription was activated with the increase of malonyl-CoA concentration.We optimized the system by changing the position of the fapO and the expression level of the FapR.We obtained one construction with highest efficiency that can increase the output range of the fluorescence signal by 8-fold with increasing malonyl-CoA concentrations,which performed better than the previously reported malonyl-CoA biosensors constructed in S.cerevisiae.The second system was constructed by fusing FapR with a transcription activator protein and inserting the fapO in the upstream activation domain of the promoter,the obtained sensing system can repress transcription with the increasing concentration of malonyl-CoA.Through optimition the biological elements comprising the biosensor including the original promoter,the position and numbers offapO,activators fused with FapR,the repression module malonyl-CoA biosensor with different efficiency were obtained.The two malonyl-CoA sensoring systems constructed in this study that can respond to malonyl-CoA concentrations but having opposite signal output trends provide a powerful tool for the regulation of malonyl-CoA metabolic pathways and the screening of genetic mutant libraries.(3)In yeast cytoplasm,Acetyl-CoA carboxylase is the only enzyme for malonyl-CoA biosynthesis.Besides regulated at the transcriptional level by the synthesis of phospholipids,acetyl-CoA carboxylase is also regulated at protein level.The calalytic ability can be reduced by protein kinase phosphorylation,thus decreasing the synthetic efficiency of malonyl-CoA.In this study,we performed a comprehensive study on the effect of Accl phosphorylation sites modification to the catalytic activity.Firstly,the site-mutant strains were screened using most efficient malonyl-CoA biosensor of activation module.Through the screening of phosphorylation mutants,we found another phosphorylation site that can affect the activity of Acc1 in addition to Ser659 and Ser1157.The addition of the S686A mutation to S659AS1157A mutation further released the negative effect of phosphorylation modification,resulting in a higher activity of the intracellular acetyl-CoA carboxylase,thus promoted the synthetic efficiency of malonyl-CoA.A three-site combinatorial mutation Acc1(S686AS659AS1157A)which increased the titer of 3-hydroxypropionic acid by 1.5-fold was obtained,and the contribution to the product was better than the previously reported Acc1(S659AS1157A)mutation.In this study,the synthesis of malonyl-CoA was enhanced by the modification of key enzyme and reducing the key consumption pathway by transcription regulation in S.cerevisiae.Two malonyl-CoA sensing systems constructed in this study provided powerful tools for further study about malonyl-CoA metabolic pathway regulation and mutant libraries screening.This work not only directly enhanced the biosynthesis of 3-hydroxypropionic acid,the establishment of these platform strains and the construction of malonyl-CoA biosensors also contribute to the synthesis of other malonyl-CoA derivative products in S.cerevisiae.In addition,the construction and optimization of malonyl-CoA biosensors also provide an important example for the construction of other metabolite biosensors. |
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