Mechanism Analysis Of Spontaneous And Efficient Accumulation Of Malonyl-Coa By Microbial Method

Malonyl-CoA is a key rate limiting precursor for the microbial synthesis of many flavonoids and polyketones.Many studies have broken the synthetic pathway by increasing intracellular malonyl-CoA supply,but current strategies all rely on the addition of expensive inducers and cannot be used in large-scale industrial production.In order to find a highly efficient and inexpensive means of increasing the intracellular malonyl-CoA supply and to get rid of dependence on the inducer,this study constructed a system that can efficiently synthesize malonyl-CoA from acetic acid and utilizes a population-based gene switch based on the Quorum sensing(QS)system to realizes the spontaneous and efficient accumulation of malonyl-CoA.The main research contents and conclusions of this paper are as follows:1.Construction of a high-efficiency accumulation pathway of malonyl-CoA using metabolic by-product acetic acid as precursorIn order to increase intracellular malonyl-CoA supply,a highly efficient accumulation pathway from acetic acid to malonyl-CoA was constructed to increase intracellular malonyl-CoA concentration.The five efflux pathways of acetyl-CoA were truncated by gene knockout to increase intracellular acetyl-CoA concentration;the conversion from acetic acid to acetyl-CoA was achieved by overexpressing acetyl-CoA synthetase from different sources to further enhancee intracellular Acetyl-CoA content,providing sufficient precursor for the synthesis of malonyl-CoA;conversion of acetyl-CoA to malonyl-CoA by overexpressing acetyl-CoA carboxylase from different sources;exploring different biotin ligases effect on acetyl-CoA carboxylase activity.We finally determined that the Acetyl-CoA synthetase Acs from Escherichia coli had the highest acetyl-CoA synthesis activity,and the acetyl-CoA carboxylases SeaccA,SeaccC,and SeaccD from Salmonella enterica has the highest malonyl-CoA synthesis activity with the help of CgbirA from Corynebacterium glutamicum.The intracellular malonyl-CoA concentration is up to 0.812 nmol/mg DCW,and the naringenin yield is up to 252.31 mg/L.2.Gene switch construction and vitality comparison based on quorum sensing systemIn order to achieve dynamic spontaneous accumulation of malonyl-CoA,a total of four sets of quorum sensing systems from Vbrio fischeri and Pseudomonas aeruginosa were reconstituted in the engineering bacteria.Promoters related to the QS system,the signal molecule protein,the receptor protein gene and the promoters upstream of the gene were cloned and assembled in the engineering bacteria.It was found that the endogenous promoter had a weaker start-up strength,and the QS system could not normal operate in the engineering bacteria;therefore,we use the T7 promoter with higher activation intensity to replace the signal molecule protein and the receptor protein promoter,and the intracellular signal molecule and the receptor protein concentration are increased under the induction of IPTG,so that the intracellular signal molecule-receptor complex concentration reached the detection threshold of the promoter and successfully initiated the expression of the reporter gene gfp,and the QS system was initially constructed successfully;in order to achieve the independent regulation of the QS system and avoide the addition of inducer.The constitutive promoter trc(lost operon),which can work normally under the condition of different carbon source was used to replace the T7 promoter with the signal molecule protein and the receptor protein,and realizes the constitutive expression of the signal molecule protein and the receptor protein.The regulated gene switch is constructed.The viability of four sets of quorum sensing systems was compared and we determine to uset he highest activation activity gene switch based on the Lux quorum sensing system of Vibrio fischeri.3.Construction of a Strictly regulated system spontaneous and efficiently synthesis malonyl coenzyme AThe experiment found that the constructed gene switch has expression leakage,in order to reduce the leakage,realizing the strict regulation of the target gene by the gene switch.So we carried out directional transformation of the promoter element.The sequence downstream of the promoter PluxI-10 region was replaced with the RBS site of the T7 promoter,and the sequence downstream of the promoter-10 region to the transcription start site was found to be associated with the binding of RNA polymerase and promoter;The sequences upstream of the promoter-35 region were truncated to different lengths to explore the optimal promoter length of the promoter,and it was found that the upstream sequence of the promoter-35 region is related to the leakage regulation of the promoter and the efficiency of transcription.We finally construct a genetic switch that is tightly regulated and has a high transcription strength.Combining the high accumulation pathway of malonyl-CoA and the strictly regulated gene switch constructed in the second chapter,the basal cells were constructed,so that the engineering bacteria did not express recombinant protein at the beginning of fermentation,and maintained a good growth state.When the OD600 reached 0.5 The high-efficiency accumulation of malonyl-CoA was spontaneously initiated,and the intracellular metabolic flux was redirected to the target product malonyl-CoA,and the intracellular malonyl-CoA concentration reached 0.325 nmol/mg DCW.The basal cells were applied to the production of flavonoids,naringenin.The production of naringenin increased from the initial 15.776 mg/L to 80.69 mg/L,an increase of 411.5%.