| Medium-chain fatty acids(MCFA)have a wide range of nutritional and chemical values,and have been used in many fields such as food,medical treatment,and biofuels.Areas.Traditional MCFA is mainly derived from tropical crops such as palm oil and coconut oil,and the production scale is small,not enough to meet market demand.Studies have been conducted on the synthesis of MCFA by microbial methods.Among them,the reverse fatty acid β-oxidation pathway(r-BOX)can synthesize the highest MCFA yield at present,but it is necessary to add IPTG inducers to turn on the target genes when the recombinant engineered strains grow to a suitable growth period expression.However,IPTG inducers cannot be used in large quantities due to their high price.At the same time,they are cytotoxic and will increase the purification cost of subsequent target products,which will hinder the large-scale production of MCFA.In order to weaken the negative effects of inducers,we are looking for a strategy that can spontaneously turn on the expression of target genes according to the growth status of the flora.This study is based on the iteration of the Vibrio fischeri Quorum sensing system(QS)and T7 expression system.The genetic circuit switch regulation laboratory obtained an engineered strain that used the reverse fatty acid-β oxidation pathway to synthesize MCFA and was modified to realize the spontaneous accumulation of MCFA and replace the use of the inducer IPTG.The main research content and conclusions of this paper are as follows:1.Regulating the spontaneous synthesis of MCFA based on the QS system of Vibrio fischeriFirst,the gene elements of the Vibrio fischeri QS system were amplified using the Vibrio fischeri genome as a template: including the signal molecule synthetase gene lux I,the receptor protein gene lux R and the promoter Plux I.Then the key pathway genes in the MCFA synthesis strain(thiolase Bkt B,3-hydroxyacyl-Co A dehydrogenase,3-hydroxyacyl-Co A dehydratase Fad B,trans-enoyl-Co A reductase Ter,thioesterase Ydi I)The T7 promoter was replaced with the Plux I promoter,and the signal molecule synthetase gene lux I and the receptor protein gene lux R were also transferred to explore the efficiency of the Vibrio fischeri QS system to regulate the synthesis of MCFA.After optimizing the carbon source and culture temperature,79.6 mg/L MCFA was synthesized without adding the inducer IPTG.2.Construction of iterative gene circuit switch of Vibrio fischeri QS system and T7 expression systemIt was observed that the output of MCFA synthesized by the QS system of Vibrio fischeri was far less than the yield of MCFA synthesized by the induction and regulation of IPTG.In order to increase the intensity of regulation of target genes,the T7 RNA polymerization in the T7 expression system was introduced into the Vibrio fischeri QS system.The enzyme element constructs an iterative gene circuit switch,which is 112.21%stronger than the control intensity of the Vibrio fischeri QS system.The action process is:as the number of bacteria increases,the number of signal molecules is increased and the receptor protein forms a complex.Then the signal molecule-receptor protein complex binds to the Plux I promoter region of Vibrio fischeri to allow the downstream T7 RNA polymerase to be expressed in large quantities.Then T7 RNA polymerase specifically recognizes and transcribes the target gene expression downstream of the T7 promoter to realize the cascade amplification of gene signals.Taking the expression of the green fluorescent protein EGFP as a characterization,it was observed that there is a high level of leakage expression in the iterative gene circuit switch,which caused the substrate to flow to the target protein synthesis pathway at the early stage of cell growth,which impaired the normal growth of the cell.In order to ensure the normal growth of the bacteria,the Plux I promoter with a total length of 214 bp for the iterative gene circuit switch was optimized by truncating 114 bp,87bp,80 bp,and 57 bp,and the promoters Plux I(1),Plux I(2),Plux I(3)were obtained.),Plux I(4).The results show that the optimized promoters after truncation have improved to varying degrees.Among them,the 87 bp Plux I(2)promoter has the largest improvement in leakage,and the leakage is reduced by 61.82% compared with the original long promoter.At the same time,the leakage is reduced by 61.82%.It retains 93.71% of the original length of the regulatory intensity,which is 61.33% of the regulatory intensity of the T7 promoter.In order to further reduce the leaky expression,the promoter was optimized for plasmid vector copy number,and the high copy number vector p CDFDuet-1 was replaced with the low copy number vector p COLADuet-1.After replacing the vector,the leakage was further reduced by84.55%.The intensity also reached 88.92% of the regulation intensity of the T7 promoter.3.Iterative gene circuit switches stably and spontaneously regulate the accumulation of medium-chain fatty acid productsIn order to overcome the dependence on the inducer IPTG in the existing fermentation process of MCFA,the iterative gene circuit switch promoter Plux I is used to express T7 RNA polymerase,and T7 RNA polymerase is then specific to the T7 promoter responsible for transcribing the key enzyme gene in the medium-chain fatty acid synthesis pathway.Sexual binding,transcription synthesis includes Bkt B(thiolase)from Ralstonia eutrop Ha,Fad B(3-hydroxyacyl-Co A dehydrogenase and 3-hydroxyacyl-Co A dehydrogenase)from Escherichia coli.Acyl-Co A dehydratase),Ter(trans-enoyl-Co A reductase)from Euglena gracilis,Ydi I(thioesterase)from Escherichia coli,the four enzymes are acetyl-Co A as The precursor substances,after condensation,reduction,dehydration,and reduction,finally realize the high-efficiency and spontaneous synthesis of medium-chain fatty acids.The fermentation conditions of the MCFA synthetic strain were optimized,and the appropriate culture temperature,culture speed,and medium p H value were explored.It was determined that the highest value can be obtained under the conditions of 200 rpm,30℃,45 m L,and medium p H value of 7.5.The production of medium-chain fatty acids was 568.4 mg/L,which was 93.16% of the production of MCFA that had previously relied on the IPTG-induced T7 promoter to regulate synthesis.It can indicate that the iterative gene circuit switch is very effective in regulating the synthesis of MCFA. |
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