Analysis Of The Synthetic Pathway Of Raspberry Ketone And Its Heterologous High-efficiency Expression In Microorganisms

Raspberry ketone is an aroma compound found in raspberries.It has important physiological functions and is widely used in food,cosmetics,agriculture,medicine,and other fields.However,the natural extraction of raspberry ketone is not only low in yield but also affected by the season and region,which cannot meet the market demand.Although the chemical synthesis of raspberry ketone has a high yield,there are problems such as environmental pollution,serious equipment corrosion,and many by-products.In recent years,research on the synthesis of raspberry ketones through microbial metabolic engineering has attracted great attention.As a kind of highly reduced substance,fatty acids can produce a large amount of important metabolic precursor acetyl-CoA through ?oxidation,and release a large amount of reducing power.Therefore,fatty acids can be used as carbon sources for the production of raspberry ketones,providing sufficient precursors and energy.This article intends to construct an E.coli cell factory that can use multiple carbon sources such as glycerol and fatty acids to generate raspberry ketones.By constructing and optimizing the synthesis pathway of raspberry ketones,genetic engineering technology is used to transform the metabolic pathways of E.coli to accumulate intermediate product C.Diacid monoacyl-Coenzyme A;At the same time,it combines with fatty acid efficient utilization module to realize the high-level synthesis of raspberry ketone and other polyketide compounds in E.coli cell factories.The main research contents are as follows:(1)Analysis and Characterization of the Synthetic Route of Raspberry Ketone.The 4-coumarate-CoA ligase(At4CL1)from Arabidopsis thaliana,the benzylacetone synthase(RpBAS)from and the raspberry ketone reductase(RiRZS1)from idaeus(RiRZS1)genes were used to construct and optimize the raspberry ketone synthesis pathway in E.coli BW25113.The medium-copy single plasmid was used to sequentially express RiRZS1,RpBAS,At4CL1,and the resulting strain CR4 was obtained.Analyzed the key enzyme benzylacetone synthase(BAS)in the synthetic pathway,and established a phylogenetic tree through amino acid sequence alignment and finally determined that the BAS raspberry ketone,which is still derived from Rheum palmatum,has the highest yield,reaching 13.1 mg/L.Overexpression of CurA(encoding dihydrocurcumin reductase)on the genome to enhance the synthesis pathway,the strain CR5 was obtained,and the yield of raspberry ketone reached 16.2 mg/L.(2)Modification of malonyl-CoA pathway.Knocked out the metabolic bypass gene poxB(pyruvate oxidase)and fabF(?-ketoacyl-ACP synthase ?)to reduce carbon loss,and made the metabolic flow to the direction of raspberry ketone synthesis as much as possible;Overexpression of acs(acetyl-CoA synthetase)on the genome enhanced the production of acetyl-CoA;Overexpression of accBCDA(acetyl-coenzyme A carboxylase complex)gene enhanced the production of malonyl-CoA;Overexpression of cgPPC(phosphoenolpyruvate carboxylase)derived from Corynebacterium glutamicum and BCDH(branched chain ?-keto acid dehydrogenase)derived from Streptomyces avermitilis on the genome enhanced the production of oxaloacetate and malonyl-CoA;The carboxylation of acetyl-CoA to synthesize malonyl-CoA and phosphoenolpyruvate to synthesize oxaloacetate was a carbon fixation reaction,therefore,the bicarbonate transporter BT derived from Synechococcus and the carbonic anhydrase CA derived from cyanobacteria were overexpressed on the genome to increase the content of HCO3-,and finally the yield of raspberry ketone was increased to 59.3 mg/L.(3)Performance optimization of raspberry ketone cell factory.To improve strain stability and raspberry ketone production,it was necessary to integrate multiple genes into the chromosome.Therefore,the NRMI(Nigri recombinase mediated integration)method of continuous integration of large fragments was optimized.This method could reduce the plasmid in the process of resistance elimination.The transfer and elimination of 119accBCDA and 119bt-ca were continuously integrated into the fadR site,the positive rate could reach 100%,and the cycle is shortened from 7 days to 3 days.At the same time,five nox mutants that could be effectively identified by Nigri and were strictly inactivated between each other were screened out,which can achieve continuous integration of chromosomes at multiple sites.The fatty acid-inducible frd3 promoter was obtained through screening and truncation,which could be used to fine-tune related genes when supplementing fatty acids as a carbon source.(4)Integration of fatty acid utilization module.Introduced fatty acid efficient utilization module,supplementing fatty acids as one of the carbon sources to provide a large amount of precursors and energy for the synthesis of raspberry ketones;Knocked out SthA and strengthen PntAB to realize the N ADH to N ADPH conversion;The medium composition and process conditions for the best level of raspberry ketone test were:1%fatty acid,3%glycerol,1%yeast extract,5mM 4-coumaric acid in the medium;The induction temperature was 30?;The concentration of the inducer was 0.01%,and the small test level of raspberry ketone reached 98.6 mg/L.The study was carried out on a 1L fermentor.In fed-batch fermentation,raspberry ketones were synthesized from soybean oil,and the maximum yield could reach 192.57 mg/L.(5)Construction of other polyketide cell factories.The synthesis pathways of Phloroglucinol,Flaviolin,TAL,and Aloesone were introduced into the chassis cells CC13 with high production of acetyl-CoA and malonyl-CoA constructed in the previous stage.The yield of PHL01 phloroglucinol reached 18.48 mg/L,which was about 4 times higher than that of the control BW-PHL.The relative content of flaviolin produced by the FLA01 strain was 7 times that of the control strain BW-FLA.The TAL yield of TAL01 strain was 1.06 mg/L,which was about 5 times higher than the control BW-TAL.The relative content of aloe ketone produced by the strain ALS01 was 5 times that of the control BW-ALS.In this study,the synthetic pathway of raspberry ketone was constructed in E.coli,and the synthesis pathway of malonyl-CoA and the pathway of fatty acid utilization were modified to realize the efficient synthesis of raspberry ketone.And used the chassis to synthesize four polyketides,providing new ideas and research basis for the production of polyketides.