Construction And Optimization Of Olivetolic Acid Metabolic Pathway In Synechocystis Sp.PCC 6803

With the rapid development of modern society,the problems of environmental pollution and energy shortage are becoming more and more serious,and the increasing greenhouse effect makes the research on resource utilization of atmospheric carbon dioxide become a hot spot.The application of cyanobacterial photosynthetic cell factories has attracted widespread attention.The carbon dioxide absorbed during the growth of cyanobacteria is driven by solar energy to synthesize high-value natural products,which not only brings economic benefits but also alleviates environmental pollution.Olivetolic acid is a plant secondary metabolite catalyzed by type III polyketone synthetase,and it is also a central intermediate in the synthesis of important compounds such as cannabidiol.Currently,olivetolic acid is mainly obtained by chemical synthesis or plant extraction,which is not only time-consuming and labor-consuming,but also pollutes the environment.In order to reduce the cost of producing olivetolic acid and relieve the environmental pressure,according to the codon preference of Synechocystis sp.PCC 6803,the codons of olivetolic acid synthase gene（TKS）,olivetolic acid cyclase gene（OAC）and hexanoyl-Co A synthase gene（AAE1）in cannabis root trichomes were optimized.This study used Synechocystis sp.PCC 6803 as the chassis,the heterologous expression of these genes in cyanobacteria cells was realized by homologous recombination,and then the expression of TKS,OAC and AAE1 genes was detected by genomic PCR amplification and RT-qPCR,and a new cyanobacteria cell factory for olivetolic acid synthesis was constructed.In order to increase the yield of olivetolic acid synthesized by cyanobacteria,we analyzed the metabolic network of cyanobacteria and optimized it.The following are the specific research contents and results:（1）Construction of olivetolic acid metabolism pathway:Synechocystis sp.PCC 6803 was used as the chassis cell,and two key genes of olivetolic acid synthesis（olivetolic acid synthase gene and olivetolic acid cyclase gene）and hexanoyl-Co A synthase gene were integrated into the genome by means of genetic engineering technology,and the light intensity promoter Ppsb A2s was used to drive the expression of the target gene,so as to obtain the first generation mutant SCY02 which synthesized olivetolic acid.Under the nutrition condition of BG11 medium supplemented with hexanote,the growth characteristics of mutant SCY02 and wild-type Synechocystis sp.PCC 6803 were compared and analyzed.The expression levels of TKS,OAC and AAE1genes in mutant SCY02 cells were identified by real-time fluorescence quantitative PCR,and the yield of olivetolic acid was detected by high performance liquid chromatography.Furthermore,the second generation mutant SCY03 for olivetolic acid synthesis was constructed by using Pcpc560 as the promoter of olivetolic acid synthesis gene.Compared with the first generation mutant SCY02,the second generation mutant SCY03 had higher expression levels of TKS,OAC and AAE1 genes and olivetolic acid production.（2）Optimization of olivetolic acid metabolism pathway in cyanobacteria:on the basis of the second generation mutant SCY03,the ADP-glucose pyrophosphorylase gene（coding slr1176）in cyanobacteria glycogen biosynthesis pathway was further knocked out,and the NADP-dependent malic enzyme gene（mae B）from Escherichia coli was inserted into the original ADP-glucose pyrophosphorylase gene site.Since the expression of mae B gene partly interferes with the tricarboxylic acid cycle of cyanobacteria,which promotes part of the carbon flux to flow into the metabolic pathway of olivetolic acid synthesis,thus promoting the increase of olivetolic acid production,and then construct the best mutant SCY05 for olivetolic acid production in this study.（3）Optimize the culture conditions of engineering cyanobacteria:The concentration of hexanote in the engineered cyanobacterial medium was adjusted to 8m M,which accelerated the biomass accumulation of SCY05 algal strain,shortened its growth cycle,achieved high-efficiency expression of exogenous genes,and increased the yield of olivetolic acid.To continue to optimize the culture conditions of engineering cyanobacteria,the mutant cyanobacteria SCY05 was continuously aerated with a carbon dioxide-air ratio of 1:19（5%CO₂,95%air）,which not only greatly increased the growth of cyanobacteria,but also pushed the yield of olivetolic acid to the highest 9.41±0.35mg/L.