| In recent years,environmental protection has become a hot topic.In agriculture,lots of agricultural pesticides have caused great harm to the environment.Therefore,reducing the use of agricultural pesticides is an important mothed to decresase ecological damage.The degradable advantages of biological insecticides are of great significance to environmental protection.And it is particularly important to study degradable biological insecticides.Cembratriene-ol is mainly found in plants and has good effects on insect resistance,but its content is scarce in plants.There are many difficulties in direct extraction from plants because of its complex extraction and high extraction cost,andit is difficult to be chemically synthesized because of its complex structures.Therefore,the synthesis of cembratriene-ol by modifying microorganisms and synthetic biology methods has aroused widespread interest.In this study,Escherichia coli and Saccharomyces cerevisiae were used as the initial strains to construct recombinant strains producing cembratriene-ol.The recombinant strains with the efficient production of cembratriene-ol were obtained via the construction of the synthesis pathway of cembratriene-ol and the metabolic modification.Systemtic optimization strategies were also used to improve the production of cembratriene-ol.The main contents are as follows:（1）We expressed two plant-derived cembratriene-ol synthases in S.cerevisiae and investigated their catalytic efficiency for the production of cembratriene-ol.The cembratriene-ol synthase CBTS*with high catalytic efficiency was obtained.Two key enzyme genes ggpps and cbts*were heterologously expressed in S.cerevisiae W303,cembratriene-ol was synthesized in S.cerevisiae and its yield reached 3.72μg·L-1.After over-expression of the key enzyme farnesyl diphosphate synthase（ERG20）,the yield of cembratriene-ol was increased by1.84-fold that of the control.The above pathwat was reconstructed in S.cerevisiae BY4741,and the yield of cembratriene-ol was increased by 3-fold that of the control.（2）The key genes of the above pathway were integrated into the genome of the S.cerevisiae to produce cembratriene-ol.The promoter Phxt1 was used to replace the initial promoter of squalene synthase（ERG9）to knockdown the competitive ergosterol biosynthetic pathway.The carbon metabolism flow can better flow to the target product,and the yield of cembratriene-ol is increased by 1.8-fold that of the control.The free plasmids with the key enzyme genes ggpps and cbts*were transferred into the strain S25 to obtain the strain S26,the cembratriene-ol yield of which reached 188.20μg·L-1.It was 26.02-fold that of the strain S25that only integrates expression and weakens the competitive pathway.Based on the compartmentalization method,based on strain S25 the key enzyme genes ggpps and cbts*were located in the mitochondria to obtain the recombinant strain S27.Its cembratriene-ol yield reached 283.98μg·L-1,which was 76.3-fold that of the initial strain.（3）Gene ggpps and cbts were heterologously expressed in E.coli to produce cembratriene-ol,the yield of which reached 1.58×10-2 mg·L-1.Compared the effect of cbts and cbts*on the production level of cembratriene-ol,it was found that cbts*was preferred for the production of cembratriene-ol in E.coli.Its cembratriene-ol yield increased by 20-fold that of the control.The key enzyme genes（1-deoxy-D-xylulose 5-phosphate synthase gene dxs and isoprenyl pyrophosphate isomerase gene idi）were overexpressed to increase the yield of cembratriene-ol,which was by increased by 3.75-fold that of the control.The yield of cembratriene-ol was further increased by 2.6-fold that of the control via enhancing expression level of genes dxs and idi using plasmids with high-copy.（4）Co-expression plasmids were used to systematically regulate the expression of key genes ggpps,cbts*,dxs and idi,and the yield of cembratriene-ol in E.coli was increased by1.53-fold that of the control.The farnesyl pyrophosphate synthase gene isp A was overexpressed to improve the production of cembratriene-ol and its yield was increased by 1.4-fold that of the control.The fermentation media and fermentation time were optimized,and the optimal glucose and yeast extraction addition method obtained was glucose:yeast extraction=2:4.The yield of cembratriene-ol in E.coli reached 53.72 mg·L-1 at 72 h,which was 3357.5-fold that of the initial strain.Meanhwhile,the MEV pathway was reconstructed in E.coli,and the DXP pathway and the MEV pathway were used to synthesize cembratriene-ol.The dual pathways significantly promoted the synthesis of cembratriene-ol,and its yield was increased by 1.13-fold that of the control at 24 h. |
