Cell Tolernce And Biosynthesis Of Pinene In Candida Glycerinogenes

Pinene is a monoterpene of plant origin,which is widely used in spices,flavorings,pharmaceuticals and fine chemicals,and especially has important applications in national defense,and thus is an important new compound.At present,pinene is mainly derived from chemical synthesis,but the steps are complicated,costly,and seriously damaging to the environment.With the development of synthetic biology,the synthesis of pinene using microorganisms as hosts has become the focus of research,however,the great toxicity of pinene to hosts has severely limited its fermentation synthesis,and the current synthesis amounts are in the range of mg/L.Candida glycerinogenes is an industrial strain with multiple resistance properties.In this paper,we take C.glycerinogenes as the research object and focus on the pinene tolerance and pinene biosynthesis of C.glycerinogenes,and the main research contents are as follows:（1）Probing the mechanism and tolerance modification based on C.glycerinogenes pinene high tolerance and transcriptomics.Through the pinene tolerance assay revealed that C.glycerinogenes had a strong pinene tolerance,which was 5 times higher than that of Saccharomyces cerevisiae.The cell membrane fluidity and permeability of C.glycerinogenes increased under pinene stress,and the transcriptome of C.glycerinogenes was found to be 5times higher than that of Saccharomyces cerevisiae;according to the transcriptome assay of pinene stress,C.glycerinogenes increased cell membrane fluidity and permeability through the regulation of cell membrane synthesis,fatty acid synthesis,lipid metabolism,carbon metabolism,and the production of a variety of other substances;according to the results of transcriptome assay,C.glycerinogenes achieved high tolerance to pinene by regulating cell membrane synthesis,fatty acid synthesis,lipid metabolism,carbon metabolism,MAPK signaling and other responses;combined with the addition of exogenous ergosterol,squalene and alginate to improve the tolerance to pinene in the bacterium,and the genes of ergosterol and alginate synthesis pathway were significantly up-regulated under pinene stress,and HMGR,Tpp and ERG6.Based on the above results,overexpression of Tpp and ERG6 genes under 200 mg/L pinene stress significantly increased intracellular alginate and ergosterol contents,and increased the amount of Cg-Tpp bacteriophage by 9%and Cg-ERG6bacteriophage by 11%,further improving the tolerance of C.glycerinogenes to pinene.（2）MVA metabolic flux analysis and its metabolic engineering to enhance the promotion of pinene synthesis.The results showed that the squalene content of C.glycerinogenes was3.6 times higher than that of S.cerevisiae,and its squalene content was 20 times higher than that of mevalonate,and the HMGR gene was up-regulated 4 times by intensifying the MVA pathway,which proved that C.glycerinogenes has a stronger MVA pathway and stronger downstream pathway for pinene synthesis.The integration of pinene synthase Pt30 into the genome of C.glycerinogenes and GC-MS showed that the engineered bacteria synthesized pinene;the N-terminal transit peptide of pinene synthase was truncated according to bioinformatics analysis.According to the bioinformatics analysis,the N-terminal transit peptide of pinene synthase was truncated,and the fusion expression of truncated pinene synthase t48Pt30 with ERG20^WW improved the catalytic efficiency of the enzyme and substrate,and the yield of pinene was increased to 2.6 mg/L.The overexpression of exogenous ACL gene promoted the synthesis of acetyl coenzyme A,the starting substrate of MVA pathway,and the overexpression of ERG10,ERG13 and t HMGR increased the flux of MVA upstream pathway and Overexpression of ERG10,ERG13 and t HMGR increased the flux of MVA upstream pathway and reduced the flux of ethanol and other byproducts synthesis by inhibiting ADH and GPD genes through antisense RNA technology,and the final pinene yield reached 6.0 mg/L.（3）Regulation of MVA pathway for pinene synthesis using osmotic stress response Hog1.Transcriptome assay results and fermentation data showed that increasing extracellular osmolarity upregulated the gene expression level of the MVA pathway of C.glycerinogenes,and at the same time slowed down the apparent accumulation of intracellular squalene endangered by osmolarity stress;in response to osmolarity stress,the synthesis of the osmoprotectant glycerol was increased,and it was also found that using glycerol as a carbon source significantly upregulated the expression level of the MVA pathway as well as the glycolytic pathway.Hog1,a key gene in response to osmotic stress regulation,was overexpressed to improve multiple stress tolerance,accelerate growth rate and sugar consumption rate,while HMGR,a key gene in the MVA pathway,was upregulated 5-fold and squalene content was increased by 33%,presumably related to squalene accumulation.When the Hog1 gene was knocked out,the cells showed the opposite trait;based on the above results overexpression of Hog1 gene and fermentation with glycerol as the carbon source,the final pinene production was increased by 29%.（4）Rational molecular modification of the pinene synthase gene Pt30 to enhance pinene synthesis.In order to improve the catalytic efficiency of pinene synthase Pt30 and substrate GPP,multiple sequence alignment and homology modeling of pinene synthase Pt30 were performed;the Pt30-1 model was molecularly docked with small molecule ligands and interaction force analysis of amino acid residues within 5（?）of the active pocket was performed,and it was found that amino acids Y348,I375,T376,I484,T486 around the active pocket In order to solve this problem,we conducted virtual screening and rational design of key amino acids of pinene synthase using bioinformatics,and found that pinene synthase Pt30was modified by point mutation of key amino acid sites.T376R modification and fusion expression with ERG20^WW resulted in a 26%increase in pinene yield.（5）The fermentation strategy for pinene synthesis was explored based on the above studies.The osmotic stress fermentation strategy and process study found that the best effect was achieved by adding 30 g/L Na Cl at 9 h of fermentation,and the pinene yield was increased by 17%;the pinene yield was increased by 41%and 69%by overexpressing exogenous phosphatase and introducing NPP synthase through non-MVA pathway,respectively,and the squalene response effect was obvious,and the system of promoting pinene synthesis through squalene response with GPP and NPP as dual substrates was constructed.The yield of pinene reached 14.6 mg/L.By analyzing the dynamic relationship between pinene,ROS and squalene,it was found that pinene synthesis caused ROS accumulation,ROS as a signaling molecule promoted squalene synthesis,while the up-regulation of MVA pathway facilitated pinene synthesis.The final fermentation yield of pinene synthesis in a 5 L fermenter was 16.4 mg/L,which was higher than the highest yield（11.7 mg/L）reported so far for S.cerevisiae.