| 2-Phenylethanol(2-PE)is one of the most important flavor compounds,with a rose-like odor.It has been utilized in many fields,such as food,tobacco and cosmetics manufacture.Recently,microbial biosynthesis of 2-PE has gained great attention,since chemically synthesized 2-PE contains carcinogenic byproducts and extracted one from flowers is costly.However,the high cytotoxicity of 2-PE limits its prosuction.In this study,a superb multiple stress-tolerant yeast,Candida glycerinogenes,was selected to improve 2-PE synthesis and uncover the underlying mechanism of 2-PE tolerance.(1)Multi-module metabolic engineering of C.glycerinogenes enhanced 2-PE production.Combining genomic metabolism network and GC-MS technology,it was confirmed that C.glycerinogenes strain possesses the Ehrlich pathway with L-phenylalanine as the precursor and the phenylpyruvate pathway with glucose as the precursor,and the former had the higher efficient 2-PE synthesis.To improve 2-PE titer,2-PE metabolic networks involved in Ehrlich pathway were stepwise rewired using metabolic engineering,including the following:(1)overexpressing L-phenylalanine permease Aap9 enhanced precursor uptake;(2)overexpressing enzymes(aminotransferase Aro9,decarboxylase Aro10,dehydrogenase Adh1and transcriptional factor Aro80)of Ehrlich pathway increased catalytic efficiency;and(3)disrupting the formation of by-product phenylacetate catalyzed by Ald2 and Ald3 and ethanol synthesis related gene pdc1 maximized the metabolic flux toward 2-PE.The final titer of2-phenylethanol reached 4.6 g·L-1,which increased 27.8%.In-situ extraction technology found that 2-PE cytotoxicity(4.6 g·L-1)was the major barrier to the high-level production.(2)Transcriptomic analysis explored the mechanism of 2-PE tolerance.Spot dilution assay and growth curves indicated that C.glycerinogenes showed higher 2-PE tolerance compared to Saccharomyces cerevisiae.C.glycerinogenes could grow at medium containing4.0 g·L-1 2-PE,which is much higher than the 2-3 g·L-1 that can completely inhibit most prokaryotic strains and fungi.Transcriptomic analysis showed that numerous genes related to ergosterol synthesis,phospholipid synthesis,fatty acid synthesis,glutathione synthesis,arginine synthesis and MAPK signal transduction pathway were up-regulated,while genes associated with ribosome proteins and central metabolic pathway were down-regulated in response to 2-PE stress.Genetic engineering of partial pathways confirmed the important role of these pathways in resist 2-PE stress.Based on the understanding of the regulation mechanism of 2-PE tolerance,genetic engineering of pressure-responsive gene element slc1to improve 2-PE tolerance.Under 3.5 g·L-1 2-PE,the biomass of the overexpression strain Cgslc1 increased 18.3%compared to the wild-type strain.(3)Screening the 2-PE stress resistance gene elements and exploring its regulatory mechanism.This study employed a genome-scale screen and successfully identified a2-PE-resistant gene gsh2 in C.glycerinogenes.Transcriptomic data showed that the expression of gsh2 was significantly up-regulated(7.9-folds)under 2-PE stress.After exogenous addition of glutathione and its degradation diethyl maleate,the 2-PE tolerance of C.glycerinogenes was respectively significant increased or reduced,indicating that contribution of gsh2 to 2-PE tolerance is largely dependent on intracellular glutathione levels.Analyzing the physiological phenotypic changes of Cggsh2 and wild-type strains under 2-PE stress,we found glutathione protects cells against 2-PE stress by reducing ROS accumulation,lipid peroxidation,and cell membrane damage.The upstream regulatory factors Hap5 was obtained by yeast one-hybrid technology and genetic engineering.Combined with bioinformatics and genetic mutation technique,it is confirmed that Hap5 activates the downstream target gene gsh2 through the CCTTA-box binding site.Through q RT-PCR technology and subcellular localization technology,we found that Hap5 transferd from the cytoplasm to the nucleus to regulate the transcriptional expression of downstream target genes,and its activation occurs at the post-translational modification level rather than the transcription level under 2-PE stress.Finally,combined with the stress resistance gene element gsh2 and its regulatory element Hap5,the molecule mechanism of C.glycerinogenes strain in response to 2-PE stress at intracellular solubles was initially obtained.Under 2-PE stress,Hap5 is activated and imported into the nucleus rapidly.Then,the nuclear Hap5 binds to the glutathione synthetase(gsh2)promoter via CCAAT box,to induce the expression of gsh2 gene.The increased gsh2expression contributes to enhanced cellular glutathione content,and consequently alleviates ROS accumulation,lipid peroxidation,and cell membrane damage caused by 2-PE toxicity.(4)Difference in extracellular and intracellular tolerance of 2-PE and its different mechanisms.The concentration of 2-PE production was higher than 2-PE addition in C.glycerinogenes,SEM showed C.glycerinogenes turned to roundness,bigger and reduced specific surface area under 2-PE addition;however,C.glycerinogenes turned to smaller and increased specific surface area during 2-PE fermentation process.The main differences of tolerance mechanism were studied by analysing the composition and molecular level of cell membrane.The extracellular addition of 2-PE resulted in down-regulation of the transcriptional expression of unsaturated fatty acid synthesis genes,and increased the proportion of saturated fatty acid in cell membrane,which strengthened the rigidity of cell membrane and reduced 2-PE enter to cell.However,during the intracellular synthesis of 2-PE,the transcriptional expression of unsaturated fatty acid synthesis genes was up-regulated,the proportion of unsaturated fatty acids was increased,and the flexibility of cell membrane was enhanced,which was accelerated to rapid efflux of 2-PE.These different mechanisms were mediated by transcriptional factors Hog1 and Swi5.C.glycerinogenes activated Hog1 and repressed Swi5 to upregulate erg5 and erg4 expression,which increased cell membrane rigidity and resisted 2-PE import under 2-PE addition situation.During 2-PE fermentation process,C.glycerinogenes activated Hog1 and repressed Swi5 to upregulated 2-PE transporter proteins Cdr1 and Acyl-Co A desaturase 1 Ole1 to increased 2-PE export,which reduced 2-PE intracellular toxicity.Based on these,fine-tuning the expression of ole1 gene increased 2-PE tolerance.(5)Improving 2-PE production based on the regulation mechanism of 2-PE tolerance.To improve the 2-PE tolerance of strain UA5p14,2-PE stress resistance gene elements slc1,gsh2or ole1 was engineered in strain UA5p14.The results showed that slc1 overexpressing strain UA5p20 showed the highest cell biomass at 3 g·L-1 2-PE,which increased 20.2%compared to strain UA5p14.The next one was gsh2 overexpressing strain UA5p21,which 14.4%higher than strain UA5p14.The 2-PE fermentation performance of strain UA5p20 was investigated.The results showed that maximum growth rate of strain UA5p20 was 1 h-1,2-PE titer was 5.0g·L-1,L-Phe conversion was 95.9%,and 2-PE productivity was 0.21g·(L·h)-1,which increased by 75.4%,38.9%,5%and 177%,respectively.The maximum titer of 2-PE by the engineered strain UA5p20 was 6.2 g·L-1 in a 5 L fermenter.q RT-PCR showed that strain UA5p20 enhanced the 2-PE tolerance and production mainly by up-regulated the expression of 2-PE synthesis pathway,central metabolic pathway and cell membrane biosynthesis pathway involed genes,which was consistent with the results of mechanism research. |
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