Transcriptome Analysis And Metabolic Engineering Of Recombinant Escherichia Coli Producing Glycolate

Glycolate is an important?-hydroxy carboxylic acid which is easily degraded and absorbed and is widely used in packaging,textile,medical cosmetics and other fields.In our preliminary study,we have constructed engineered strain Mgly545,which produced 16.66 g·L^-1 glycolate in M9inorganic salt medium by using glucose as carbon source.However,M9 medium is suitable for the glycolate production rather than cell growth.The low biomass caused the low efficiency of glycolate production.Thus,Mgly545 could not reach a balance between cell growth and glycolate production.To solve those challenges,we mainly conducted research from the following three aspects:?1?In order to achieve efficient accumulation of glycolate in rich medium,this study explored the effects of different nitrogen sources and ICDH knockout on glycolate production.The addition of organic nitrogen sources could improve the cell growth in Mgly545,however,it caused excessive accumulation of acetate and decrease of glycolate production,which was not conducive to industrial production.The Mgly625 strain?with the ICDH deletion in Mgly545?kept the high glycolate production when grown under tryptone and yeast extract,reaching to 0.81 g glycolate/OD?2.6-fold higher than Mgly545?.?2?Three samples including the fermentation samples of Mgly545 in inorganic and organic nitrogen sources and the fermentation sample of Mgly625 in organic nitrogen sources were subjected to transcriptomics.The results showed that the significant up-regulation of acetyl-CoA synthetase?acs?in Mgly625 enhanced the utilization of acetate and reduced the accumulation of acetate.The up-regulation of phosphoenolpyruvate carbosylase kinase?pck?in Mgly625 improved the PEP replenishing pathway to recycle PEP for glycolate production.The intracellular NADPH/NADP+of three transcripts was mansured and transcriptional level of genes related to NADPH anabolism was compared.It was found that the organic nitrogen sources could enhance the pentose phosphate pathway and provide more NADPH for glycolate synthesis.To further study Mgly625,the significant changed genes related to N-regulation,oxidative stress response and iron transport were analyzed.Glutamate and serine were found to increase the biomass and productivity respectively.Meanwhile,overexpressing the arginine transport gene argT?Mgly6252?accelerated the cell growth rate and increased the biomass.Further,the presence of Fe²⁺also speed up the cells growth and compensate for the lack of reducing equivalents.Finally,the titer of glycolate in Mgly6252 reached 22.43 g·L^-1 in 5 L bioreactor.?3?In order to simplify the yield measurement of glycolate and lay a foundation for high-throughput screening of mutant strain,a glycolate biosensor was constructed in this study.In Escherichia coli,the glycolate could induce transcriptional regulator GlcC and enhance the transcriptional level of glcD.We constructed a biosensor plasmid combining the P_glcD and the fluorescence protein gene sfGFP,which could monitor the yield of glycolate by measuring the fluorescence intensity of cell.We studies the sensitivity and specificity of glycolate biosensor.The result showed that the low-copy biosensor pGBS-L was better in responding the different concentrations of glycolate,and the detection limit was 0-50 mM.In real-time monitoring of glycolate yield,fluorescence intensity showed a better linear relationship with glycolate production?R²=0.9082?.The response and detection limit are further improved by optimizing the RBS between P_glcD and sfGFP.In addition,the study showed that ArcA inhibited the expression of fluorescence protein under micro-aerobic condition.In high-throughput screen,biosensor could effectively screen out the mutant strains with higher glycolate yield and provided a new insight for the detection of metabolite production in fermentation engineering.