Impact And Underlying Molecular Mechanisms Of Temperature And Medium Composition On Ethanol Fermentation Of Zymomonas Mobilis

As a clean and renewable energy alternative to fossil fuels,bioethanol has a large demand and a broad market,but its production is restricted by many aspects,such as fermentation strains,raw materials and environmental conditions.Zymomonas mobilis is model ethanologenic bacterium with many excellent properties,such as high glucose absorption rate,high ethanol yield,high glucose and ethanol tolerance,resistant to a wide range of temperature（24～45°C）and p H（p H4.0～8.0）,and generally regarded as safe（GRAS）.Besides Saccharomyces cerevisiae,Z.mobilis is another model ethanologen.Ethanol fermentation is affected by carbon,nitrogen,and inorganic salts in fermentation media,and environmental conditions such as temperature,p H,and oxygen content.In this study,effects of temperature on cell growth,morphology,glucose uptake and ethanol production of Z.mobilis wild-type strain ZM4 was investigated,and RNA-Seq was used to examine the effect of temperature on the overall gene expression profile.In addition,a recombinant strain ZM4＿EGFP expressing the reporter gene EGFP was constructed,and the effect of temperature on the expression of exogenous EGFP proteins was also investigated.Subsequently,bioinformatics methods were used to select heat-responsive genes,which were then characterized by genetic studies to evaluate the association of candidate genes with high-temperature resistance.The results showed that temperature had a significant effect on cell growth and morphology of ZM4 or ZM4＿EGFP.With the increase of temperature,cell biomass decreased gradually,and cell shape became longer correspondingly.Temperature affected the transcriptional profile of ZM4 with 478 genes up-regulated and 481 genes down-regulated in ZM4 cultured at 45℃compared with those at 30℃.The results of flow cytometry and Western blot showed that high temperature of 45℃was not favorable for the expression of exogenous protein EGFP,but a lower temperature range（36～40℃）was more suitable for the expression of EGFP.High-temperature-resistant candidate genes were selected from RNA-Seq results,and then characterized by genetic studies through replacing the original promoter of the target gene with an inducible Ptet promoter or increasing the copy number of the target gene.The results showed that genes ZMO0236,ZMO1335,gro ESL,ZMO0994 and foreign gene csp L were associated with thermotolerance,and overexpression of these genes enhanced the thermotolerance of ZM4.Uncoupling between cell growth and ethanol production was observed during fermentation that glucose in the medium was not completely consumed and ethanol production did not reach the maximum titer even though cell growth reached the stationary phase,which significantly decreased ethanol productivity.Effects of supplementation of additional nitrogen sources in rich medium（RM）and replacement of original nitrogen sources in RM and minimal medium（MM）on Z.mobilis were examined in order to address this problem.The results illustrated that nitrogen sources such as yeast extract,peptone and corn steep liquor produced by different companies affected cell growth,glucose utilization and ethanol production of Z.mobilis.Among nitrogen sources investigated,YE^BD,YE^SG,and 5%CSL are nitrogen sources that are able to couple cell growth with ethanol production.Concentration of major ionic elements in these nitrogen sources were then quantified using the inductively coupled plasma optical emission spectroscopy（ICP-OES）.The experimaental results demonstrated that magnesium ion in the medium is one of the key factors affecting cell growth,glucose consumption and ethanol production.The effect of magnesium on gene expression was further investigated using RNA-Seq transcriptomics,and the results showed that survival of ZM4 was maintained under low magnesium ion pressure by down-regulating the expression of genes related to protein secretion and up-regulating the expression of genes related to stress.Using the method of replacing the original promoter of the target gene with a Ptet inducible promoter,four Mg²⁺transporter related mutants were constructed to further study the relationship between the mechanism of Mg²⁺transport and environmental stress.The results exhibited that ZM4＿0770 was sensitive to magnesium ion changes and ZM4＿0484 cell morphology became larger under induced conditions.RNA-Seq results of these two mutants showed that overexpression of ZMO0770 caused the overexpression of gene encoding cell wall hydrolase Sel B,which may disrupt cell wall structure and lead to reduced cell viability under low magnesium ion stress.Overexpression of ZMO0484 caused differential expression of five genes adjacent to ZMO0484.The effect of overexpression of ZMO0484 is similar to mre B deletion,possibly by promoting the expression of peptidoglycan hydrolase leading to softer and rougher cell walls and ultimately larger cell size.Finally,based on the inducible CRISPR-dCas12a system constructed earlier in our laboratory,a library of p EZ-cr RNA editing plasmids was constructed,which contains a total of68,253 cr RNAs designed for the whole genome of Z.mobilis including 54,259 in gene coding regions,11,994 in intergenic regions,and 2,000 negative controls.The plasmid library was then electroporated into ZM＿dCas12a mutant strain to obtain the ZM＿dCas12a＿E1006A＿cr RNA library（hereinafter abbreviated as“ZM＿dCas12a library”）.Based on the previous research and preliminary experimental results,RM was used as the control,and media of 8 mg/L Mg²⁺,p H4,and 40°C were selected as stress conditions.Under these conditions,ZM＿dCas12a library strains showed little change in the growth with or without the addition of 0.8μg/m L of tetracycline,and there were almost no differences among specific growth rates of the strains.RNA-Seq results of the ZM＿dCas12a library strains showed that the expression value of dCas12a in the ZM＿dCas12a library under four conditions of RM,40°C,8 mg/L Mg²⁺,and p H4 induced by 0.8μg/m L tetracycline was 1.47±0.23,1.22±0.28,1.23±0.19,and 20.36±0.31folds than those without tetracycline induction（0μg/m L）,respectively.By comparing RNA-Seq data of ZM4 and ZM＿dCas12a library strains under different stress conditions,two strains shared a small number of up-regulated and down-regulated genes under different stress conditions.The results indicated that gene ZMO0374 encoding fructansucrase was sensitive to changes in magnesium ions in the medium,while ZMO1063 encoding phage shock protein A and ZMO1237 encoding lactate dehydrogenase are likely to be related tohigh-temperature resistance.In conclusion,this study not only systematically studied the effect of temperature and nitrogen source in nutrient elements on fermentation performance of Z.mobilis at molecular level,but also identified and characterized genes for high-temperature resistance.In addition,a genome-wide CRISPRi system was established and applied to study the genes and regulatory networks under stress conditions such as high temperature,low magnesium ions,and acidic p H,and the results from these studies under various stress conditions can help identify essential genes as well as general and condition-specific stress responsive genes and regulatory network to facilitate future development of of Z.mobilis as a synthetic chassis for sustainable biochemical production.At the same time,the combination of RNA-Seq transcritpomics,genome-wide CRISPRi-Seq phenomics,and bioinformatics analysis provides guidance on investigating stress responses in other microrganisms.