Analysis Of Biodetoxification Mechanism Of Phenolic Aldehyde Inhibitors And The Construction Of The Key Gene Device Library

Phenolic aldehydes derived from the pretreatment step of biorefinery processing chain include 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, representing p-hydroxyphenyl （H）, syringyl （S）, and guaiacyl （G） groups, respectively. These compounds are one of the three major inhibitor groups generated from lignocellulose pretreatment, furans, weak organic acids, and phenolics. Different from furan aldehydes （furfural and 5-hydroxymethylfurfural） and weak acids （acetic acid, formic acid, and levulinic acid）, phenolic aldehydes contain numerous sub-groups, behave poor water solubility, and are slowly biodegraded due to the aromatic rings. As an indispensable procedure, biodetoxification is a novel idea for inhibitor removal by the unique microorganisms with the strong inhibitor degradation and tolerance. The biodetoxification mechanism of furan aldehydes and weak acids are well studied in the previous works. However, biodetoxification of phenolic aldehydes are rarely concerned. In the thesis, the metabolic pathway and tolerance mechanism of phenolic aldehydes for the filamentous biodetoxification fungus Amorphotheca resinae ZN1 and the ethanologenic bacterium Zymomonas mobilis ZM4 were elucidated using RNA-Seq and DNA microarray; the key genes relating to phenolic aldehydes biodetoxification was screened and expressed in Z. mobilis ZM4, and it also investigated the phenolic aldehydes conversion and ethanol fermentability of the consolidated bioprocessing strain; finally, according to transcriptome and proteome data, the key gene device library relating to bacteria, yeast, and fungus were contructed for the biodetoxification of phenolics, furans, and weak acids inhibitors.In the first part, it aimed at elucidating the molecular mechanism of phenolic aldehyde inhibitors conversion into their acids and alcohols for A. resinae ZN1. Based on RNA-Seq technique, it developed the transcriptome of phenolic aldehydes conversion of A. resinae ZN1. 534,1576, and 1261 genes were differentially expressed during the conversion of 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, respectively. Oxidoreduction and transport were the main biological process during phenolic aldehydes convresion for A. resinae ZN1 by GO analysis. According to the predicted metabolic pathway of phenolic aldehyde inhibitors, alcohol dehydrogenase, aryl-alcohol dehydrogenase, and aldehyde reductase were the key enzymes for the reduction pathway of phenolic aldehydes, and aldehyde dehydrogenase was in charge of the oxidation of phenolic aldehydes.In the second part, it carried out the transcriptome using DNA microarray under the stress of phenolic aldehydes for Z. mobilis ZM4 to make the bioconversion mechanism clear. 442,67, and 306 genes differentially expressed under the stress of 4-hydroxybenzaldehyde, syringaldehyde, and vanillin, respectively. Reduction, transport, and regulation were the main mechanism of phenolic aldehyde inhibitors conversion for Z. mobilis ZM4. It identified 72 key genes relating to the reduction of phenolic aldehydes for Z. mobilis ZM4, including ZM01116 encoding oxidoreductase and ZMO1885 encoding NADH:flavin oxidoreductase/NADH oxidase significantly differentially expressed under the three phenolic aldehydes stress. According to genome map location,272 differentially up-regulated and 560 differentially down-regulated genes involved with 36 and 63 gene clusters under the stress of at least two phenolic aldehyde inhibitors, respectively.In the third part, it developed the robustness engineering in chassis Z. mobilis ZM4 to invesitigate the key genes relating to phenolic aldehyde conversion. It tried to strengthen the nature reduction pathway and reconstruct the oxidation pathway in Z. mobilis ZM4 in order to improve phenolic aldehyde inhibitors conversion and cellulosic ethanol productivity. It found that the NAD+-ALDH （PP₂680） from P. pulida KT2440 obviously improved phenolic aldehyde inhibitors conversion and cellulosic ethanol fermentation. Compared with the control, ethanol titer, ethanol productivity, and ethanol yield were separately increased by 63.7%,100.0%, and 106.3% in 15%（w/w） solid content corn stover at 24 h. The purified protein PP₂680 oxidized furfural,4-hydroxybenzaldehyde, vanillin, furfural, and 5-hydroxymethylfurfural into 4-hydroxybenzoate, vanillate, furoic acid, and 2,5-furandicarboxaldehyde in vitro. There was no phenolic acid and furan acid in fermentation system, although PP₂680 obviously impoved aldehyde inhibitor conversion and cellulosic ethanol fermentation. The co-expression of NAD+-ALDH （PP2680） and NADH-ADH （ZMO1696） improved aldehyde inhibitor conversion and cellulosic ethanol fermentability, and the indirect proof of cofactor anaplerotic reaction may mainly lead to fementability improvement for PP₂680. Unexpectedly, heterologous expression of PP₂680 gene in Z. mobilis ZM4 improved the gene expression level of alcohol dehydrogenase of ED pathway and H+ transport ATPase in oxidative phosphorylation.In the fourth part, it focused on the construction of the device library of the key genes relating to lignocellulos-derived inhibitors biodetoxification in order to provide synthetic biology tools for the inhibitor robustness engineering. According to transcriptome and proteome data under inhibitor stress, it constructed the device library of key genes for phenolics, furans, and weak acids biodetoxification for the bacteria （including B. subtilis, C beijerinckii, C. glutamicum, E. coli, L. brevis, P. putida, T. pseudethanolicus, and Z. mobilis）, yeast （including Pichia stipites and S. cerevisiae）, and filamentous fungus （A. resinae）. It found that the key genes of phenolics biodetoxification mainly involved with oxidoreduction, transport, and regulation; the key genes of furans biodetoxification involved in oxidoreduction, transport, regulation, and oxidation stress; the key genes of weak acid biodetoxification involved with central carbon metabolism, transport, and regulation; lignocellulose hydrolysate-tolerance mainly involved with transport and regulation, except for the versatile biological process for its complex components. Based on the contructed device library of the key genes for inhibitor biodetoxification, it predicted the ultimate degradation pathway of phenolic aldehydes in Z. mobilis ZM4 and found that the oxidation of phenolic aldehydes into phenolic acids （such as procatechuate and 3-O-methylgallate） was the key metabolic branch point.Conclusively, it intensively analyzed the biodetoxification mechanism of phenolic aldehyde inhibitors of the biodetoxification and fermentation strains at molecular biology level. It investigated phenolic aldehyde inhibitors detoxification and ethanol fermentation for the robustness engineering strain Z. mobilis ZM4. It constructed the device library of the key genes for the biodetoxification of phenolics, furans, and weak acids for bacteria, yeast, and fungus. This study would provide the synthetic biology gene device library and the consolidated bioprocess engineering platform for the inihibitor conversion robustness and fermentability strenthening.