Tolerance Mechanism Elucidation And Metabolic Engineering Improvement Of Biorefinery Fermentation Strains

Tolerance to inhibitors generated during pretreatment is one of challenges for the efficient bioconversion of lignocelluloses biomass.While acetic acid is the most important acidic inhibitor released from the hydrolysis of acetyl groups in hemicelluloses,and p-benzoquinone is a newly identified quinone inhibitor released from the degradation of ligin,Na+is accumulated with the neutralization after the pretreatment.The existence and accumulation of these inhibitors in lignocellulose hydrolysate shows strong inhibition on microbial cell growth and product fermentation.It is crucially important to remove acetic acid,sodium ion and p-benzoquinone from the pretreated lignocellulose feedstock for achieving the high fermentation yield of biofuels and biochemicals.Biodetoxification of inhibitor using the specific microorganisms with inhibitor degradation ability prior to enzymatic hydrolysis and fermentation,or development of robust fermention strains with high inhibitor tolerance is the promising way to overcome the inhibition of acetic acid,sodium ion and p-benzoquinone.The filamentous fungus Amorphotheca resinae ZN1 is the most important biodetoxification strain with the advantages of zero waste water generation,zero energy consumption and high fermentation yield.This study elucidated the biological metabolism and tolerance response of acetic acid by A.resinae ZN1.Acetic acid metabolism by A.resinae ZN1 in the submerged liquid culture and solid state culture with varying glucose and xylose showed that acetic acid was consumed as a prior substrate to glucose and xylose by A.resinae ZN1,and the consumption is highly accelerated by solid state culture.Acetic acid metabolism pathway and tolerance response in A.resinae ZN1 were investigated by genome-wide RNA-Seq analysis and qRT-PCR analysis.Acetic acid was found to be metabolized through the tricarboxylic acid cycle when glucose existed in the medium,while through the two cycles of both the tricarboxylic acid cycle and glyoxylate cycle when there was no sugar in the medium.The tolerance response of A.resinae ZN1 to acetic acid included various biological processes such as activation of ions transport,increase in amino acids uptake and biosynthesis,as well as induction of ergosterol biosynthesis and energy generation.Zymomonas mobilis ZM4 is an imporatant industrial strain for cellulosic ethanol fermentation,but it is sensitive to metal ions.This study screened and overexpressed the genes responsible for the transmembrane uptake and/or efflux of metal ions in Z.mobilis ZM4 for increasing its sodium ion tolerance.The ZMO0119 gene encoding Na+/H+ antiporter was identified to be highly effective for reducing intracellular sodium ion concentration of Z mobilis by improving the sodium ion transport capacity.Overexpression of ZMO0119 gene in Z.mobilis ZM4 reduced the intracellular sodium ion content by 43.7%compared to the control strain,significantly improved the tolerance of Z.mobilis cells to sodium ion stress.and accelerated the cell growth,glucose consumption,and cellulosic ethanol production by Z.mobilis from the dry acid pretreated and biodetoxified corn stover feedstock containing high sodium ion content.p-Benzoquinone is a lignin derived strong inhibitor in pretreatment of lignocellulose to biorefinery fermentation strains.Different from the well acknowledged inhibitors of furan aldehydes,weak acids,and phenolics,its inhibitory behaviors,tolerance mechanism and detoxification strategy had not been clearly elucidated.This study identified the existence of high p-benzoquinone in various dry dilute acid pretreated lignocellulose feedstocks and examined the inhibitory behaviors of p-benzoquinone on different biorefinery strains.p-Benzoquinone was identified to be an inevitable byproduct of lignocellulose acid based pretreatment,and severely inhibited the cell growth and product fermentation of different yeasts and bacteria strains by inducing reactive oxygen species generation,destroying cell outer membrane integrity and inhibiting glycolysis and product fomiation process directly at a low level of 0.02-0.3 g/L.The p-benzoquinone degradation by different biorefinery fermentation strains were examined at the metabolic level and the genes responsible for p-benzoquinone conversion were mined at the transcriptional level.The p-benzoquinone tolerance was found to be well closely related to the conversion of p-benzoquinone to the non-toxic hydroquinone,various genes encoding oxidoreductase genes,reductase,dehydrogenase and hydroxylase were identified to be responsible for p-benzoquinone conversion in Z.mobilis.The overexpression of the key genes of ZM01696,ZMO1576,ZMO1949,ZMO1984 and ZMO1399 significantly improved the tolerance of Z.mobilis to p-benzoquinone,and accelerated the cellulosic ethanol production in the p-benzoquinone containing medium and in the lignocellulose hydrolysates.In conclusion,in order to overcome the inhibition of acetic acid,sodium ion and p-benzoquinone on biorefinery strain,the acetic acid metabolism behavior,degradation and tolerance mechanism in A.resinae ZN1 were elucidated at the metabolic level and the molecular biology level;The robustness engineering strain Z mobilis ZM4 with improved sodium ion tolerance and ethanol production was constructed through rational genetic modification;The inhibitory behaviors,degradation and tolerance mechanism of p-benzoquinone as well as the key genes responsible for p-benzoquinone tolerance were investigated based on the metabolic level analysis and the gene transcription level analysis.This study provided the important synthetic biology basis for the future modifications with enhanced inhibitor tolerance and cellulosic ethanol fermentability by biorefinery fermentation strains.