Molecular Mechanisms Of Enhanced Ethanol Tolerance Associated With Hfq Overexpression And Exploration Of Argonaute Endonuclease In Zymomonas Mobilis

Industrial microbial strains are the pivotal of biomanufacturing and bioeconomy.Zymomonas mobilis is a non-model ethanol natural production strain.Due to the unique physiological characteristics,Z.mobilis is regarded as one of the excellent chassis cells for the construction of microbial cell factories for lignocellulosic biomass ethanol and other biochemicals.However,in the production of cellulosic ethanol and other biochemicals,the growth rate and fermentability of Z.mobilis are usually inhibited by the toxic compounds produced during the pretreatment and enzymatic hydrolysis as well as the toxic end-products such as ethanol and isobutanol.Therefore,improvement of the tolerance and robustness of microbial cell factories is of great significance for economic biochemical production.It has been reported that overexpression of hfq helps Z.mobilis tolerate a variety of hydrolysate inhibitors,but the underlying molecular mechanism has not been revealed yet.Despite that endogenous and exogenous genome-editing tools using CRISPR-Cas systems have been developed in Z.mobilis,more efficient and effective genome editing tools are still needed.In this study,Z.mobilis ZM4 was used as a model to explore the molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression.The recombinant strains with hfq overexpression（ZM4-hfq）or hfq deletion（ZM4-Δhfq）were constructed and evaluated in terms of cell growth,sugar utilization,and ethanol production.Corresponding to the better cell growth,ZM4-hfq exhibited faster glucose consumption and ethanol production than ZM4 under8%ethanol stress and hfq deletion in Z.mobilis significantly decreased the ethanol tolerance.The effect of ethanol stress on global transcriptional differences was evaluated by RNA-Seq and bioinformatics methods.The presence of ethanol made many genes differentially expressed,which were mainly associated with biogenesis of cell wall and membrane,metabolism,transcription,and general stress responses.hfq overexpression helps negatively regulate the flagellar related genes and conserve energy for cell growth and ethanol stress responses.Moreover,hfq overexpression enhances sulfate assimilation and cysteine biosynthesis and eliminates the oxidative stress induced by ethanol inhibition.In addition,Hfq protein interactions were identified by a new protein interaction analysis method YESS（Yeast Endoplasmic Reticulum Sequestration Screening,YESS）system.Hfq may indirectly modulate the Rpo H through its interaction with Dna K/J and subsequently regulates the Rpo H-mediated heat shock responses under ethanol stress.Moreover,combining with Co-IP-MS and YESS system analysis,ZMO1690,ZMO0989 and other proteins were identified to be associated with the enhanced ethanol tolerance by overexpressing hfq in Z.mobilis.To address the problem of lacking efficient gene-editing tools in Z.mobilis,the potential of development of Agos-based genome editing tools in Z.mobilis was further explored.A prokaryotic Ago（p Ago）from a hyperthermophilic archaeon Thermococcus thioreducens（Ttd Ago）was characterized in vitro,and results showed that Ttd Ago has a typical DNA-guided DNA endonuclease activity,and the efficiency and accuracy of cleavage are modulated by temperature,divalent ions,as well as the phosphorylation and length of g DNAs and their complementarity to the DNA targets.The cleavage activity of Ttd Ago requires divalent metal ions such as Mn²⁺or Mg²⁺.Ttd Ago cleaved DNA at a wide range of temperatures from 30°C to 95°C and had good DNA cleavage activity at 70～80°C.For Ttd Ago,a minimum of 16 nt g DNA is required.Ttd Ago prefers to use 5’P-g DNA as a guide strand with no obvious preference for the 5’end-nucleotide of the guide.Ttd Ago has a high tolerance for mismatches between the guide and target strands,and generates double-stranded DNA breaks in double-stranded DNA utilizing a g DNA or a pair of g DNAs.Furthermore,Ttd Ago had cell toxicity to Z.mobilis,which led to a decreased growth rate and final biomass.Interestingly,the overexpression of hfq contributed to the toxicity reduction.The preliminary results of this study thus indicated that Ttd Ago may have genome editing activity in Z.mobilis although further investigation is needed.In conclusion,this study systematically investigated the underlying mechanism of enhanced ethanol tolerance by hfq overexpression and identified genes and pathways related to ethanol stress tolerance,which provides more genetic targets for robust stain construction in Z.mobilis.Furthermore,the catalytic mechanism of Ttd Ago and its in vivo editing activity in Z.mobilis were explored,which broadens the understanding of prokaryotic Ago proteins and lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.At the same time,this study established an alternative YESS system to investigate protein-protein interactions in Z.mobilis,which provides a new strategy for the exploration of prokaryotic protein-protein interactions.