Development Of Genetics Tool Of Cellulolytic Clostridia

Lignocellulosic biomass is the most abundant biopolymer on earth,yet its recalcitrance to hydrolysis has severely hampered its exploitation for renewable energy and materials.In nature,anaerobic and cellulolytic Clostridia,such as Ruminiclostridium cellulolyticum and Ruminiclostridium papyrosolvens,can efficiently digest plant cell wall polysacharides into fermentalbe sugars thanks to their extracellular multienzymatic complexes called cellulosomes.They are considered to be promising consolidated bioprocessing candidates for producing renewable green chemicals from cellulose to replace petroleum-based products.However,their metabolic engineering is crucial for their potential as chassis for synthetic biology.The genetic modification of these candidate is relatively lacking,especially in R.papyrosolvens.For example,the methylation of plasmid transformed into R.cellulolyticum in vitro and the off-target phenomenon of Clos Tron technology.These defects seriously hinder the genetic engineering modification of cellulolytic clostridia and biotransformation technology of lignocellulose.In our study,a plasmid in-vivo methylation system was developed which made the transformation of R.cellulolyticum more easily.On the other hand,the activity of four inducible promoters in R.papyrosolvens was analyzed and compared.The research details were as following:I.Development of an in-vivo methylation system of plasmids in R.cellulolyticumThe Restriction-Modification system as biological immune system,commonly exists in prokaryotes.Microorganisms imploying it can distinguish self/non-self components and protect from foreign DNA.R.cellulolyticum has a type II restriction enzyme Cce I,which results in the transformation inability of foreign DNA.Thus,the foreign plasmid need to be methylatd by Cce I methyltransferase?M.Cce I,Ccel₂867?in vitro before transformation.However,Cce I methyltransferase is expensive and easily inactivated.In order to overcome the inadequacy of plasmid methylation in vitro,we successfully cloned the Cce I methyltransferase gene from R.cellulolyticum and constructed an engineering strain of Escherichia coli that can express functionally express M.Cce I.Using this engineered strain,the plasmids were methylated in vivo,and successfully transformed into R.cellulolyticum.Based on this method,the restriction endonuclease gene cce I in R.cellulolyticum was knocked out.A difficult point in this study was the functional expression of the methyltransferase gene in E.coli.E.coli has type IV restriction enzymes,such as Mcr A,Mcr BC,which can specifically recognize and cleave methyl-modified DNA.So,the DNA methyltransferase gene is a toxic gene to E.coli.In this study,we found that E.coli HST08 express the Cce I methyltransferase from R.cellulolyticum.II.Screening and application of inducible promoters in R.papyrosolvensCompared with R.cellulolyticum,R.papyrosolvens has a higher degree of evolution of cellulosome,suggesting a more complete evolution of its gene function.However,R.papyrosolvens still lacks genetic manipulation system,especially elements of gene experesion,such as inducible promoters.Therefore,we first compared two exogenous Clostridial inducible promoters?arabinose-induced promoter Para from C.acetobutylicum and lactose-induced Plac from C.perfringens?and two endogenous inducible promoters?cellobiose induced Pcel and xylan induced Pxyl?in R.papyrosolvens.The results showed that both of the endogenous xylan-inducible promoter Pxyl and exogenous lactose-inducible promoter Plac with high specificity and stringency are suitable for R.papyrosolvens.Pxyl have the shorter time to be induced but Plac have the lower concentration of inducer.Imploying them,we successfully construct a maz F-based counterselection system,which can be used to increase the mutant screening efficiency via induction of plasmid curing.This system can easily screen mutants by inducing plasmids curing.In summary,we developed a plasmid in-vivo methylation system for rapid and low-cost transformation of R.cellulolyticum,and a counterselection system based on Maz EF imploying two highly specific and stringent inducible promoters in R.papyrosolvens.These works will thus increase the genetic manipulation capability for engineering cellulolyitc Clostridia.