The Construction Of Gluconobacter Oxydans Gene Editing System

Gluconobacter oxydans is widely used in industry due to its ability of oxidizing carbohydrate rapidly.At present,gene editing method in G.oxydans is homologous recombination,but this method has low efficiency and complicated operation,which makes it very difficult to manipulate genome,therefore limiting the application of G.oxydans.In recent years,the clustered regularly interspaced short palindromic repeats(CRISPR)system has been widely used in genome manipulation,which enriches gene manipulation methods and improves the efficiency of genome editing greatly.In this study,the CRISPR system was applied in G.oxydans,where Cas proteins from different sources were expressed to construct a two-plasmid gene manipulating system,which has achieved gene knockout,transcription repression and transcription activation in G.oxydans.Through the optimization of expression vectors and promoters,a gene knockout system has been constructed to improve gene knockout efficiency.Then through the optimization of cr RNA repeat sequences,the efficiency of gene transcription repression has been improved.And through the selection of activation domains,the CRISPRa system has been built up and optimized.The development of the CRISPR system in G.oxydans fills up the shortage of gene editing methods,and provides an effective gene manipulation tool for G.oxydans in metabolic engineering.Major results achieved in this study are as below:(1)Constructing a CRISPR gene editing system in G.oxydans.By expressing the Cas9protein from Streptococcus pyogenes in G.oxydans constructing the sg RNA expression cassette,and opyimizing the promoters and expression vectors of Cas9 and sg RNA gene operating system was constructed,which is suitable for G.oxydans.Besides,through heterologous expression of recombinase and optimization of the length of homologous fragments,the gene editing efficiency of G.oxydans was increased to 20.0%.(2)In order to achieve the transcriptional regulation in G.oxydans,a d Cpf1 mediated CRISPR interference(CRISPRi)gene transcription repression system was constructed.By expressing the inactivated Cpf1 protein(d Cpf1)and cr RNA containing 19 nt repeats in G.oxydans,the system shows an effective repression level in gene transcription which repression level for single genes can reach up to 97.9%.It also can be applied in the repression of multiple genes at the same time and shows strong repression ability.The system was applied to the metabolism pathway of L-sorbose and respiratory chain,and determined the effect of cytochrome bo₃ oxidase on cell growth.(3)Based on the transcription repression of G.oxydans,the inactivated Cpf1 protein was fused with four activation domains to construct a CRISPR-a Cpf1 mediated gene transcription activation system.By searching for locus near the transcription start site of the promoter,the transcription activation cr RNA was constructed,which guid the activation domain to the promoter-10 to-35 region,recruiting RNA polymerase,and increasing the transcription level of fluorescent protein by 53%.