Study On The Genetic Engineering Of Xanthan Gum Degradation Gene Cluster

Xanthan gum is a water-soluble polysaccharide secreted by Xanthomonas campestris,Modification the xanthan molecular structure is expected to provide new materials for biomedicine and food industry,and has high application value and development prospect.Currently,the biodegradation pathway of xanthan gum has not been fully elucidated,hampers rational modification of xanthan.Microbacterium sp.XT11 is a xanthan-degrading microbactium which can use xanthan gum as the sole carbon source for growth.The results of the previous genomic data analysis showed that there is a gene cluster closely related to xanthan gum biodegradation in the XT11 genome,The elucidation of the structure and expression regulation characteristics of the gene cluster was valuable for revealing the molecular mechanism of xanthan gum biodegradation.it was beneficial to resoving the molecular mechanism of xanthan gum biodegradation.In this study,xanthan-degrading strain Microbacterium sp.XT11 was used as starting strain,and the xanthan degradation related gene cluster was extracted from the genome of the XT11 strain using the transformation assisted recombination(TAR).The gene cluster size is about 17 kb,contains the endglucanase gene,glucosidase gene,mannosidase gene,xanthan lyase gene,a transcriptional regulatory element and ABC type sugar transport system coding genes.Using the efficient homologous recombination mechanism of Saccharomyces cerevisiae and GFP reporter gene optimized the screening efficiency,our work successfully linked the gene cluster to the B.subtilis-E.coli shuttle expression plasmid pAX01 to obtain the recombinant expression plasmid pALHC.The construction of the recombinant expression plasmid pALHC is valuable for subsequent heterologous expression and structural analysis of gene cluster.Subsequently,we investigated the transcription and expression of each element in xanthan degradation cluster by reverse transcription PCR and real-time quantitative PCR.The results showed that the endglucanase was expressed and regualated as one transcript and the other four protein-coding genes(ABC-type sugar transporter,Glucosidase,Mannase,and Xlyase)belong to one transcription unit.This result indicated indicates that the xanthan degradation gene cluster may be regulated by various trans-acting factors.The gene expression analysis showed that the expression level of each gene in the xanthan degradation gene cluster increased in xanthan medium compared with that in glucose medium.However,within the the latter transcription unit,there may be an endogenous promoter in front of the mannosidase coding gene,resulting in increased expression level.Finally,based on gene transcription and expression results,the promoter prediction online software Softberry and MEME suite were used to predict possible promoter sequences and regulatory motifs in the gene cluster,respectively.The results showed that three potential promoters exist in the upstream of the endoglucanase-encoding gene,the ABC-type sugar transporter gene,and the mannosidase-encoding gene,respectively.The common regulatory motif of three promoters was TCNATNCT.Electrophoretic mobility shift assay(EMSA)was used to capture the trans-acting factors involved in the regulation of each element expression in the gene cluster.Recovery of bound proteins and LC-MS analysis suggested 6 possible regulatory proteins,including single-stranded DNA binding protein(LX1-1GL000807),hypothetical protein:transcriptional regulator K(LX1-1GL000216),RpiR family transcriptional regulator(LX1-1GL000455),cold shock protein: DNA binding protein(LX1-1GL001153),hypothetical protein:DNA-directed RNA polymerase,sigma subunit(LX1-1GL000256),cold shock protein: DNA binding protein(LX1-1GL000925).Our work is valuable for revealing the xanthan degradation pathway and rational modifying of xanthan molecules.