Optimization Of Heterogeneously Xylanase B Expressed Engineering Strain And Study Of Secretory Mechanism Of Heterologous Protein In Kluyveromyces Lactis

As a good host strain for protein heterologous expression, Kluyveromyces lactis was widely used for large-scale protein production in the food industry and the pharmaceutical industry. In the previous study, the xylanase B heterologous expression strain K. lactis GKX31 was constructed. It was found that the expression level of cell synthesis related gene KIGAS1-1 and molecular chaperone genes KlERO1, KIKAR2 and unfolded protein response （UPR） related gene KIGCN4 were significant changed while the protein heterologous expression level increased. In this study, gene deletion and gene over expression strategy were used for studying the affection of protein heterologous expression by the host strains changes.The K. lactis KlGas1-1 and KlGas1-2 proteins are classified as β-1,3-glucanosyltransferases and show 67% and 64% homology with β-1,3-glucanosyltransferase Gasl from S. cerevisiae S288c. Comparative amino acid sequences and predicted protein structures of KlGasl-1, K1Gas1-2, and Gas1, each of these proteins contains a Glyco_hydro₇2 domain （glucanosyltransferase domain） and X8 domain （involved in carbohydrate binding）. We conclude that the products of K1GAS1-1 and K1GASl-2 are β-1,3-glucanosyltransferases, and involved in K. lactis cell wall synthesis.KIGAS1-1 deletion strain K. lactis K1-1, KlGASl-2 deletion strain K. lactis K1-2 and two genes double deletion strain K. lactis KD were constructed using homologous double exchange strategy. It was found that single gene deletion did not affect cell growth and cell morphology. While the double genes deletion resulted growth lagged and cell shape turned into sphere with abnormal expansion. By the transmission electron microscopy observation, it was found that the KlGAS1-1 deletion decrease the thickness of cell wall, and KlGAS1-2 deletion result an opposite phenotype change, which increase the thickness of cell wall. Transcription patterns of six KIGas family genes were evaluated in GKX31 and the deletion mutants. KlGAS1-1, KlGAS1-2, and KlGAS3 are KlGas family genes most highly expressed in K. lactis. The β-1,3-glucanosyltransferase genes KlGAS1-1, KlGAS1-2 and other K1Gas family genes in K. lactis display transcription complementation that affects cell morphology, thickness of cell wall and cell growth.KlGASl-1 deletion and KlGASl-2 deletion could affect the increasing heterologous protein secretion in K. lactis. KlGASl-1 deletion and two genes double deletion could improve the secretion of heterologous protein （improved by 17.7% and 11.5%）. KlGAS1-2 deletion could reduce the secretion of heterologous protein （reduced by 35.6%）. KlGASl-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion-and transport-related genes. The findings indicate that increased transcription of secretion- and transport-related genes in K. lactis similarly enhances heterologous protein secretion, and K1-1 is a "super-secretor" of heterologous protein, KIGAS1-1 deletion was a more effective cell wall engineering approach than the other two deletion treatments for increasing heterologous protein secretion in K. lactis.The KIER01, KIKAR2, KIGCN4 over expression strain K. lactis GKERO, K. lactis GKKAR, K. lactis GKGCN were constructed using over expression vector pRS426-Gk which constructed by "DNA Multimer method". Comparision of over expression strains and original strain in phenotypic differences such as cell growth and xylanase B activity, it was found that the over expression of KlERO1, KIKAR2 and KIGCN4 resulted growth lagged. The over expression of KIERO1, KIKAR2 could improve the secretion of heterologous protein （improved by 33.6% and 14.1%）, and over expression of KIGCN4 could not improve the secretion of heterologous protein. It was found that in the strains with lower protein heterologous secretion level, the transcription levels of KICOG6, KlIMH1 and KICUP5 were increased. It indicated that the Golgi-to-vacuole mis-sorting of protein is the bottleneck limiting factor for heterologous protein secretion level improvement in K. lactis., the three genes will be modified for improving the heterologous protein secretion, in further studies.