title: Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.
abstract: β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 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. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Zhao
authors.initials: Z
authors.lastname: Lian
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Jing-Bo
authors.initials: JB
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Chi
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Guo-Hua
authors.initials: GH
authors.lastname: Guan
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, P.R. China.
authors.forename: Ji-Lun
authors.initials: JL
authors.lastname: Li
mesh_terms: D002473:Cell Wall / Q000648:ultrastructure; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics / Q000378:metabolism; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D043364:Endo-1,4-beta Xylanases
keywords: GAS1; Kluyveromyces lactis; heterologous protein secretion; β-1,3-glucanosyltransferase