A novel, lactase-based selection and strain improvement strategy for recombinant protein expression in Kluyveromyces lactis

Microbial Cell Factories

Research

Claudia Simon¹  Sven-Erik Behrens¹  Melanie Wagner²  Thomas Klose²  Osita F Onuma²  Jan Baumann²  Jorrit-Jan Krijger²  Katja Schulze²  Christian Reinsch²  Karin D Breunig² 

[1] Institute of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, 06120, Halle, Germany;Institute of Biology, Martin-Luther University Halle-Wittenberg, 06120, Halle, Germany;

关键词: Lactis;    Expression Cassette;    Bovine Viral Diarrhea Virus;    Heterologous Gene;    Oxazolone;   

DOI  :  10.1186/1475-2859-11-112

 received in 2011-12-20, accepted in 2012-05-17,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThe Crabtree-negative yeast species Kluyveromyces lactis has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its LAC genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the LAC4 promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of K. lactis by homologous recombination was hampered by the high rate of non-homologous end-joining.ResultsSelection for growth on lactose was applied to target the insertion of heterologous genes downstream of the LAC4 promoter into the K. lactis genome and found to yield high numbers of positive transformants. Concurrent reconstitution of the β-galactosidase gene indicated the desired integration event of the expression cassette, and β-galactosidase activity measurements were used to monitor gene expression for strain improvement and fermentation optimization. The system was particularly improved by usage of a cell lysis resistant strain, VAK367-D4, which allowed for protein accumulation in long-term fermentation. Further optimization was achieved by increased gene dosage of KlGAL4 encoding the activator of lactose and galactose metabolic genes that led to elevated transcription rates. Pilot experiments were performed with strains expressing a single-chain antibody fragment (scFvox) and a viral envelope protein (BVDV-E2), respectively. scFvox was shown to be secreted into the culture medium in an active, epitope-binding form indicating correct processing and protein folding; the E2 protein could be expressed intracellularly. Further data on the influence of protein toxicity on batch fermentation and potential post-transcriptional bottlenecks in protein accumulation were obtained.ConclusionsA novel Kluyveromyces lactis host-vector system was developed that places heterologous genes under the control of the chromosomal LAC4 promoter and that allows monitoring of its transcription rates by β-galactosidase measurement. The procedure is rapid and efficient, and the resulting recombinant strains contain no foreign genes other than the gene of interest. The recombinant strains can be grown non-selectively in rich medium and stably maintained even when the gene product exerts protein toxicity.

【 授权许可】

Unknown   
© Krijger et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.