| Summary: | <p>Abstract</p> <p>Background</p> <p>The Crabtree-negative yeast species <it>Kluyveromyces lactis</it> has been established as an attractive microbial expression system for recombinant proteins at industrial scale. Its <it>LAC</it> genes allow for utilization of the inexpensive sugar lactose as a sole source of carbon and energy. Lactose efficiently induces the <it>LAC4</it> promoter, which can be used to drive regulated expression of heterologous genes. So far, strain manipulation of <it>K. lactis</it> by homologous recombination was hampered by the high rate of non-homologous end-joining.</p> <p>Results</p> <p>Selection for growth on lactose was applied to target the insertion of heterologous genes downstream of the <it>LAC4</it> promoter into the <it>K. lactis</it> 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 <it>KlGAL4</it> 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 (scFv<sub>ox</sub>) and a viral envelope protein (BVDV-E2), respectively. scFv<sub>ox</sub> 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.</p> <p>Conclusions</p> <p>A novel <it>Kluyveromyces lactis</it> host-vector system was developed that places heterologous genes under the control of the chromosomal <it>LAC4</it> 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.</p>
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