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Background  The 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.    Results  Selection 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 (scFv ox ) and a viral envelope protein (BVDV-E2), respectively. scFv ox  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.    Conclusions  A 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.

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