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A high yield of lactic acid per gram of glucose consumed and the absence of additional metabolites in the fermentation broth are two important goals of lactic acid production by microrganisms. Both purposes have been previously approached by using a Kluyveromyces lactis yeast strain lacking the single pyruvate decarboxylase gene ( KlPDC1 ) and transformed with the heterologous lactate dehydrogenase gene ( LDH ). The LDH gene was placed under the control the KlPDC1 promoter, which has allowed very high levels of lactate dehydrogenase (LDH) activity, due to the absence of autoregulation by KlPdc1p. The maximal yield obtained was 0.58 g g −1 , suggesting that a large fraction of the glucose consumed was not converted into pyruvate. In a different attempt to redirect pyruvate flux toward homolactic fermentation, we used K. lactis LDH transformant strains deleted of the pyruvate dehydrogenase (PDH) E1α subunit gene. A great process improvement was obtained by the use of producing strains lacking both PDH and pyruvate decarboxylase activities, which showed yield levels of as high as 0.85 g g −1 (maximum theoretical yield, 1 g g −1 ), and with high LDH activity.

The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the Kl PDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the Kl PDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The l ac Z gene from Escherichia coli was cloned downstream of the Kl PDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii , and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii ; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor α prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.