The fermentation of both glucose and xylose is important to maximize ethanol yield from renewable biomass feedstocks. In this article, we analyze growth, sugar consumption, and ethanol formation by the yeast Kluyveromyces marxianus UFV-3 using various glucose and xylose concentrations and also under conditions of reduced respiratory activity. In almost all the conditions analyzed, glucose repressed xylose assimilation and xylose consumption began after glucose had been exhausted. A remarkable difference was observed when mixtures of 5 g L(-1) glucose/20 g L(-1) xylose and 20 g L(-1) glucose/20 g L(-1) xylose were used. In the former, the xylose consumption began immediately after the glucose depletion. Indeed, there was no striking diauxic phase, as observed in the latter condition, in which there was an interval of 30 h between glucose depletion and the beginning of xylose consumption. Ethanol production was always higher in a mixture of glucose and xylose than in glucose alone. The highest ethanol concentration (8.65 g L(-1)) and cell mass concentration (4.42 g L(-1)) were achieved after 8 and 74 h, respectively, in a mixture of 20 g L(-1) glucose/20 g L(-1) xylose. When inhibitors of respiration were added to the medium, glucose repression of xylose consumption was alleviated completely and K. marxianus was able to consume xylose and glucose simultaneously.
Here, we present the draft genome sequence of Kluyveromyces marxianus CCT 7735 (UFV-3), including the eight chromosomes and the mitochondrial genomic sequences.
In several organisms used for recombinant protein production, integration of the expression cassette into the genome depends on site-specific recombination. In general, the yeast Kluyveromyces lactis shows low gene-targeting efficiency. In this work, two K. lactis ku80⁻ strains defective in the non-homologous end-joining pathway (NHEJ) were constructed using a split-marker strategy and tested as hosts for heterologous gene expression. The NHEJ pathway mediates random integration of exogenous DNA into the genome, and its function depends on the KU80 gene. KU80-defective mutants were constructed using a split-marker strategy. The vectors pKLAC1/Plg1 and pKLAC1/cStpPlg1 were used to evaluate the recovered mutants as hosts for expression of pectin lyase (PNL) and the fusion protein streptavidin-PNL, respectively. The transformation efficiency of the ku80⁻ mutants was higher than the respective parental strains (HP108 and JA6). In addition, PNL secretion was detected by PNL assay in both of the K. lactis ku80⁻ strains. In HP108ku80⁻/cStpPlg1 and JA6ku80⁻/Plg1 cultures, the PNL extracellular specific activity was 551.48 (±38.66) and 369.04 (±66.33) U/mg protein. This study shows that disruption of the KU80 gene is an effective strategy to increase the efficiency of homologous recombination with pKLAC1 vectors and the production and secretion of recombinant proteins in K. lactis transformants.
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