Isolation, nucleotide sequence, and physiological relevance of the gene encoding triose phosphate isomerase from Kluyveromyces lactis

Abstract

Lack of triose phosphate isomerase activity (TIM) is of special interest because this enzyme works at an important branch point of glycolytic flux. In this paper, we report the cloning and sequencing of the Kluyveromyces lactis gene encoding TIM. Unlike Saccharomyces cerevisiae DeltaTPI1 mutants, the K. lactis mutant strain was found to be able to grow on glucose. Preliminary bioconversion experiments indicated that, like the S. cerevisiae TIM-deficient strain, the K. lactis TIM-deficient strain is able to produce glycerol with high yield.

FIG. 1

Confirmation of gene disruption by Southern analysis. Lane 1, DNA from the control strain; lane 2, DNA from the disrupted strain; lane 3, Dig-labelled DNA molecular weight marker III (Boehringer Mannheim).

FIG. 2

Growth curves of the K. lactis control strain (closed circles) and ΔTPI1 (open squares) strains. The values are the average of two independent experiments with a standard deviation lower than 5%. A, YNB–2% glucose; B, YNB–2% lactose; C, YNB–0.5% ethanol–2% glucose; D, YEP–2% glucose. OD, optical density.

FIG. 3

Production of glycerol and glucose utilization by the K. lactis control strain (closed circles and squares, respectively) and the ΔTPI1 strain (open circles and squares, respectively). Experimental results are the average of two independent experiments with a standard deviation lower than 10%. Glu, glucose; Gly, glycerol (g/l).