The ‘petite‐negative’ yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity

Abstract

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcA delta strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low-affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.

A dual signalling pathway for the hypoxic expression of lipid genes, dependent on the glucose sensor Rag4, is revealed by the analysis of the KlMGA2 gene in Kluyveromyces lactis

“…Micolonghi et al (2011). The eventual accumulation of metabolites with repressive activity, like ethanol (Bianchi et al, 1996) or carbon dioxide/HCO 2 3 (M. M. Bianchi, unpublished data), did not affect the induction of transcription of the hypoxic reporter gene KlPDC1. The hypoxic condition in the bioreactor was achieved by closing the inlet air valve: oxygen concentration in the medium dropped to zero within 10 min.…”

“…Yeast total RNA was extracted from cultures grown in YPD medium to OD 600 1 using the hot phenol protocol (Köhrer & Domdey, 1991) and quantified by measuring the absorption at 260 nm. Northern blot assays were performed by electrophoresis in agarose/formaldehyde gels and transferred to membranes following a standard procedure as described by Bianchi et al (1996). Probes were obtained by PCR using GDK genomic DNA as a template and the primers reported in Table S1.…”

“…The KlPDC1 gene, encoding the unique K. lactis pyruvate decarboxylase enzyme, is tightly regulated at the transcriptional level and is induced by hypoxia (Bianchi et al, 1996;Destruelle et al, 1999;Camattari et al, 2007): its mutation generates Rag 2 strains. In order to find out whether KlMga2 was involved in KlPDC1 transcriptional regulation, hypoxic KlPDC1 expression was assayed in the mutant strain (Fig.…”

Influence of Mutations in Hexose‐Transporter Genes on Glucose Repression in Kluyveromyces Lactis

“…A number of RAG genes have been cloned and characterised at the molecular level. Among them are RAGI encoding a low-affinity glucose transporter (Goffrini et al, 1990;WLsolowski-Louvel et al, 19921, RAG2 encoding phosphoglucose isomerase (Wksolowski-Louvel et al, 1988a;Goffrini et al, 1991), RAG5 encoding hexokinase (Prior et al, 1993) and RAG6 encoding pyruvate decarrboxylase (Bianchi et al, 1997). The association of the Rag-phenotype with mutations in these genes indicated that the dependence on respiration results from the impairment of glycolysis or a reduction of the glycolytic flux (Goffrini et al, 1991).…”

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

“…In the case of K. lactis, a Crabtree-negative yeast, the growth rate of a PDC-null mutant was comparable to that of a wild-type strain, 16) but the yield of a strain expressing L-LDH and lacking KlPDC1, which was reported by Porro et al, 10) was still far from the maximum theoretical yield: 0.58 lactic acid produced/ g-glucose consumed rather than 1 g/g. Bianchi et al modified the strain by additional disruption of KlPDA1, encoding the pyruvate dehydrogenase (PDH; EC 1.2.4.1) E1 subunit, which converts pyruvic acid into acetylcoenzyme A.…”