RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis

ABSTRACT 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 aKluyveromyces 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 theKlPDC1 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 usedK. 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.

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Replacement of a Metabolic Pathway for Large-Scale Production of Lactic Acid from Engineered Yeasts

Applied and Environmental Microbiology ◽

10.1128/aem.65.9.4211-4215.1999 ◽

1999 ◽

Vol 65 (9) ◽

pp. 4211-4215 ◽

Cited By ~ 98

Author(s):

Danilo Porro ◽

Michele M. Bianchi ◽

Luca Brambilla ◽

Rossella Menghini ◽

Davide Bolzani ◽

...

Keyword(s):

Lactic Acid ◽

Large Scale ◽

Kluyveromyces Lactis ◽

Lactic Acid Production ◽

Pyruvate Decarboxylase ◽

Polymer Materials ◽

Efficient Production ◽

Scale Production ◽

High Productivity ◽

Large Scale Production

ABSTRACT Interest in the production of l-(+)-lactic acid is presently growing in relation to its applications in the synthesis of biodegradable polymer materials. With the aim of obtaining efficient production and high productivity, we introduced the bovinel-lactate dehydrogenase gene (LDH) into a wild-type Kluyveromyces lactis yeast strain. The observed lactic acid production was not satisfactory due to the continued coproduction of ethanol. A further restructuring of the cellular metabolism was obtained by introducing the LDH gene into aK. lactis strain in which the unique pyruvate decarboxylase gene had been deleted. With this modified strain, in which lactic fermentation substituted completely for the pathway leading to the production of ethanol, we obtained concentrations, productivities, and yields of lactic acid as high as 109 g liter−1, 0.91 g liter−1 h−1, and 1.19 mol per mole of glucose consumed, respectively. The organic acid was also produced at pH levels lower than those usual for bacterial processes.

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Autoregulation of the Kluyveromyces lactis pyruvate decarboxylase gene KlPDC1 involves the regulatory gene RAG3

Microbiology ◽

10.1099/mic.0.078543-0 ◽

2014 ◽

Vol 160 (7) ◽

pp. 1369-1378 ◽

Cited By ~ 3

Author(s):

Daniela Ottaviano ◽

Chiara Micolonghi ◽

Lorenza Tizzani ◽

Marc Lemaire ◽

Micheline Wésolowski-Louvel ◽

...

Keyword(s):

Environmental Factors ◽

Nuclear Localization ◽

Transcriptional Activity ◽

Kluyveromyces Lactis ◽

Regulatory Gene ◽

Pyruvate Decarboxylase ◽

Genetic Screen ◽

Gene Products ◽

Transcription Level ◽

Autoregulatory Mechanism

In the yeast Kluyveromyces lactis, the pyruvate decarboxylase gene KlPDC1 is strongly regulated at the transcription level by different environmental factors. Sugars and hypoxia act as inducers of transcription, while ethanol acts as a repressor. Their effects are mediated by gene products, some of which have been characterized. KlPDC1 transcription is also strongly repressed by its product – KlPdc1 – through a mechanism called autoregulation. We performed a genetic screen that allowed us to select and identify the regulatory gene RAG3 as a major factor in the transcriptional activity of the KlPDC1 promoter in the absence of the KlPdc1 protein, i.e. in the autoregulatory mechanism. We also showed that the two proteins Rag3 and KlPdc1 interact, co-localize in the cell and that KlPdc1 may control Rag3 nuclear localization.

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The 'petite-negative' yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity

Molecular Microbiology ◽

10.1046/j.1365-2958.1996.346875.x ◽

1996 ◽

Vol 19 (1) ◽

pp. 27-36 ◽

Cited By ~ 100

Author(s):

Michele M. Bianchi ◽

Lorenza Tizzani ◽

Monika Destruelle ◽

Laura Frontali ◽

Micheline Wesolowski-Louvel

Keyword(s):

Kluyveromyces Lactis ◽

Single Gene ◽

Pyruvate Decarboxylase ◽

Decarboxylase Activity ◽

Petite Negative Yeast ◽

Gene Expressing

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Pyruvate decarboxylase from Kluyveromyces lactis

European Journal of Biochemistry ◽

10.1046/j.1432-1033.2002.03006.x ◽

2002 ◽

Vol 269 (13) ◽

pp. 3256-3263 ◽

Cited By ~ 25

Author(s):

Florian Krieger ◽

Michael Spinka ◽

Ralph Golbik ◽

Gerhard Hübner ◽

Stephan König

Keyword(s):

Kluyveromyces Lactis ◽

Pyruvate Decarboxylase

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The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

Eukaryotic Cell ◽

10.1128/ec.3.3.589-597.2004 ◽

2004 ◽

Vol 3 (3) ◽

pp. 589-597 ◽

Cited By ~ 17

Author(s):

Michele Saliola ◽

Paola Chiara Bartoccioni ◽

Ilaria De Maria ◽

Tiziana Lodi ◽

Claudio Falcone

Keyword(s):

Succinate Dehydrogenase ◽

Glyoxylate Cycle ◽

Alternative Pathway ◽

Kluyveromyces Lactis ◽

Carbon Sources ◽

Pyruvate Decarboxylase ◽

Northern Analysis ◽

Gene Encoding ◽

Dehydrogenase Gene ◽

High Sequence Conservation

ABSTRACT We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.

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