Efficient Homolactic Fermentation by Kluyveromyces lactis Strains Defective in Pyruvate Utilization and Transformed with the Heterologous LDH Gene

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 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) E1␣ 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.

Efficient Production ofl-Lactic Acid by Metabolically EngineeredSaccharomyces cerevisiaewith a Genome-Integratedl-Lactate Dehydrogenase Gene

“…Bianchi et al used K. lactis strains lacking either PDC activity or PDC and PDH activities, transformed with the LDH gene placed under the control of the promoter of KlPDC1 gene and cloned into stable multicopy vector. Transgenic K. lactis strains showed remarkable improvement under the fed-batch condition (3).…”

Genetic Engineering ofCandida utilisYeast for Efficient Production ofL-Lactic Acid

“…16) Additional inactivation of PDH activity was required to increase the yield of L-lactic acid in K. lactis, 9) although this was accompanied by a significant growth defect, which is not considered desirable, particularly in fermentation processes. In C. utilis, however, inactivation of PDC activity alone was sufficient to obtain high yields of L-lactic acid, the yield being 95.1% (103.3 g/l; >99:9% e.e.)…”

“…This relatively high optimal temperature is also advantageous because lactic acid at high concentrations easily becomes solidified in the medium due to low solubility. Usually, fermentation of S. cerevisiae and K. lactis is limited at lower temperatures from 30 to 32 C. [5][6][7][9][10][11][12][13][14] The pH value of the medium also appeared to be important for the production of L-lactic acid, since a previous study showed that adjustment of pH was effective for the high expression of PDC in C. utilis.…”

“…4) Decreasing PDC activity has been found to boost the production of L-lactic acid in Saccharomyces cerevisiae [4][5][6][7][8] and Kluyveromyces lactis. 9,10) In the case of S. cerevisiae, which is known as a Crabtree-positive yeast, and which has three PDC genes (ScPDC1, ScPDC5, and ScPDC6), reduction/inactivation of PDC activity causes a severe growth defect. Increasing the copy number of L-LDH and the use of EMS-generated mutants in which the growth defect is suppressed has resulted in better strain performance.…”

Microbial conversion of sugars from plant biomass to lactic acid or ethanol

“…This is more critical as we attempt to replace petroleum-based plastics with renewable PLA-based plastics, which would require production of the base chemical in large quantities and lignocellulosic biomass as the feedstock for fermentation. Biocatalysts currently being developed to reduce the cost of production of optically pure isomers of lactic acid include Kluyveromyces (Bianchi et al, 2001;Porro et al, 1999), Saccharomyces (van Maris et al, 2004;Saitoh et al, 2005), Pichia (Ilmen et al, 2007), Rhizopus (Skory, 2000) and Lactobacillus (Liu et al, 2007); however, Escherichia coli stands out as an excellent microbial biocatalyst for this purpose (Dien et al, 2001;Grabar et al, 2006). E. coli is a well studied bacterium that uses glycolysis via the Embden-Meyerhoff-Parnas (EMP) pathway to convert hexose sugars into a mixture of acids (lactic, acetic, formic and succinic) and ethanol (Clark, 1989).…”