Cloning, characterisation, and heterologous expression of the Candida utilis malic enzyme gene

Abstract

The Candida utilis malic enzyme gene, CME1, was isolated from a cDNA library and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1,926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of approximately 70.2 kDa. The inferred amino acid sequence suggested a cytosolic localisation for the malic enzyme, as well as 37 and 68% homologies with the malic enzymes of Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. Expression of the CME1 gene was subject to carbon catabolite repression and substrate induction, similar to the regulatory mechanisms observed for the C. utilis dicarboxylic acid permease. The CME1 gene was successfully expressed in S. cerevisiae under control of the S. cerevisiae PGK1 promoter and terminator. When coexpressed with the S. pombe malate permease gene (mae1), it resulted in a recombinant S. cerevisiae strain able to completely degrade 90% of the extracellular L-malate within 24 h.

The Biochemistry of Malic Acid Metabolism by Wine Yeasts – A Review

“…Cloning and molecular analysis of the C. utilis malic enzyme gene, CME1, revealed a high degree of homology with the malic enzyme genes of S. pombe and S. cerevisiae (Saayman et al, 2006). The C. utilis malic enzyme uses either NAD + or NADP + for the decarboxylation of oxaloacetate, but only NADP + for the decarboxylation of L-malate.…”

“…Malic enzymes are widely distributed in nature and have been reported in the yeasts S. pombe (Viljoen et al, 1994), Rhodotorula glutinis (Fernández et al, 1967), Z. bailii (Kuczynski and Radler, 1982), S. cerevisiae (Boles et al, 1998) and C. utilis (Saayman et al, 2006 (Temperli et al, 1965), the S. cerevisiae malic enzyme uses both NAD + and NADP + as electron acceptor, with NAD + being favoured (Kuczynski and Radler, 1982). The C. utilis malic enzyme uses either NAD + or NADP + for the decarboxylation of oxaloacetate, but only NADP + for the decarboxylation of L-malate (Saayman et al, 2006).…”

Biological Demalication and Deacetification of Musts and Wines: Can Wine Yeasts Make the Wine Taste Better?

“…The rather low malate consumption can be explained by the absence of an active L-malate carrier, low substrate affinity and the mitochondrial location of the malic enzyme [46][47][48]. The malic enzyme, under fermentation conditions is regulated by the fermentative glucose metabolism that causes mitochondrial deterioration [20].…”

“…The bacterial mleA genes from Oenococcus oeni and the mae1 genes (malate permease) from Schizosaccharomyces pombe yeast were cloned and co-expressed in S. cerevisiae [19]. The co-expression of the Candida utilis malic enzyme gene (cme1) and S. pombe malate transporter gene (mae1) in S. cerevisiae resulted in complete malate consumption [20]. However, only the work of Husnik et al [19] was productive; the authors were able to construct a stable genetically engineered wine S. cerevisiae yeast, carrying the mae1 and mleA genes.…”

Targeting Demalication and Deacetification Methods: The Role of Carboxylic Acids Transporters

“…Moreover, the malic enzyme, located in the mitochondria, has low substrate affinity (Km=50 mM) [11,12] and under fermentation conditions, is regulated by the fermentative glucose metabolism that causes mitochondrial deterioration [13]. These biochemical characteristics make the yeast demalication activity has being strain dependent [14][15][16].…”