Lactose Inducible Expression of Transcription Factor Gene SEF1 Increases Riboflavin Production in the Yeast Candida famata

Abstract

Riboflavin (vitamin B2) is required for synthesis of the flavin coenzymes: riboflavin-5’-phosphate (flavin mononucleotide) and flavin adenine dinucleotide. Riboflavin is important biotechnological commodity with annual market around 250 million US dollars. It is mostly used as component of feed premixes for animals (80%), in food industry as food colorant, in medicine and component of multivitamin mixtures and as drug for treatment of some diseases. Over the past two decades, the microbial production of riboflavin by fermentation completely replaces the chemical synthetic route. The main producers of riboflavin in industry are engineered strains of the bacterium Bacillus subtilis and of the mycelial fungus Ashbya gossypii. Flavinogenic yeast Candida famata has great biosynthetic potential. Using combination of classical selection and metabolic engineering (overexpression of SEF1, RIB1 and RIB7 genes coding the positive regulator, the first and the last structural enzymes of riboflavin synthesis) resulted in the construction of genetically stable strain of C. famata that produces 16 gram of riboflavin per liter in bioreactor. However, the productivity of riboflavin biosynthesis remains still insufficient for industrial production of this vitamin. Studies of transcriptional regulation of genes involved in riboflavin synthesis and using of strong promoters of C. famata for construction of efficient producers of vitamin B2 are areas of both scientific and industrial interest. Aim. The aim of the current work was to improve riboflavin oversynthesis by the available C. famata strains in synthetic and natural lactose-containing media. Methods. The plasmid DNA isolation, restriction, ligation, electrophoresis in agarose gel, electrotransformation, and PCR were carried out by the standard methods. Riboflavin was assayed fluorometrically using solution of synthetic riboflavin as a standard. The cultivation of yeasts was carried out in YNB or YPD media containing different source of carbon and on whey. Results. The strains of C. famata expressed additional copy of central regulatory gene SEF1 under control of the promoter of LAC4 gene (coding for β–galactosidase) C. famata were constructed. The influence of SEF1 gene expression under control of lactose inducible promoter of CfLAC4 gene on riboflavin production was studied. It was shown that the C. famata strains containing “pLAC4_cf-SEF1_cf” expression cassette revealed 1.6-2.1-fold increase in riboflavin yield on lactose when compared to the parental strain. The riboflavin production constructed strains on whey reached 1.69 gram per liter in flask batch culture. Conclusions. The constructed strains containing additional copy of SEF1 gene under the control of LAC4 promoter is a perfect platform for development of industrial riboflavin production on by-product of dairy industry, whey.