Cheese whey supports high riboflavin synthesis by the engineered strains of the flavinogenic yeast Candida famata

Abstract

Background Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales exceeding nine billion US dollars. It is used primarily as a component of feed premixes, a food colorant, a component of multivitamin mixtures and medicines. Currently, industrial riboflavin production uses the bacterium, Bacillus subtilis, and the filamentous fungus, Ashbya gossypii, and utilizes glucose and/or oils as carbon substrates. Results We studied riboflavin biosynthesis in the flavinogenic yeast Candida famata that is a genetically stable riboflavin overproducer. Here it was found that the wild type C. famata is characterized by robust growth on lactose and cheese whey and the engineered strains also overproduce riboflavin on whey. The riboflavin synthesis on whey was close to that obtained on glucose. To further enhance riboflavin production on whey, the gene of the transcription activator SEF1 was expressed under control of the lactose-induced promoter of the native β-galactosidase gene LAC4. These transformants produced elevated amounts of riboflavin on lactose and especially on whey. The strain with additional overexpression of gene RIB6 involved in conversion of ribulose-5-phosphate to riboflavin precursor had the highest titer of accumulated riboflavin in flasks during cultivation on whey. Activation of riboflavin synthesis was also obtained after overexpression of the GND1 gene that is involved in the synthesis of the riboflavin precursor ribulose-5-phosphate. The best engineered strains accumulated 2.5 g of riboflavin/L on whey supplemented only with (NH4)2SO4 during batch cultivation in bioreactor with high yield (more than 300 mg/g dry cell weight). The use of concentrated whey inhibited growth of wild-type and engineered strains of C. famata, so the mutants tolerant to concentrated whey were isolated. Conclusions Our data show that the waste of dairy industry is a promising substrate for riboflavin production by C. famata. Possibilities for using the engineered strains of C. famata to produce high-value commodity (riboflavin) from whey are discussed.

Construction of the advanced flavin mononucleotide producers in the flavinogenic yeast Candida famata

“…Recently, we have shown that the engineered strains of C. famata are characterized by robust growth on lactose and cheese whey and riboflavin overproduction in these media. Expression of SEF1 transcription activator under control of LAC4 promoter increased riboflavin production on whey (Ruchala et al, 2022). We decided to check if introduction of SEF1 under control of lactose‐induced LAC4 promoter activates FMN synthesis in strains with overexpressed FMN1 gene.…”

“…Reciprocally, the D. hansenii FMN1 gene was expressed under control of C. famata TEF1 promoter in the strain AF‐4/pLAC4‐SEF1 containing additional copy of SEF1 gene under the control of LAC4 promoter (Ruchala et al, 2022). The presence of the introduced gene was confirmed by PCR using corresponding pairs of primers (Supporting Information: Figure ).…”

“…Additionally, the plasmid with gene C. famata SEF1 gene under control of LAC4 promoter (Ruchala et al, 2022) was introduced in transformants, containing the additional copy of FMN1 gene, obtained on the basis of the recipient strains BRP and BRP/RFE1 (Supporting Information: Table ). Resulted transformants containing an additional copy of gene SEF1 under the control of the LAC4 promoter were characterized by increased production of FMN during cultivation in the medium with lactose (Figure 3).…”

“…promoter (Ruchala et al, 2022). The presence of the introduced gene was confirmed by PCR using corresponding pairs of primers (Supporting Information: Figure S1D).…”

“…Additionally, the plasmid with gene C. famata SEF1 gene under control of LAC4 promoter (Ruchala et al, 2022) was introduced in transformants, containing the additional copy of FMN1 gene, obtained on the basis of the recipient strains BRP and BRP/RFE1…”