Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts.

BACKGROUND: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known. RESULTS: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L- 1 (C. famata), and that of roseoflavin was 5 mg L- 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L- 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all. CONCLUSIONS: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.

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

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 ß-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.

Expression of yeast homolog of the mammal BCRP gene coding for riboflavin efflux protein activates vitamin B2 production in the flavinogenic yeast Candida famata.

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2 ) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.