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

Abstract

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B 2) 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 GTPcyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.

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

“…Next experiment shows data on riboflavin synthesis by the strain BRP/RFE1 of C. famata with overexpression of the homolog of the mammalian riboflavin efflux protein, named as riboflavin excretase ( RFE1 ) in the background of strain BRP with overexpression of genes SEF1, RIB1 and RIB7 [ 27 ]. It was found that whey without nitrogen source supported active riboflavin production though riboflavin titer was below that accumulated in YNB/glucose or YPD media.…”

“…Therefore, we suggested that expression of SEF1 gene under control of LAC4 promotor could be used for activation of riboflavin synthesis on whey. As parental strains, BRP/RFE1 with overexpression of RFE1 gene coding for riboflavin excretase [ 27 ] and BRPI with activation of purine nucleotide synthesis de novo [ 28 ] were used. Resulted strains were named BRP/RFE1/pLAC4-SEF1 and BRPI/pLAC4-SEF1 (Additional file 1 : Table S1).…”

“…Recombinant C. famata strains used throughout this work (Additional file 1: Table S1) have been described previously [15,16,[26][27][28][29]. New recombinant strains were constructed by introduction of plasmids pNTC/ pLAC4_cf-SEF1_cf (Additional file 1: Fig.…”

Genome Sequence and Analysis of the Flavinogenic Yeast Candida membranifaciens IST 626

“…Riboflavin transport in and out of the cell is still poorly characterized in flavinogenic yeasts, but two riboflavin permeases and one riboflavin excretase were described in P. guilliermondii (reviewed in [ 63 ]). More recently, a riboflavin excretase Rfe1 from C. flareri was also identified based on homology with D. hansenii DEHA2C03784p [ 64 ]. Using the same homology approach, we identified a putative riboflavin excretase in the genome sequence of C. membranifaciens IST 626 ( Table 4 ).…”

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

“…Recombinant C. famata strains (Supporting Information: Table ) FP, AF4/pLAC4‐SEF, BRP, BRP/RFE1 used as recipients through this work have been described previously (Dmytruk et al, 2014; Tsyrulnyk et al, 2020, 2021; Yatsyshyn, Ishchuk, et al, 2009). Cultivation of C. famata strains in liquid media was carried out in the shakers (200 rpm) at 30°C in 10 mL of liquid media in 100 mL Erlenmeyer flasks for 96 h. The following cultivation media were used: YPD (0.5% yeast extract, 1% peptone and 2% glucose); YNB with 0.2% of yeast extract and 5% glucose; YNB with 0.2% of yeast extract and 5% lactose; cheese whey with 5% of lactose and 0.3% (NH 4 ) 2 SO 4 .…”