Riboflavin Overproduction Research Articles

Riboflavin overproduction from diverse feedstocks with engineered Corynebacterium glutamicum

Riboflavin overproduction byCorynebacterium glutamicumwas achieved by screening synthetic operons, enabling fine-tuned expression of the riboflavin biosynthetic genesribGCAH.The synthetic operons were designed by means of predicted translational initiation rates of each open reading frame, with the best-performing selection enabling riboflavin overproduction without negatively affecting cell growth. Overexpression of the fructose-1,6-bisphosphatase (fbp) and 5-phosphoribosyl 1-pyrophosphate aminotransferase (purF) encoding genes was then done to redirect the metabolic flux towards the riboflavin precursors. The resulting strain produced 8.3 g l-1of riboflavin in glucose-based fed-batch fermentations, which is the highest reported riboflavin titer withC. glutamicum. Further genetic engineering enabled both xylose and mannitol utilization byC. glutamicum, and we demonstrated riboflavin overproduction with the xylose-rich feedstocks rice husk hydrolysate and spent sulfite liquor, and the mannitol-rich feedstock brown seaweed hydrolysate. Remarkably, rice husk hydrolysate provided 30% higher riboflavin yields compared to glucose in the bioreactors.

Riboflavin overproduction on lignocellulose hydrolysate by the engineered yeast Candida famata.

Lignocellulose (dry plant biomass) is an abundant cheap inedible residue of agriculture and wood industry with great potential as a feedstock for biotechnological processes. Lignocellulosic substrates can serve as valuable resources in fermentation processes, allowing the production of a wide array of chemicals, fuels, and food additives. The main obstacle for cost-effective conversion of lignocellulosic hydrolysates to target products is poor metabolism of the major pentoses, xylose and L-arabinose, which are the second and third most abundant sugars of lignocellulose after glucose. We study the oversynthesis of riboflavin in the flavinogenic yeast Candida famata and found that all major lignocellulosic sugars, including xylose and L-arabinose, support robust growth and riboflavin synthesis in the available strains of C. famata. To further increase riboflavin production from xylose and lignocellulose hydrolysate, genes XYL1 and XYL2 coding for xylose reductase and xylitol dehydrogenase were overexpressed. The resulting strains exhibited increased riboflavin production in both shake flasks and bioreactors using diluted hydrolysate, reaching 1.5g L-1.

Induction of Oxidative Stress in Sirtuin Gene-Disrupted Ashbya gossypii Mutants Overproducing Riboflavin.

AgHST1 and AgHST3 genes encode sirtuins that are NAD+-dependent protein deacetylases. According to previous reports, their disruption leads to the overproduction of riboflavin in Ashbya gossypii. In this study, we investigated the potential causes of riboflavin overproduction in the AgHST1Δ and AgHST3Δ mutant strains of A. gossypii. The generation of reactive oxygen species was increasd in the mutants compared to in WT. Additionally, membrane potential was lower in the mutants than in WT. The NAD+/NADH ratio in AgHST1Δ mutant strain was lower than that in WT; however, the NAD+/NADH ratio in AgHST3Δ was slightly higher than that in WT. AgHST1Δ mutant strain was more sensitive to high temperatures and hydroxyurea treatment than WT or AgHST3Δ. Expression of the AgGLR1 gene, encoding glutathione reductase, was substantially decreased in AgHST1Δ and AgHST3Δ mutant strains. The addition of N-acetyl-L-cysteine, an antioxidant, suppressed the riboflavin production in the mutants, indicating that it was induced by oxidative stress. Therefore, high oxidative stress resulting from the disruption of sirtuin genes induces riboflavin overproduction in AgHST1Δ and AgHST3Δ mutant strains. This study established that oxidative stress is an important trigger for riboflavin overproduction in sirtuin gene-disrupted mutant strains of A. gossypii and helped to elucidate the mechanism of riboflavin production in A. gossypii.

Predictive Modeling of Riboflavin Production in Lactiplantibacillus plantarum MTCC 25432 Using Fuzzy Inference System.

Riboflavin (Vitamin B2) is an essential vitamin and a microbial metabolite produced by some lactic acid bacteria (LAB). This investigation aims to study the overproduction of riboflavin in selected Lactiplantibacillus plantarum strain by using the one factor at a time (OFAT) tool coupled with the Fuzzy Inference System (FIS) and its validation through fermentative production in semi-defined media. Out of three Lactiplantibacillus strains used in this study, the maximum riboflavin producing strain was selected based on its ability to grow and produce higher levels of riboflavin. In results, Lactiplantibacillus plantarum strain MTCC 25432 was able to produce 346 µg/L riboflavin in riboflavin deficient assay medium and was investigated further. By using the OFAT-fuzzy FIS system, casamino acid in the range of 5-20 g/L, GTP 0.01-0.04 g/L, sodium acetate 5-15 g/L, and glycine 5-15 g/L were used to predict their effect on riboflavin production. The conditions optimized with modeling showed a 24% increment in riboflavin production (429 µg/L) by Lactiplantibacillus plantarum MTCC 25432 vis-a-vis the unoptimized counterpart (346 µg/L). In conclusion, an FIS-based predictive model was effectively implemented to estimate the riboflavin within an acceptable limit of 3.4%. Riboflavin production enhancing effects observed with various levels of sodium acetate, casamino acid, and GTP could be useful to re-design matrices for riboflavin production.

Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening.

Bacillus subtilis is one of the commonly used industrial strains for riboflavin production. High-throughput screening is useful in biotechnology, but there are still an insufficient number of articles focusing on improving the riboflavin production of B. subtilis by this powerful tool. With droplet-based microfluidics technology, single cells can be encapsulated in droplets. The screening can be carried out by detecting the fluorescence intensity of secreted riboflavin. Thus, an efficient and high-throughput screening method suitable for riboflavin production strain improvement could be established. In this study, droplet-based microfluidics screening was applied, and a more competitive riboflavin producer U3 was selected from the random mutation library of strain S1. The riboflavin production and biomass of U3 were higher than that of S1 in flask fermentation. In addition, the results of fed-batch fermentation showed that the riboflavin production of U3 was 24.3 g/L, an 18% increase compared with the parent strain S1 (20.6 g/L), and the yield (g riboflavin/100 g glucose) increased by 19%, from 7.3 (S1) to 8.7 (U3). Two mutations of U3 (sinRG89R and icdD28E) were identified through whole genome sequencing and comparison. Then they were introduced into BS168DR (parent of S1) for further analysis, which also caused riboflavin production to increase. This paper provides protocols for screening riboflavin-producing B. subtilis with droplet-based microfluidics technology and reveals mutations in riboflavin overproduction strains.

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Weissella cibaria riboflavin-overproducing and dextran-producing strains useful for the development of functional bread.

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5’ untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by (i) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and (ii) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads.

Riboflavin synthesis from gaseous nitrogen and carbon dioxide by a hybrid inorganic-biological system

Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.g., ammonia) inputs are required. Gas-fixing microorganisms that can grow on H2 from renewable water splitting and gaseous CO2 and N2 offer a renewable path to overcoming these limitations but confront challenges owing to the scarcity of genetic engineering in such organisms. Here, we demonstrate that the hydrogen-oxidizing carbon- and nitrogen-fixing microorganism Xanthobacter autotrophicus grown on a CO2/N2/H2 gas mixture can overproduce the vitamin riboflavin (vitamin B2). We identify plasmids and promoters for use in this bacterium and employ a constitutive promoter to overexpress riboflavin pathway enzymes. Riboflavin production is quantified at 15 times that of the wild-type organism. We demonstrate that riboflavin overproduction is maintained when the bacterium is grown under hybrid inorganic-biological conditions, in which H2 from water splitting, along with CO2 and N2, is fed to the bacterium, establishing the viability of the approach to sustainably produce food and nutrients.

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

BackgroundRiboflavin 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.ResultsWe 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.ConclusionsOur 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.

Spontaneous Riboflavin-Overproducing Limosilactobacillus reuteri for Biofortification of Fermented Foods.

Riboflavin-producing lactic acid bacteria represent a promising and cost-effective strategy for food biofortification, but production levels are typically insufficient to support daily human requirements. In this study, we describe the novel human isolate Limosilactobacillus reuteri AMBV339 as a strong food biofortification candidate. This strain shows a high natural riboflavin (vitamin B2) overproduction of 18.36 μg/ml, biomass production up to 6 × 1010 colony-forming units/ml (in the typical range of model lactobacilli), and pH-lowering capacities to a pH as low as 4.03 in common plant-based (coconut, soy, and oat) and cow milk beverages when cultured up to 72 h at 37°C. These properties were especially pronounced in coconut beverage and butter milk fermentations, and were sustained in co-culture with the model starter Streptococcus thermophilus. Furthermore, L. reuteri AMBV339 grown in laboratory media or in a coconut beverage survived in gastric juice and in a simulated gastrointestinal dialysis model with colon phase (GIDM-colon system) inoculated with fecal material from a healthy volunteer. Passive transport of L. reuteri AMBV339-produced riboflavin occurred in the small intestinal and colon stage of the GIDM system, and active transport via intestinal epithelial Caco-2 monolayers was also demonstrated. L. reuteri AMBV339 did not cause fecal microbiome perturbations in the GIDM-colon system and inhibited enteric bacterial pathogens in vitro. Taken together, our data suggests that L. reuteri AMBV339 represents a promising candidate to provide riboflavin fortification of plant-based and dairy foods, and has a high application potential in the human gastrointestinal tract.

From Brown Seaweed to a Sustainable Microbial Feedstock for the Production of Riboflavin.

The increasing global demand for food and energy production encourages the development of new production strategies focused on sustainability. Often, microbial bioprocesses rely on food or feed competitive feedstocks; hence, there is a trending need for green substrates. Here, we have proven the potential of brown seaweed biomass as microbial feedstock on account of its content of mannitol and the glucose polymer laminarin. Our host, Corynebacterium glutamicum, was engineered to enable access to mannitol as a carbon source through the heterologous expression of the mannitol-specific phosphotransferase system and the mannitol-1-phosphate-5-dehydrogenase from Bacillus subtilis. Overproduction of riboflavin was coupled with mannitol and glucose consumption via constitutive overexpression of the biosynthetic riboflavin operon ribGCAH from C. glutamicum. Brown seaweed extract and brown seaweed hydrolysate from Laminaria hyperborea, containing mannitol and glucose, were used as a carbon source for flask and bioreactor fermentations. In a seaweed-based fed-batch fermentation, the riboflavin final titer, yield, and volumetric productivity values of 1,291.2 mg L−1, 66.1 mg g−1, and 17.2 mg L−1 h−1, respectively, were achieved.

Co-Overexpression of RIB1 and RIB6 Increases Riboflavin Production in the Yeast Candida famata

Riboflavin or vitamin B2 is a water-soluble vitamin and a precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which play a key role as enzyme cofactors in energy metabolism. Candida famata yeast is a promising producer of riboflavin, as it belongs to the group of so-called flavinogenic yeasts, capable of riboflavin oversynthesis under conditions of iron starvation. The role of the particular structural genes in the limitation of riboflavin oversynthesis is not known. To study the impact of overexpression of the structural genes of riboflavin synthesis on riboflavin production, a set of plasmids containing genes RIB1, RIB6, and RIB7 in different combinations was constructed. The transformants of the wild-type strain of C. famata, as well as riboflavin overproducer, were obtained, and the synthesis of riboflavin was studied. It was found that overexpression of RIB1 and RIB6 genes coding for enzymes GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase, which catalase the initial steps of riboflavin synthesis, elevated riboflavin production by 13–28% relative to the parental riboflavin-overproducing strains.

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

The ascomycetous yeast Candida membranifaciens has been isolated from diverse habitats, including humans, insects, and environmental sources, exhibiting a remarkable ability to use different carbon sources that include pentoses, melibiose, and inulin. In this study, we isolated four C. membranifaciens strains from soil and investigated their potential to overproduce riboflavin. C. membranifaciens IST 626 was found to produce the highest concentrations of riboflavin. The volumetric production of this vitamin was higher when C. membranifaciens IST 626 cells were cultured in a commercial medium without iron and when xylose was the available carbon source compared to the same basal medium with glucose. Supplementation of the growth medium with 2 g/L glycine favored the metabolization of xylose, leading to biomass increase and consequent enhancement of riboflavin volumetric production that reached 120 mg/L after 216 h of cultivation. To gain new insights into the molecular basis of riboflavin production and carbon source utilization in this species, the first annotated genome sequence of C. membranifaciens is reported in this article, as well as the result of a comparative genomic analysis with other relevant yeast species. A total of 5619 genes were predicted to be present in C. membranifaciens IST 626 genome sequence (11.5 Mbp). Among them are genes involved in riboflavin biosynthesis, iron homeostasis, and sugar uptake and metabolism. This work put forward C. membranifaciens IST 626 as a riboflavin overproducer and provides valuable molecular data for future development of superior producing strains capable of using the wide range of carbon sources, which is a characteristic trait of the species.

Effects of sirtuins on the riboflavin production in Ashbya gossypii.

This study focuses on sirtuins, which catalyze the reaction of NAD+-dependent protein deacetylase, for riboflavin production in A. gossypii. Nicotinamide, a known inhibitor of sirtuin, made the color of A. gossypii colonies appear a deeper yellow at 5mM. A. gossypii has 4 sirtuin genes (AgHST1, AgHST2, AgHST3, AgHST4) and these were disrupted to investigate the role of sirtuins in riboflavin production in A. gossypii. AgHST1∆, AgHST3∆, and AgHST4∆ strains were obtained, but AgHST2∆ was not. The AgHST1∆ and AgHST3∆ strains produced approximately 4.3- and 2.9-fold higher amounts of riboflavin than the WT strain. The AgHST3∆ strain showed a lower human sirtuin 6 (SIRT6)-like activity than the WT strain and only in the AgHST3∆ strain was a higher amount of acetylation of histone H3 K9 and K56 (H3K9ac and H3K56ac) observed compared to the WT strain. These results indicate that AgHst3 is SIRT6-like sirtuin in A. gossypii and the activity has an influence on the riboflavin production in A. gossypii. In the presence of 5mM hydroxyurea and 50µM camptothecin, which causes DNA damage, especially double-strand DNA breaks, the color of the WT strain colonies turned a deeper yellow. Additionally, hydroxyurea significantly led to the production of approximately 1.5 higher amounts of riboflavin and camptothecin also enhanced the riboflavin production even through the significant difference was not detected. Camptothecin tended to increase the amount of H3K56ac, but the amount of H3K56ac was not increased by hydroxyurea treatment. This study revealed that AgHst1 and AgHst3 are involved in the riboflavin production in A. gossypii through NAD metabolism and the acetylation of H3, respectively. This new finding is a step toward clarifying the role of sirtuins in riboflavin over-production by A. gossypii.Key points• Nicotinamide enhanced the riboflavin production in Ashbya gossypii.• Disruption of AgHST1 or AgHST3 gene also enhanced the riboflavin production in Ashbya gossypii.• Acetylation of H3K56 led to the enhancement of the riboflavin production in Ashbya gossypii.

Biotechnological Strategies of Riboflavin Biosynthesis in Microbes

Riboflavin is an essential micronutrient for humans and must be obtained exogenously from foods or supplements. Numerous studies have suggested a major role of riboflavin in the prevention and treatment of various diseases. There are mainly three strategies for riboflavin synthesis, including total chemical synthesis, chemical semi-synthesis, and microbial fermentation, the latter being currently the most promising strategy. In recent years, flavinogenic microbes have attracted increasing attention. Fungi, including Eremothecium ashbyii and Ashbya gossypii , and bacteria, including Bacillus subtilis , Escherichia coli , and lactic acid bacteria, are ideal cell factories for riboflavin overproduction. Thus they are good candidates for enhancing the level of riboflavin in fermented foods or designing novel riboflavin bio-enriched foods with improved nutritional value and/or beneficial properties for human health. This review briefly describes the role of riboflavin in human health and the historical process of its industrial production, and then highlights riboflavin biosynthesis in bacteria and fungi, and finally summarizes the strategies for riboflavin overproduction based on both the optimization of fermentation conditions and the development of riboflavin-overproducing strains via chemical mutagenesis and metabolic engineering. Overall, this review provides an updated understanding of riboflavin biosynthesis and can promote the research and development of fermented food products rich in riboflavin.

Recent Advances in Construction of the Efficient Producers of Riboflavin and Flavin Nucleotides (FMN, FAD) in the Yeast Candida famata.

The approaches used by the authors to design the Candida famata strains capable to overproduce riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are described. The metabolic engineering approaches include overexpression of SEF1 gene encoding positive regulator of riboflavin biosynthesis, IMH3 (coding for IMP dehydrogenase) orthologs from another species of flavinogenic yeast Debaryomyces hansenii, and the homologous genes RIB1 and RIB7 encoding GTP cyclohydrolase II and riboflavin synthase, the first and the last enzymes of riboflavin biosynthesis pathway, respectively. Overexpression of the abovementioned genes in the genetically stable riboflavin overproducer AF-4 obtained by classical selection resulted in fourfold increase of riboflavin production in shake flask experiments.Overexpression of engineered enzymes phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase catalyzing the initial steps of purine nucleotide biosynthesis enhances riboflavin synthesis in the flavinogenic yeast C. famata even more.Recombinant strains of C. famata containing FMN1 gene from D. hansenii encoding riboflavin kinase under control of the strong constitutive TEF1 promoter were constructed. Overexpression of the FMN1 gene in the riboflavin-producing mutant led to the 30-fold increase of the riboflavin kinase activity and 400-fold increase of FMN production in the resulting recombinant strains which reached maximally 318.2mg/L.FAD overproducing strains of C. famata were also constructed. This was achieved by overexpression of FAD1 gene from D. hansenii in C. famata FMN overproducing strain. The 7- to 15-fold increase in FAD synthetase activity as compared to the wild-type strain and FAD accumulation into cultural medium were observed. The maximal FAD titer 451.5mg/L was achieved.

SEF1 and VMA1 Genes Regulate Riboflavin Biosynthesis in the Flavinogenic Yeast Candida Famata

Abstract—Riboflavin (vitamin B2) is an important component of the diet of living organisms since it is a precursor of flavin coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide) involved in numerous enzymatic reactions. It is known that flavinogenic yeast C. famata is able to perform riboflavin overproduction under conditions of iron deficiency, but the regulation of this process remains unknown. It was shown that the deletion of the SEF1 gene (encoding transcription activator) blocked the ability for riboflavin overproduction under conditions of iron deficiency. It was determined that SEF1 promoters of other flavinogenic yeasts (Candida albicans and Candida tropicalis) fused with SEF1 ORF of C. famata can restore the overproduction of riboflavin in the sef1Δ mutant. The disruption of the VMA1 gene (encoding the vacuolar ATPase subunit A) led to overproduction of riboflavin in C. famata in iron complete medium.

Effect of Gene SFU1 on Riboflavin Synthesis in Flavinogenic Yeast Candida famata

Abstract—Riboflavin or vitamin B2 is a necessary component for all living organisms since it is the precursor of flavin coenzymes FMN (flavin mononucleotide) and FAD (flavin adenine dinucleotide), which are involved in numerous enzymatic reactions. Flavinogenic yeast Candida famata overproduces riboflavin under iron starvation; however, regulation of this process is poorly understood. Regulatory gene SEF1 encoding the transcription activator has been identified. Its deletion blocks yeast’s ability to overproduce riboflavin under iron starvation. It is known that, in the pathogenic flavinogenic yeast C. albicans, Sfu1 (GATA-type transcription factor) represses SEF1. It is demonstrated in this study that deletion of the SEF1 gene in wild type C. famata leads to overproduction of riboflavin.

Cloning of Genes Sef1 and Tup1 Encoding Transcriptional Activator and Global Repressor in the Flavinogenic Yeast Meyerozyma (Candida, Pichia) guilliermondii

Abstract—Two Meyerozyma (Candida, Pichia) guilliermondii genes coding for homologs of transcriptional factor Sef1p of Candida famata and Tup1p of Candida albicans were identified, cloned, and deleted. Deletion of a homologue of Sef1p transcriptional factor in M.(P.) guilliermondii completely blocked over-synthesis of riboflavin under conditions of iron deficiency. The results of genetic complementation analysis suggest that previously reported rib83 mutants and newly constructed knock-out strains belong to the same complementation group and are defective in the same SEF1 gene. Inactivation of the identified homolog of the TUP1 gene in M.(P.) guilliermondii wild-type strain led to 1.5-fold increase in cellular iron content and 1.5–1.7-fold increase in riboflavin production. Introduction of a plasmid-borne copy of the TUP1 gene did not restore metabolic defects of the riboflavin overproduction and iron accumulation in mutant strain M. (P.) guilliermondii m3, bearing the hit1 mutation. The obtained results suggest that both transcription factors Sef1p and Tup1p are involved in the regulation of iron acquisition and riboflavin biosynthesis by yeast belonging to the CUG-clade. The molecular mechanism of action Tup1p on riboflavin biosynthesis in M.(P.) guilliermondii required further elucidation.

Development of new dominant selectable markers for the nonconventional yeasts Ogataea polymorpha and Candida famata.

Nonconventional yeast Candida famata and Ogataea polymorpha are interesting organisms for basic and applied studies. O. polymorpha is methylotrophic thermotolerant yeast capable of xylose alcoholic fermentation whereas C. famata is capable of riboflavin overproduction. Still, the new tools for molecular research of these species are needed. The aim of this study was to develop the new dominant selective markers for C. famata and O. polymorpha usable in metabolic engineering experiments. In this work, the BSD gene from Aspergillus terreus coding for blasticidin S deaminase, O. polymorpha AUR1 gene required for sphingolipid synthesis and IMH3 gene, which encodes IMP dehydrogenase, were tested as the new dominant selective marker genes. Our results showed that AUR1 and IMH3 genes could be used as dominant selective markers for O. polymorpha with frequencies of transformation of 40 and 20 transformants per microgram of DNA, respectively. The IMH3 gene was successfully used as the marker for construction of O. polymorpha strains with increased ethanol production from xylose due to overexpression of TAL1, TKL1 and AOX1 genes. The BSD gene from A. terreus, conferring resistance to blasticidin, was found to be efficient for selection of C. famata transformants.