This dissertation clarifies metabolic engineering applications to reveal new correlations of regulation in riboflavin synthesis by flavinogenic yeasts Candida famata. The data can be further applied to create stable superproducers of riboflavin.
Riboflavin (vitamin B2, or lactoflavin) plays a crucial role in the metabolism of living organisms. It is a precursor of flavin cofactors flavin mononucleotide (FMN) and flavinadine dinucleotide (FAD). Decreased concentration of vitamin B2 in cells leads to inhibition of their proliferation, inflammatory diseases such as infectious skin inflammation, cheilitis, glossitis, sepsis, cataracts, and migraines.
Yeasts, as unicellular eukaryotes, have significant prerogatives in biotechnological processes compared to fungi and bacteria. The genome modification of these organisms by using metabolic engineering has already shown the existence of an interesting and promising area of research. Thus, the aim of this work is to identify and study the principles of action of new factors involved in the biosynthesis of riboflavin by the flavinogenic yeast C. famata.
We have constructed the C. famata strains with a damaged gene for the synthesis of the vacuolar ATPase subunit. Obtaining deletion mutants allowed us to demonstrate that the VMA1 gene has a regulatory effect on the formation of riboflavin by them. The resulting strain produces 27 times more riboflavin than the original strain by one gram of cells. These yeasts showed a temperature-sensitive phenotype. The growth and flavinogenesis of the strain deteriorated significantly when cultivated at 35°C. It was found that the parental strain of C. famata is more thermotolerant and, in response to increasing temperature, starts to synthesize more riboflavin than when grown at 28 °C.
We also examine the influence of SEF1 gene promoters from flavinogenic and non-flavinogenic yeasts on the initiation of riboflavin formation. We found that SEF1 promoters from the flavinogenic yeast C. albicans and, to a lesser extent, the non-flavinogenic yeast C. tropicalis, fused to the ORF of the SEF1 gene from C. famata, affect the ability to restore riboflavin overproduction in sef1Δ. This method of overexpression of the gene allowed to increase in the level of metabolite synthesis by 20 times.
We have discovered that due to additional enhancement of GND1 gene expression it is possible to increase the content of riboflavin in the culture fluid by 2, 1.5 and 1.3-fold, compared to the original strains L20105, AF-4 and BRP. We have shown that overexpression of the ZWF1 gene inhibits riboflavin biosynthesis and growth processes of mutants.
For the first time we describe the positive effect of derepression of the 6-phosphogluconate dehydrogenase gene on the synthesis of riboflavin by flavinogenic yeast was shown.
Mutants that overexpressed both genes of PPP, namely L20105/ZWF1/GND1, AF-4/ZWF1/GND1 and BRP/ZWF1/GND1 were characterized only by a minor increase in the content of flavins (no more than 1.05 times compared to the parent strains).
We found that overexpression of the SOL3 gene can also affect riboflavin oversynthesis. The increase in vitamin yield per gram of biomass from 1.05 to 1.5 times. This data suggests that overexpression of the SOL3 gene in combination with GND1 and/or ZWF1 may lead to significantly greater accumulation of the riboflavin precursor Ru5P, and thus further stimulate riboflavin production.
We also confirmed that mutants with induced gene expression of the enzyme 6-phosphogluconate dehydrogenase tend to synthesize even greater amounts of riboflavin on a medium with lactose as a single source of carbon. Thus, the cultivation of strains on milk whey, in which lactose is the only source of carbon, contributed to an even more intense formation of vitamin B2. In general, changes in the culture medium allowed to increase the level of synthesis by 1.4, 1.7 and 1.7 for strains L20105/GND1, AF-4/GND1 and BRP/GND1, compared to synthesis on mineral medium with glucose.
We were first, who investigated the localization of the transporter protein Rfe1 (RiboFlavin Excretase) in the yeast C. famata. Binding a fluorescent label to this protein revealed that Rfe1 is located in the plasma membrane. We have proven that Rfe1 does not function in the nucleus.
The presented experimental data are important for understanding certain mechanisms of control over riboflavin biosynthesis. Fortunately, in the modern world, researchers are gradually discovering new regulatory factors of flavinogenesis. Our contribution to this progress is important and may, in the future, contribute to the creation of new stable overproducers of riboflavin, and thus increase the profitability of production processes. In particular, the application of whey as a substrate for the growth of strains provides greater opportunities to make the process cheaper and more environmentally friendly as the whey is usually disposed of as waste from the dairy industry.
