RF deficiency may lead to increased risk of cardiovascular disease, impairment of iron metabolism and night blindness. Flavins are used for the treatment of ariboflavinosis, a condition marked by lesions in the corners of the mouth, on the lips, and around the nose and eyes, or as general health supplements in the case of malnutrition [ 21 ].
Supplementation with RF helps with the treatment of lactic acidosis [ 23 ]. FAD is also considered potential treatment for diseases such as Friedreich ataxia [ 25 ] and chronic granulomatous disease [ 26 ]. As excellent redox coenzymes, FMN and FAD are important biochemical reagents with significant application potential in the enzyme industry.
Nowadays, the industrial production of RF is exclusively accomplished by microbial fermentation without the involvement of chemical synthesis. Chemical synthesis of RF essentially consisted of six to eight chemical steps starting from d -glucose or D-ribose. Please refer to earlier literature for more information about chemical methods of RF synthesis [ 2 , 3 ]. The industrial strains used for RF production mainly are mainly derived from the bacterium Bacillus subtilis B.
The yeast Candida famata C. As the engineered RF production strains are typically acquired by a combination of mutagenesis and genetic engineering, the genetic background is often complex and can be perplexing.
For example, B. All plants and fungi, as well as most bacteria, are capable of producing RF [ 1 ]. Fungi such as the flavinogenic A. The biotechnological engineering of flavin overproducing strains is always accomplished by overexpression of genes in the relevant pathways, suppression of competing pathways, and disruption of regulatory genes responsible for feedback inhibition.
Schematic overview of the relevant pathways for flavin production. As the precursor of RF, GTP serves as the donor of the pyrimidine ring and the nitrogen atoms of the pyrazine ring, as well as the ribityl side chain of the vitamin [ 39 ]. R5P is further phosphorylated to form PRPP, which in turn is converted into GTP via a series of enzyme-catalyzed reactions of the purine biosynthesis pathway.
While similar, the biochemical pathways of GTP synthesis from Ru5P in bacteria and fungi show a different preference for carbon sources.
Fungi such as A. A shunt also converts a part of the acetyl-CoA into OAA, which is further transformed into glycine for purine synthesis. By contrast, bacteria such as B.
Detailed reviews of the overall pathway of RF synthesis from carbon precursors can be found elsewhere [ 2 , 27 ]. A schematic of the RF synthesis pathway is shown in Fig. The final step in RF biosynthesis reaction VII involves dismutation of two molecules of DrL including an exchange of a 4-carbon unit, which results in a molecule of RF and the regeneration of a molecule of ArP.
This step of the reaction is catalyzed by RF synthase. A mutant of B. Earlier publications offer detailed reviews of the biosynthesis pathway of RF and related enzymes [ 1 , 2 , 42 , 43 ]. The mechanism of ArPP dephosphorylation was unclear until relatively recently. In , researchers found that enzymes from the haloacid dehalogenase HAD superfamily catalyze the dephosphorylation of ArPP.
Individual deletion of ybjI or yigB did not result in RF auxotrophy [ 45 ]. Just like in E. Moreover, the deletion of individual ArPP phosphatase genes does not lead to RF auxotrophy, indicating the existence of multiple redundant isoenzymes.
Generally, the overexpression of genes responsible for the synthesis of RF is an efficient method for enhancing RF production. Industrial RF producer strains of B. The monofunctional RFKs show sequence and structural homology with the C-terminal module of the bifunctional enzymes.
Both monofunctional and bifunctional enzymes have been modified for improved activity by mutation of key residues. Nowadays, commercial RF production is exclusively accomplished by microbial fermentation [ 2 ]. The industrial RF production strains are mostly derived from the bacterium B. Nevertheless, other species are also engineered for the production of RF, including C. The industrial RF production strains were typically developed by a combination of mutagenesis and several rounds of genetic engineering.
The mutagenesis process consists of several screening steps for resistance to different antimetabolites. Structural analogues of RF, roseoflavin RoF r , different purine analogues such as 8-azaguanine Az r , thioguanine and 8-azaxanthine, as well as decoyinine Dc r and the glutamine antagonist methionine sulfoxide [ 52 , 76 , 77 , 78 ], have been successfully used for the screening of RF overproducing B.
Although A. The RF over-producer A. Metabolic engineering is the science of rewiring the metabolism of cells to enhance the production of native metabolites or to endow cells with the ability to produce new products [ 85 ].
Metabolic engineering involves the continuous improvement of cells through several rounds of genetic engineering [ 86 ]. Many RF overproducing strains have been constructed through metabolic engineering Table 1. According to the metabolic pathway, the strategies of metabolic engineering for RF production can be classified into modifications of the RF synthetic pathway, the purine pathway, the central carbon metabolism, the synthesis of glycine, cell-scale optimization, and so on.
In general, metabolic engineering for enhanced RF production was achieved by overexpressing the synthetic pathways of RF or its precursors, which was accomplished by the combination of direct gene duplication, replacement of the native promoter with a strong one, disruption of competing pathways, or modification of regulatory genes. Other strategies focused on the improvement of the host traits, for example by reducing the maintenance metabolism.
Overexpression of the RF synthesis pathway is a proven effective method for enhancing RF production, and has been achieved through multiple methods. Overexpression of genes responsible for RF synthesis or the RF operon is one of the most common methods to improve the synthesis of RF.
Strains with multiple copies of the RF operon on an integrative vector produced approximately tenfold more RF [ 52 ]. The heterologous expression of the rib operon from Bacillus cereus B. In the flavinogenic fungus A. Accordingly, overexpression of the RIB genes resulted in a significant increase in the RF yield [ 53 ]. When expressed in wild-type E.
The EC10 operon was also expressed from a low copy number plasmid to alleviate the metabolic burden of heterologous expression [ ].
Duplication of some or all genes involved in the synthetic pathways of RF is an obvious choice for enhanced RF production. However, the expression of genes responsible for the synthesis of RF can also be improved without increasing their copy number.
Overexpression of the RF operon was also achieved by replacing the native rib promoters with strong constitutive promoters from the SPO1 phage or the P15 promoter to eliminate the feedback inhibition of the natural RF operon [ 52 ]. GTP, which is produced by the purine pathway, is a key precursor for the synthesis of RF. Consequently, increased expression of the purine pathway can effectively enhance the production of RF.
Overexpression of the purine pathway has been accomplished by mutagenesis and metabolic engineering. The mutagenesis method is suitable for the breeding of B.
Mutants resistant to purine analogues, decoyinine or methionine sulfoxide showed a deregulation of purine synthesis and increased flux from inosine monophosphate IMP to guanosine monophosphate GMP [ 52 , , ]. Metabolic engineering of the purine pathway includes the overexpression of enzyme-coding genes, as well as the disruption of regulatory genes and competing pathways, especially the pyrimidine pathway.
Improved flux through the purine pathway has been accomplished by various metabolic engineering strategies. Firstly, the overexpression or mutation of key enzymes can be used to increase synthesis of the key purine GTP. Secondly, purine synthesis was also increased by derepression of the regulator. Furthermore, the purine pathway of A. Moreover, purine synthesis was also improved by blocking the competing pyrimidine biosynthesis pathway, which led to improved RF production [ 97 ].
Optimization of the expression of genes involved in the AMP branch, which competes for purinogenic precursors, also resulted in higher RF production [ 53 ]. While the overexpression of the purine pathway is an efficient strategy for increasing RF production, the carbon flux through the purine pathway itself is limited by the flux of metabolites coming from the pentose phosphate PP pathway.
Consequently, a further increase of RF production requires enhancing the metabolic flux through the PP pathway. Accordingly, the overexpression of the genes implicated in the PP pathway facilitated the production of RF. For instance, increased expression of the zwf gene encoding the glucosephosphate dehydrogenase in B.
Similarly, the co-overexpression of mutant zwf and gnd encoding 6-phosphogluconate dehydrogenase from Corynebacterium glutamicum C. Gluconeogenesis GNG produces glucose from certain non-carbohydrate carbon sources, and enhancing it can also stimulate the production of RF. Overexpression of gapB encoding NADPH-dependent glyceraldehydephosphate dehydrogenase , fbp encoding fructose-1,6-bisphosphatase and pckA encoding phosphoenolpyruvate carboxykinase in B.
The deregulated expression of the gluconeogenetic genes gapB and pckA was also accomplished by knockout of the genetic repressor CcpN [ ]. Similarly, a knockout of the pfkA gene encoding 6-phosphofructokinase I resulted in a downregulation of the EMP instead of a complete blockage [ ]. This avoided problems of inefficient synthesis of glycine, which is an important precursor in purine synthesis. The supplementation of the precursor glycine in the medium was helpful for the production of RF by A.
As a precursor of purine biosynthesis, glycine is also a limiting factor for RF production in A. Overexpression of the GLY1 gene encoding threonine aldolase under the control of the TEF promoter and terminator, together with threonine supplementation in the culture medium, led to a tenfold increase of threonine aldolase specific activity and ninefold increase of RF production [ ].
This was explained by an enhancement of the intracellular availability of glycine. Disruption of the SHM2 , gene encoding serine hydroxymethyltransferase, caused a redistribution of carbon fluxes away from serine and toward glycine, which resulted in a significant increase of RF production in A.
Expression of the AGX1 gene encoding alanine-glyoxylate aminotransferase from Saccharomyces cerevisiae S. The optimization of the whole cell chassis for RF production has been implemented in several aspects.
Firstly, the optimization of electron transport contributed to the overproduction of RF. Redirection of electron flow to more efficient proton pumping branches within respiratory chains is a generally applicable metabolic engineering strategy for improving product and biomass yields [ ]. A knockout of cytochrome bd oxidase cydC deletion in B. Secondly, enhanced export of RF may also be useful to increase the RF yield.
Although the transport of RF out of the cell has not been studied in detail, the RF over-producing microorganisms can efficiently passively excrete or actively secrete RF, leading to accumulation in a medium.
Heterologous expression of the codon optimized ribMopt gene from Streptomyces davaonensis S. Moreover, enhanced RF production can also be achieved by engineering an improved robust host. As most of the industrially viable RF overproducers were constructed by a combination of classical mutagenesis and genetic engineering, it is necessary to pinpoint the exact genetic characteristics that are responsible for the overproduction of RF.
Transposon-tagged mutagenesis, omics techniques and metabolic flux analysis have been applied to reveal the relationship between specific genetic characteristics and the RF overproduction phenotype. In addition, some novel strategies have also been successfully utilized to improve RF production. The industrial RF producer B. About 10, random, transposon-tagged mutants were generated and screened. Then, the transposon insertion sites of both RF overproducing and deficient mutants were analyzed.
Subsequently, a novel target, the repressor CcpN, was revealed by reverse engineering. Transcriptome analysis was also applied to understand the genetic changes in an RF over-producing strain of B. It was found that the pur operon and other PurR-regulated genes were all downregulated in the overproducer. Another integrated whole-genome and transcriptome sequence analysis of an RF-overproducing B.
Notably, this was the first report that a mutation in yvrH RQ can deregulate the purine pathway for improved RF production. Unlike the bacterium B.
During growth, the TCA cycle was highly active, whereas the flux through gluconeogenesis and the PP pathway was rather low. Yeast extract was the main carbon donor for anabolism, while vegetable oil selectively contributed to the amino acids glutamate, aspartate, and alanine. Finally, optimization of the fermentation process and medium composition is vital for achieving an industrially viable level of RF production. This approach has been applied in various RF producers such as B.
Genetic engineering and chemical analogues screening approaches have been applied for improved RF production of LAB. Detailed introductions can be found elsewhere [ 1 , 14 , 15 ]. The LAB is widely used in the food industry, especially in the dairy industry, which makes it an advantage of RF producing LAB for the food fortification happens in situ.
Recently, researchers have isolated Lactobacillus species, which were capable of overproducing RF, from dairy and nondairy sources as well as plant sources [ 19 ].
The RF production of these strains was about 2. In another study, as many as 60 Lactobacilli were screened for the ability of RF overproducing via screening of the genes responsible for RF synthesis by a polymerase chain reaction PCR -based method [ 16 ]. Among the Lactobacilli screened, the presence of genes responsible for RF synthesis was strain-specific across different species. The L. The other isolates showed incomplete rib structural genes or absence of related genes. The isolates possessing incomplete rib structural genes could not survive in the riboflavin-deficient medium RAM.
The study also showed that the Lactobacilli isolated from human faeces and fermented bamboo shoots possessed maximum RF production. The RF producing Lactobacilli isolated was evaluated to be potential probiotic and development of RF bio enriched probiotic food [ 17 ]. The soymilk fermented by a RF-producing L. Moreover, the L.
Incorporation of Lactococcus lactis N8 L. The screened Andean LAB strains might be useful for the production of cereal-based kefir-like RF-enriched beverages in situ [ ]. The quinoa pasta fermented with the LAB L. Early methods for the production of FAD were based on the whole-cell biocatalytic conversion of its precursors. Among the tested microorganisms, bacteria and actinomycetes were able to accumulate detectable amounts of FAD, while yeasts and molds were not.
However, the addition of the expensive FMN precursor and the low rate of its conversion to FAD precluded the industrialization of this process. To overcome these disadvantages, a mutant of S. The yield of FAD was stimulated by the addition of D-cycloserine due to improved permeability of the cell membrane. The titer of FAD reached 0. In subsequent studies C. The maximum FMN titer reached 3. However, RF yielded 1.
The cloned FAD synthetase gene of C. Consequently, the FAD synthetase of C. The FAD synthetase activity of the recombinant E. In the whole-cell biocatalytic production of FAD using C. Among the phosphate compounds tested, only the metaphosphate with no adenylyl moiety could promote the phosphorylation of RF to FMN without inducing the concomitant accumulation of FAD. A method for producing flavin nucleotides was patented in [ 32 ]. In the method, a whole or part of a gene, or a gene mutated at the 23 rd glycine residue encoding the enzyme which retained RFK and had no or reduced FMNAT activity, was overexpressed in E.
In an example, a 10 ml reaction mixture produced In this case, the production of FMN-Na 2 reached When the cells of E. The flavinogenic yeast C. Recombinant strains of C. Thus, it was reasonable to utilize it industrially, and C. The isolated transformants had 3—8 copies of the recombinant FMN1 gene integrated into the genome. The integration of the FMN1 gene was also applied in the C. Vitamins are complex organic compounds required in trace amounts for normal functions of an organism.
However, mammals cannot produce many vitamins on their own and these have to be externally obtained from dietary supplements and feed additives. Over the last few decades, large-scale production of vitamins by microorganisms has been carried out and more than half of the commercially produced vitamins are fed to domestic animals Ledesma-Amaro et al.
Riboflavin vitamin B2 is a water-soluble vitamin, which is produced by all plants and most microorganisms and is essential for growth and reproduction of humans and animals Revuelta et al. Riboflavin performs its biochemical function as a precursor for the coenzymes, flavin adenine dinucleotide FAD and flavin mononucleotide FMN , which are mostly involved in redox reactions of all organisms.
These flavocoenzymes participate in the metabolism of carbohydrates, lipids, ketone bodies, and proteins from which living organisms derive most of their energy. Additionally, riboflavin promotes the conversion of tryptophan into niacin and vitamins B6 and B9 into their active forms, as well as the mobilization of iron.
Therefore, the recommended dietary allowance RDA for human and animal nutrition are 0. Industrial production of riboflavin can be performed by both chemical synthesis and fermentation. The fermentation route allows the production of vitamin B2 in a single step, which is cost-effective.
In contrast, chemical processes are multistage and expensive. Thus, nowadays, the fermentative production of riboflavin is economically and ecologically more feasible and has completely replaced chemical synthesis. The world market for riboflavin production for human and animal use has more than doubled in 13 years, from t a —1 in to t a —1 in Schwechheimer et al.
Over the last few decades, several groups of researchers have reported successful achievements in the construction of genetically modified strains of species, such as Escherichia coli, B. More frequently, such strategies have led to the overexpression of structural and regulatory genes involved in the synthesis of riboflavin or that of its precursors; consequently, this has improved strain productivity and yield of the industrial fermentation product Perkins et al.
However, there are still unresolved issues caused by various nonspecific reactions in riboflavin biosynthesis, which are not yet completely understood. The present review summarizes the latest scientific studies that have investigated microorganism-derived riboflavin synthesis using different methods, such as media component optimization, mutations and screening, genetic engineering, and biocatalyst conversion, to improve the production of vitamin B2 and its precursors.
The application of these studies is highlighted by references to recent patents related to scientific and industrial developments in microbial riboflavin production. The first commercial microbiological production of riboflavin using bacteria was performed with Clostridium acetobutylicum by acetone-butanol fermentation, where riboflavin was formed as a byproduct Leviton, Later, several species of fungi, such as Eremothecium ashbyii , A.
However, these microorganisms accumulated riboflavin slowly and at a low concentration, which were not satisfactory for commercial production of riboflavin. Since then, numerous experiments have been performed on riboflavin biosynthesis optimization via microbial strain improvement using biological, genetic, and bioinformatics approaches.
The fermentative production of riboflavin is naturally carried out by the wild-type flavinogenic ascomycetes, such as E. Among them, A. However, E. Modern approaches guided by genetic manipulations and medium supplementation have led to riboflavin overproduction in these organisms Table 1.
Althofer et al. Increased riboflavin production was shown for the A. Park et al. Using genetic techniques and supplement optimization, A. Among Candida strains, the mutant C. The maximum amounts of riboflavin produced by the yeast C.
As the biosynthesis process depends on the addition of nitrogen sources, such as glycine and hypoxanthine, selection for strains resistant to the adenine antimetabolite, 4-aminopyrazolo 3, 4-d pyrimidine, improved production Park et al. Threonine demonstrated a ninefold stimulation in a strain with a cloned threonine aldolase gene, responsible for converting threonine to glycine Abbas and Sibirny, Among other fungi, the filamentous Aspergillus niger , A.
The Japanese inventors have developed a riboflavin production process using non-flavinogenic yeast Saccharomyces cerevisiae grown in the presence of calcium acetate and zinc ions. The productivity of this process was up to 3. Some flavogenic yeast mutants of P. The recombinant strain XS-3 produced three times more riboflavin 3. Daneshazari et al. However, riboflavin biosynthesis has been most studied on the nonpathogenic bacterium, B.
Bacillus subtilis is capable of producing riboflavin precursors, inosine and guanosine, in the purine pathway, which could be converted metabolically into riboflavin. However, riboflavin overproduction has been achieved by obtaining mutants with overexpression of certain genes and resistance to purine analogs azaguanine, decoyinine, and methionine sulfoxide, or the riboflavin analog roseoflavin, as the B. Strains B. Oraei et al. Li et al.
Wu et al. Recently, the availability of advanced genetic engineering technology, combined with process development and optimization, could allow certain bacteria such as Salmonella typhimurium , C. Mycobacterium phlei was able to produce small quantities of riboflavin from beet molasses Abd-Alla et al.
Under optimized conditions, the engineered strain accumulated Succinate-utilizing Rhizobium sp. The use of lactic acid bacteria LAB is a common practice in the dairy industry, and the addition of riboflavin-producing strains to fermented products, such as fermented milk, yogurt, and cheese, increases riboflavin concentrations, which is economically viable.
Recent study on riboflavin biosynthesis during food fermentation in dairy products showed that fermentation of cow milk with Lactococcus lactis and Propionibacterium freudenreichii ssp. Burgess et al. Thakur et al. According to Jayashree et al. Guru and Viswanathan reported that Lactobacillus acidophilus produces higher riboflavin levels compared with L.
Sybesma et al. Thus, LAB are attractive riboflavin producers having the potency to extend their biosynthetic capacity by modern biotechnology methods Thakur et al. Presently, two major overproducers of commercial riboflavin include the yeast-like mold, A. Research on riboflavin biosynthesis demonstrated that characteristic features of most enzymes and steps involved in the riboflavin pathway are mostly similar between prokaryotes and plants, whereas fungi use a somewhat different pathway and enzymes Abbas and Sibirny, Most knowledge on riboflavin biosynthesis today has been obtained in considerable detail for two major industrial producers: the filamentous fungus A.
Figure 1. Schematic diagram of the riboflavin pathways in Ashbya gossypii and Bacillus subtilis. RibU originally known as ypaA — riboflavin transmembrane transporter, its substrates are FMN and riboflavin analogue roseoflavin; RibT, N-acetyltransferase GCN5 earlier predicted as transporter , which transfers the acetyl group from acetyl coenzyme A AcCoA to a variety of substrates unknown. Produced or consumed cofactors are shown with curved arrows near the Rib enzymes.
The genome of A. The six riboflavin biosynthetic genes encoding riboflavin enzymes in A. The gene RIB1 in A. The overexpression of ribAB in B. Notably, in B. The next step might be the dephosphorylation of ArPP Figure 1. However, the dephosphorylation mechanism as well as phosphatase that catalyzes conversion of ArPP into ArP remains to be elucidated in the riboflavin biosynthesis pathway, though much investigative work has been performed on the origin of the four carbons of ArP.
A specific phosphatase, catalyzing ArPP dephosphorylation, has been found in planta Arabidopsis among eight enzymes from the haloacid dehydrogenase HAD superfamily, whereas the search for similar enzymes with promiscuous functions in B. It is important to note that the product of ribAB in B. Thereafter, both branches of the riboflavin biosynthetic pathway merge into one Figure 1. The enzyme is encoded by RIB4 in A.
For A. A variety of inducers, effectors, inhibitors, and signal molecules affect metabolite overproduction in microorganisms that provide positive or negative regulation of enzyme catalyzing metabolic reactions, through regulatory genes responsible for feedback inhibition transcription and translation levels or allosteric effects on some enzymes posttranslational level Sanchez and Demain, Regulation of the riboflavin biosynthetic pathway is not completely understood for several riboflavin-producing microorganisms.
However, most studies have unraveled regulatory mechanisms behind riboflavin overproduction linked to nutritional and oxidative stress in microorganisms Schlosser et al. As is well known, wild-type microorganisms possess metabolic regulatory systems to prevent an overproduction of riboflavin.
The regulation of riboflavin synthesis occurs mostly at the level of its very slow biosynthetic enzymes; thus, it is necessary to induce a strong and stable expression of their encoding genes, which is achieved by stress response, nutrition, or pathway regulation at a certain phase of microbial growth Acevedo-Rocha et al. Schlosser et al. A more recent study reported that there was no significant increase at the transcriptional level for all RIB genes except RIB4 during the riboflavin biosynthetic phase Ledesma-Amaro et al.
In addition, the flavinogenic activity of A. It has been suggested that at elevated temperatures a specific repressor of riboflavin biosynthesis is activated, although no direct evidence has been presented. The interaction of endogenous riboflavin with light induces oxidative DNA damage in cells by emerging reactive oxygen species ROS , but exogenous riboflavin was shown to protect A.
It is probable that riboflavin protects spores of fungi and attracts insects to their dispersal Aguiar et al. With the induction of riboflavin secretion, enzyme activity involved in detoxification of ROS, e. However, Silva et al. The overproduction of riboflavin by A. In yeasts, Yap1 absence renders cells hypersensitive to oxidants generated by superoxide anion radicals.
Genome expression is operated by Yap transcription factors, which have the ability to act as both inducers and repressors. Studies on different Yap factors in S. Yap3 and Yap7 seem to be involved in hydroquinone and nitrosative stresses, respectively Silva et al.
Flavinogenic yeasts and bacteria have strains that overproduce riboflavin under iron-restrictive conditions, probably due to either the direct role of riboflavin as an electron donor for iron reduction or as a cofactor for enzyme activity.
The maximum amount of riboflavin produced by the yeast C. Its mutant was defective for riboflavin oversynthesis in the iron-deficient medium due to the mutated transcription factor gene SEF1. Similar data on an iron-deficient growth medium were obtained with mutant P.
In bacteria, the transcriptional ferric uptake regulator Fur is the main regulator of iron homeostasis Cisternas et al. Research conducted by Vasileva et al. Iron starvation was shown to induce secretion of riboflavin in Methylocystis sp.
In the fungus A. Some species of flavinogenic yeasts overproduce riboflavin in iron-sufficient media containing n-alkanes as the sole carbon source, but mechanisms of these stimulatory effects remain unknown Sugimoto et al.
The riboflavin yield is also markedly dependent on the type and initial concentrations of carbon and nitrogen sources, as well as supplementation of primary or intermediate precursors for biosynthesis. Several studies were conducted on enhancing riboflavin production by supplementation. However, for overproduction, A. Industrial waste materials, such as oil discharged by oil refinery plants, grape-must, beet molasses, peanut seed cake, and whey, have also been employed in riboflavin production but with limited success.
However, researchers are hopeful about riboflavin biosynthesis in A. As an appropriate supply of carbon source stimulating riboflavin production, B. Supplementation of glycine during fermentation with A. Notably, E. However, feedback inhibition of important enzymes in their biosynthetic pathways and toxic effects from their excess inhibited cell growth Lim et al. During the improvement of riboflavin production by Sugimoto et al.
Xanthine was suggested as an intermediate precursor because of purine structure similarities. However, in experiments with guanine auxotrophs Aerobacter aerogenes , C. Evidently, the availability of the immediate riboflavin precursor GTP synthesized from amino acids, tetrahydrofolate derivatives, and CO 2 via serine, threonine, and glyoxalate cycles is a major rate-limiting factor for riboflavin overproduction.
Practically all upregulated reactions during the trophic phase of A. However, an excess of extracellular purines represses the transcription of genes required for ATP and GTP synthesis by feedback inhibition of the de novo purine pathway.
Similarly, excess serine and threonine have the same influence. Therefore, riboflavin overproduction in A. Transcriptionally downregulated reactions were mostly used in relation to biomass formation, prevention of riboflavin consumption, and glycine degradation Lim et al.
Regulation of metabolic pathways by supplementation of structural analogs of metabolites antimetabolites inhibiting metabolic reactions is used to search for limiting steps of biosynthesis and ways to overcome them, including development of strain antimetabolite resistance Schmidt et al.
Antimetabolites, such as tubercidin blocking purine biosynthesis in C. Thus, oxalate resistance downregulated the expression of aldose reductase and methionine synthase that allows the strain to intracellularly accumulate glycine. Overexpression of malate synthase from the natural oxalate-resistant A.
However, this was the first study that described the natural isolation of riboflavin overproducer Sugimoto et al. The mutation of A. For B. Exposure to the riboflavin analog roseoflavin isolated from Streptomyces davawensis was found to lead B. Roseoflavin negatively affects FMN-specific rib operon regulators FMN riboswitches and flavoenzymes in bacteria, and is used together with multiple copies of rib operon genes to select their overproducing strains Acevedo-Rocha et al.
In contrast to fungi, riboflavin synthesis regulation in B. It is a highly conserved RNA motif selective for the cofactor FMN, which modulates the expression of the FMN synthesis-associated genes mostly transporters in response to elevated concentrations of corresponding cellular metabolites Meyer et al. The B. Two additional internal promoters P2 and P3 of the rib operon are located in the regions of ribE , ribH , and ribT genes Figure 2. The ribU gene encodes a transmembrane transporter for exogenous riboflavin uptake and flavin metabolism Figure 2 Rodionova et al.
Thus, proteins for transport and biosynthesis are synthesized in parallel to ensure availability of the vitamin Hemberger et al.
Figure 2. Scheme of the riboflavin biosynthesis regulation in B. The hairpins symbols denote confirmed transcription terminators. In addition, the regulatory function in B. RibR , an RNA-binding protein that is also not part of the rib operon, is believed to act as a regulatory protein as it seems to interfere with the FMN riboswitch function Higashitsuji et al. The gene ribR encodes a monofunctional flavokinase as a part of the transcription unit consisting of 12 genes, whose products are involved in sulfur uptake and degradation.
The ribR induction and repression occurred under methionine or taurine, and MgSO 4 , respectively Pedrolli et al. Recently, it has been shown that when sulfur is present, ribR expression increases to block FMN riboswitches, the FMN demand of the cell increases, and the rib operon is expressed even with high FMN levels Higashitsuji et al.
By modulating ribF expression through mutations in its ribosome binding site and optimizing fermentation conditions, riboflavin production was improved by fold up to a yield of In a study conducted by Wang et al. However, the exact regulation mechanism of riboflavin in E. The ribT function remained unknown until recent research showed that its enzyme is a member of GCN5-related N-acetyltransferase, which transfers the acetyl group from acetyl-CoA to a variety of substrates Srivastava et al.
The rib operon has also been studied in Bacillus amyloliquefaciens , Bacillus halodurans, Bacillus abortus, Vibrio vulnificus, Shewanella oneidensis, Actinobacillus pleuropneumoniae , C. In Photobacterium phosphoreum and Photobacterium leiognathi , riboflavin genes are localized within the lux operon Vitreschak et al.
In contrast, E. The regulatory RFN-elements are found on the chromosome of numerous, but not all, bacterial species. Interestingly, no clear phylogenetic distribution was found for these genes. Species can either have both transporter and biosynthesis genes L. Notably, the RFN element was not found in front of all transport units encoding the presumed riboflavin transporter Wels et al.
Only spirochetes, mycoplasmas, and rickettsia have neither riboflavin genes nor RFN elements Vitreschak et al. Attempts to improve microbial riboflavin-producing strains were made by both metabolic and genetic engineering, which include the following: 1 random mutagenesis by chemical exposure and UV irradiation Matsuyama et al.
Duplications, insertions, deletions, modifications, substitutions, upregulations, and downregulations of genes directly or indirectly associated with riboflavin biosynthesis were often combined by manipulation with nutritional and other growth factors Ledesma-Amaro et al. Numerous physiological and genetic methods have been developed to enhance production of defined secondary metabolites, allowing for an increase in riboflavin yield.
Mutations of key genes and non-coding regions in microbial genomes has facilitated overproducing strain development Park et al. Improvement of the producer most often begins with random mutagenesis and routine screening for mutants by qualitative and quantitative determination of riboflavin Table 2. Screening of mutants may include determining the productivity of up to several thousand colonies after each round of mutagenesis Park et al.
This approach is particularly useful when there are no data on which specific gene or region of the genome would result in the desired phenotype upon mutation. The random ninefold upregulation of genes involved in purine and riboflavin pathways was reached after the use of lagging-strand-biased mutagenesis disparity mutagenesis toward A. Table 2. Genetic modification methods used for the riboflavin-producing strains.
However, random mutagenesis may not reveal a mechanism for increasing strain productivity that is additionally unstable in contrast to site-directed mutagenesis, which implies the presence of a target nucleotide sequence with a known function.
By site-directed mutagenesis, it is possible to obtain stable and reproducible mutants with predictable gene expression regulation related to riboflavin biosynthesis Table 3. Site-directed mutagenesis is often applied to a strain obtained by random mutagenesis to optimize growth and create an overproducer.
Bacterial and fungal riboflavin biosynthetic pathways, as well as molecular-genetic strategies and toolboxes for riboflavin-producing capability improvement are different Figure 1 and Table 3. Riboflavin synthetic genes have been studied more extensively in E. Table 3. Strategies and genetic tools for the riboflavin-producing strains improvement. The parent B. Skip to content The Nutrition Source. Harvard T. The Nutrition Source Menu. Search for:. Vitamin B2 and Health Because riboflavin assists many enzymes with various daily functions throughout the body, a deficiency can lead to health problems.
Migraines Riboflavin works to reduce oxidative stress and inflammation of nerves, which are contributors to migraine headaches. A randomized controlled trial of 55 adults with migraines were given either mg daily of riboflavin or a placebo and followed for four months.
The authors noted that a beneficial effect of riboflavin did not start until after the first month, and showed maximum benefit after three months of use. A systematic review of 11 clinical trials on riboflavin as a prophylactic treatment for migraines found mixed results.
The dose for adults was typically mg daily, and for children mg daily, given for three months. There were no negative side effects observed from the supplements. Cardiovascular disease Because riboflavin assists many enzymes with various daily functions throughout the body, a deficiency can lead to health problems. References U. Department of Health and Human Services.
Institute of Medicine. Food and Nutrition Board. Prophylaxis of migraine headaches with riboflavin: a systematic review. Journal of clinical pharmacy and therapeutics.
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