riboflavin production by fermentationriboflavin production by fermentation

riboflavin production by fermentation28 May riboflavin production by fermentation

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis. Fact. Biochemistry 56, 62116220. Biotechnol. However, the industrial process of riboflavin biosynthesis in B. subtilis is still dependent on several barely resolved issues, including RibR-regulation of FMN riboswitches limiting its production; unknown riboflavin pathway phosphatases; flavin reactivity damaging cells; and the absence of a transport system to export actively flavins in contrast to that of A. gossypii (Acevedo-Rocha et al., 2019). Biotechnol. Figure 2. Fermentation of riboflavin for A. gossypii is performed at the optimum temperature range of 2630C in fed-batch fermenters (100 m3), with the initial pH of the culture medium as approximately 6.57.5, in aerobic conditions for 68 days until the yield peaks (Stahmann et al., 2000; Schwechheimer et al., 2016). The next step might be the dephosphorylation of ArPP (Figure 1). (2010), the efficient riboflavin-producing strain L. fermentum MTCC 8711 showed 2.29 mg/L of riboflavin in MRS broth after 24 h (Jayashree et al., 2010). doi: 10.1007/s10529-011-0833-z, Kavitha, S., and Chandra, T. S. (2014). Biotechnol. Multivitamin production in Lactococcus lactis using metabolic engineering. Sanchez, S., and Demain, A. L. (2008). Gloor, A. Frontiers | Improving the Production of Riboflavin by Introducing a 3, 169176. Hohmann, H.-P., Humbelin, M., van Loon, A., and Schurter, W. (2001). Time-course profiles of riboflavin production, biomass accumulation and glucose . doi: 10.1099/13500872-142-2-411. Daneshazari, R., Roayaei, M., Hossein, N., and Ghezelbash, G. (2013). Xanthine was suggested as an intermediate precursor because of purine structure similarities. 2016 ). The recombinant strain XS-3 produced three times more riboflavin (3.6 mg/L) compared to the wild-type strain ATCC 9058/L2 (1.2 mg/L) under the conditions described. B. After preliminary experiments of nitrogen source selection, the two-level Plackett-Burman (PB) design was implemented to screen medium . Bioresour. doi: 10.1016/0378-1119(88)90185-0. doi: 10.1111/j.1751-7915.2007.00015.x. Riboflavin and iron are the primary cofactors, each one assisting approximately 17% of cofactor-requiring enzymes. Hubei Guangji Pharmaceuticals (China) uses the fermentation method with B. subtilis proline-resistant strains that produce up to 26.5 g/L of riboflavin in 70 h (Schwechheimer et al., 2016). In bacteria, the transcriptional ferric uptake regulator Fur is the main regulator of iron homeostasis (Cisternas et al., 2018). Genome expression is operated by Yap1-8 transcription factors, which have the ability to act as both inducers and repressors. Thereafter, DARPP is converted into 5-amino-6-ribityl-amino-2,4(1H,3H)pyrimidinedione (ArP) by sequential reactions of deamination, side chain reduction, and dephosphorylation (Figure 1). B-Group vitamin production by lactic acid bacteria - current knowledge and potential applications. Fungal Genet. A novel process for riboflavin production using a recombinant Bacillus subtilis strain has been developed. 1, 4955. 12, 12931301. Microbial cell factories for the sustainable manufacturing of B vitamins. Biol. Riboflavin is a crucial micronutrient that is a precursor to coenzymes flavin mononucleotide and flavin adenine dinucleotide, and it is required for biochemical reactions in all living cells. (2007). Bioeng. Optimization of effective minerals on riboflavin production by Bacillus subtilis subsp. Nucleic Acids Res. Riboflavin is a B-vitamin, also known as vitamin B 2, and is an essential component of the diets of humans and higher animals.Conversion to its coenzyme derivatives: flavin mononucleotide or riboflavin phosphate (FMN) and flavin adenine dinucleotide (FAD) enables it to participate in many . Bacterial Strains Which Overproduce Riboflavin. (2017). 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., 2018; Figure 2). (2005) and Silva et al. Riboflavin-Producing Microorganisms and Culture Conditions. 13:104. Metabolic flux analysis in Ashbya gossypii using 13C-labeled yeast extract: industrial riboflavin production under complex nutrient conditions. However, riboflavin biosynthesis has been most studied on the nonpathogenic bacterium, B. subtilis, which has become a model organism among industrial riboflavin-producing strains due to its ability to secrete large amounts of protein directly into the medium in a short time (Sauer et al., 1996; Perkins et al., 1999a,b; Lee et al., 2004a,b; Wu et al., 2007). 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., 1996; Park et al., 2007; Tajima et al., 2009). Jimnez, A., Hoff, B., and Revuelta, J. L. (2019a). Examples of such bacterial overproducers are genetically engineered B. subtilis, A. gossypii, E. ashbyii, and C. famata riboflavin production strains. The ribU gene encodes a transmembrane transporter for exogenous riboflavin uptake and flavin metabolism (Figure 2) (Rodionova et al., 2017). Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis. Abstract Riboflavin is widely regarded as an essential nutrient that is involved in biological oxidation in vivo. Biol. J. Ind. doi: 10.1021/acs.biochem.7b00669. Scientific Opinion on the safety and efficacy of vitamin B2 (riboflavin) produced by Ashbya gossypii for all animal species based on a dossier submitted by BASF SE. It was hypothesized that riboflavin might be involved in the nonenzymatic reduction of weakly soluble Fe3+ to Fe2+ due to the use of mechanisms for iron assimilation, distinguished from most flavinogenic yeasts that do not overproduce riboflavin under conditions of iron limitation (Dmytruk and Sibirny, 2012). Microbiol. J. Biol. Exposure to the riboflavin analog roseoflavin isolated from Streptomyces davawensis was found to lead B. subtilis to spontaneous mutations and constitutive riboflavin overproduction (Ludwig et al., 2018). Adv. Technol. down-stream processing and comparison of the composition of riboflavin produced by fermentation or chemical synthesis W Bretzel, W Bretzel grid.417570. 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. doi: 10.1590/s1517-83822011000300030, Sybesma, W., Burgess, C., Starrenburg, M., van Sinderen, D., and Hugenholtz, J. Research conducted by Vasileva et al. A shift in carbon flux from -oxidation to the riboflavin biosynthetic pathway was proved by a twofold increase in ADE1, RIB1, and RIB5 protein synthesis, and in gene expression of gluconeogenesis and pentose phosphate cycles, but it was observed that the downregulation of pathways were related to carbon source assimilation, energy generation, and glycolysis at the riboflavin-producing phase (Table 3; Park et al., 2011). (2015). A., Vetting, M. W., Li, X., Almo, S. C., Osterman, A. L., and Rodionov, D. A. It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), needed as coenzymes for a wide variety of enzymes in the intermediate metabolism (Stahmann et al. Riboflavin - an overview | ScienceDirect Topics Cell. Thereafter, it is transformed into homokaryotic spores by a Cre (recombinase)-expressing plasmid to introduce the deletion or mutation into target genes for their functional study, as well as metabolic improvement (Aguiar et al., 2014; Table 3). Acevedo-Rocha, C. G., Gronenberg, L. S., Mack, M., Commichau, F. M., and Genee, H. J. Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Metab. The addition of activated bleaching earth containing 75 g/L rapeseed oil and oxygen-enriched air to the mutated strain ZP4 culture increased riboflavin concentration to 8.7 g/L after 5 days cultivation (Park et al., 2007; Table 1). Most knowledge on riboflavin biosynthesis today has been obtained in considerable detail for two major industrial producers: the filamentous fungus A. gossypii and the Gram-positive bacterium B. subtilis (Figure 1). (2019). The overexpression of ribAB in B. subtilis resulted in 25% greater riboflavin, indicating the biosynthesis rate-limiting step (Hmbelin et al., 1999). Abstract Ashbya gossypii Bacillus subtilis, and Candida spp. Yeasts bioproducts prospection from different brazilian biomes. Over the past two decades, the microbial production of riboflavin by fermentation completely replaced the chemical synthesis . Appl. Deregulation of purine pathway in Bacillus subtilis and its use in riboflavin biosynthesis. Nowadays, riboflavin production by microbial fermentation has completely replaced chemical synthesis (Revuelta and Wittmann, 2015). Site-directed mutagenesis is often applied to a strain obtained by random mutagenesis to optimize growth and create an overproducer. Tr. Metabolic engineering of riboflavin production in Cell. (2006). doi: 10.1007/s10295-009-0647-3, Suryadi, H., Yoshida, N., Yamada-Onodera, K., Katsuragi, T., and Tani, Y. doi: 10.1016/j.ymben.2003.11.002. Riboflavin, a water-soluble and yellow solid, the so called vitamin B 2, is an essential vitamin that is required by all animals, plants and bacteria. Riboflavin is a feed additive, food additive and clinical drug, with a significant annual demand of nearly 8000 t. Fermentation using recombinant Bacillus subtilis is currently one of the most important industrial production method for riboflavin. 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. Enzyme Microb. The productivity of this process was up to 3.4 g/L riboflavin without impurity problems, compared to the molasses-grown cells (Matsuyama et al., 1987; Table 1). Metabolic engineering of Escherichia coli for the production of riboflavin Among 15 variables, glucose, NaNO3, K2HPO4, ZnSO4, and MnCl2 were identified as the most crucial factors for riboflavin production (Wu et al., 2007). Metabolic engineering of Escherichia coli for the production of riboflavin Environ. (2004). The Merck company began riboflavin production by employing A. gossypii in 1974 (Stahmann et al., 2000). Its mutant was defective for riboflavin oversynthesis in the iron-deficient medium due to the mutated transcription factor gene SEF1. 7, 673678. Highly effective riboflavin production strains were constructed by introducing additional copies of ribDGEABH genes controlled by strong native or strength-evolved synthetic bacterial and phage promoters (Lee, 2015; Cisternas et al., 2018; Han et al., 2019). (2015) on metabolic engineering of the pentose phosphate, glycine, and purine pathways of A. gossypii describe the phosphoribosyl pyrophosphate (PRPP) synthetase and PRPP amidotransferase (ADE4) gene overexpression, which increased the carbon flux through the pentose phosphate and purine/GTP biosynthetic pathways (Table 3; Jimnez et al., 2008; Silva et al., 2015). Lactic acid bacteria as a cell factory for riboflavin production. Supplementation of glycine during fermentation with A. gossypii and Candida sp. A multiple-engineered Ashbya strain that produces up to 523 mg/L of riboflavin was generated. 85, 19071914. In 2001, BASF and Takeda (Japan) formed a joint venture and optimized the biotechnological production of vitamin B2 using the fed-batch method with vegetable oils as carbon sources and soy/corn products as nitrogen sources (Igami and Sugaya, 2016). However, these microorganisms accumulated riboflavin slowly and at a low concentration, which were not satisfactory for commercial production of riboflavin. The B. subtilis rib operon consists of five genes, ribDG, ribE, ribAB, ribH, ribT, forming one transcription unit (mRNA), and contains the regulatory region ribO, untranslated leader region, that is located with the major promoter P1 (transcription start) upstream of the first structural gene in the operon (Figure 2) (Sklyarova et al., 2012). sgRNA expression is driven by regulatory sequences from the A. gossypii SNR52 gene, which is transcribed by RNA polymerase III. However, in experiments with guanine auxotrophs Aerobacter aerogenes, C. guilliermondii, and Corynebacterium sp., lacking xanthine monophosphate (XMP) aminase, it was proven that the main precursor was guanine or a guanine nucleotide and the conversion of adenine, hypoxanthine, and xanthine to riboflavin passed through one of them (Abbas and Sibirny, 2011). Cre-loxP-based system for removal and reuse of selection markersin Ashbya gossypii targeted engineering. Open Access Article Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics Screening by Fan Xu 1,2,3,, Chuan Liu 2,3,4,, Miaomiao Xia 2, Shixin Li 2,5, Ran Tu 2, Sijia Wang 6, Hongxing Jin 1,* and Dawei Zhang 2,3,4,* 1 Cisternas, I. S., Salazar, J. C., and Garca-Angulo, V. A. For example, carbon dioxide emissions and the use of non-renewable resources are reduced by 80% and water emission by 66% each year. U.S.A. 112, 1405414059. 108, 325329. Conclusions Riboflavin production in A. gossypii is limited by a low transcription activity of the RIB genes. Riboflavin: Fermentation, Harvesting and Uses - Microbiology Notes Iron starvation was shown to induce secretion of riboflavin in Methylocystis sp., a methanotrophic bacterium (Vasileva et al., 2012). The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism. Microorganisms | Free Full-Text | Characterization of a Riboflavin (2004, 2006) characterized riboflavin synthesis in L. lactis subsp. Recent. Industrial Production of Vitamin B2 by Microbial Fermentation doi: 10.1128/jb.00722-08, Zhu, Y., Chen, X., Chen, T., Shi, S., and Zhao, X. Microbial production of riboflavin using riboflavin overproducers, Ashbya gossypii, Bacillus subtilis, and Candida famata. 7:23. doi: 10.1186/1475-2859-7-23, Mateos, L., Jimenez, A., Revuelta, J. L., and Santos, M. A. Mycobacterium phlei was able to produce small quantities of riboflavin from beet molasses (Abd-Alla et al., 2016). 11:119. doi: 10.1186/1472-6750-11-119, Higashitsuji, Y., Angerer, A., Berghaus, S., Hobl, B., and Mack, M. (2007). 70, 57695777. Biotechnol. Biotechnol. A crossing over recombination insertion in the chromosome contained the chloramphenicol resistance gene cat (ENA ID: LT622644) and disrupted the gene recA (recE), encoding a multifunctional protein for homologous recombination and DNA repair. doi: 10.1128/aem.70.10.5769-5777.2004. Figure 1. doi: 10.1002/yea.2929, Duan, Y. X., Chen, T., Chen, X., and Zhao, X. M. (2010). Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis. The last step is dismutation of DRL by the riboflavin synthase translated from ribE in B. subtilis and RIB5 in A. gossypii to form riboflavin and ArP, which is recycled in the riboflavin biosynthetic pathway (Figure 1). J. Biol. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production. Riboflavin is a byproduct of the acetone butanol fermentation as carried out by microorganisms such as Clostridium butylicum, C. acetobutylicum. A dual control mechanism synchronizes riboflavin and sulphur metabolism in Bacillus subtilis. Appl. 88, 705716. The hairpins symbols denote confirmed transcription terminators. Modern approaches guided by genetic manipulations and medium supplementation have led to riboflavin overproduction in these organisms (Table 1). doi: 10.1007/s00253-011-3325-0, Park, E. Y., Zhang, J. H., Tajima, S., and Dwiarti, L. (2007). Szczesniak, T., Karabin, L., Szczepankowska, M., and Wituch, K. (1971). (2019). Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii. (1998). Currently, owing to their higher efficiency, lower cost and lower environmental impact fermentation processes have replaced chemical synthesis methods for the industrial production of riboflavin. The genetically engineered B. subtilis strain VNIIGenetika 304/pMX45 produced 4.5 g/L of riboflavin after 25 h of fermentation, but was not stable due to the presence of repeated chromosomal and episomal copies of the rib operon (Lee, 2015). New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Antimetabolites, such as tubercidin blocking purine biosynthesis in C. famata, and itaconate and oxalate inhibiting isocitrate lyase for fatty acid use in Aphis gossipii are employed for selection of fungal riboflavin overproducing strains (Park et al., 2007; Tajima et al., 2009; Stahmann et al., 2000; Aguiar et al., 2015). The alternative pentose phosphate pathway of riboflavin biosynthesis includes the catalytic conversion of Ribu5P into 3,4-dihydroxy-2-butanone-4-phosphate (DHBP) by DHBP synthase. Thus, nowadays, the fermentative production of riboflavin is economically and ecologically more feasible and has completely replaced chemical synthesis. doi: 10.1007/s00253-006-0482-7, Li, Z., Yin, G., and Chen, T. (2013). RibR, a possible regulator of the Bacillus subtilis riboflavin biosynthetic operon, in vivo interacts with the 5-untranslated leader of Rib mRNA. Kato, T., and Park, E. Y. 174, 23072325. The genetically improved B. subtilis riboflavin overproducing strains seem to be used for fermentation products placed on the EU market as a feed additive. Patent US2477812. Appl. Mater. Microbiol. doi: 10.1038/nchem.2301, Monschau, N., Sahm, H., and Stahmann, K. P. (1998). The developed carbon-limited fed-batch method applied to industrial strains was available to recycle fermented biomass to obtain carbon and nitrogen supplies for a new fermentation cycle (Schwechheimer et al., 2016). PATENT US6322995B1. Microbiol. Sequencing the B. subtilis rib operon gave rise to new approaches for construction of novel riboflavin-producing strains (Hohmann et al., 2010). LA took the lead in writing the manuscript. In Photobacterium phosphoreum and Photobacterium leiognathi, riboflavin genes are localized within the lux operon (Vitreschak et al., 2002). Nucleic Acids Res. Microbiol. The improved CRISPR/Cas9n mediated multiplexing system reached an efficiency of 65% for three-point mutations in ribA, B, and H genes (Liu et al., 2019). 6:740. doi: 10.3389/fmicb.2015.00740, Thakur, K., Tomar, S. K., and De, S. (2015). 47:e40. Riboflavin production by Ashbya gossypii. 9, 441451. Cell. Proc. By site-directed mutagenesis, it is possible to obtain stable and reproducible mutants with predictable gene expression regulation related to riboflavin biosynthesis (Table 3). 3, 2934. Therefore, riboflavin overproduction in A. gossypii achieved via deregulation of the purine pathway at different levels to increase the glycine pool for GTP. Sci. The reasons for the physiological role of riboflavin overproduction by Candida sp. Additionally, overexpression of the inosine-5-monophosphate dehydrogenase (IMPDH) gene increased metabolic flux through the guanine pathway and ultimately enhanced riboflavin production by 40% compared to the wild-type A. gossypii (Table 3; Buey et al., 2015). J. Bacteriol. Production of Vitamin B2 (Riboflavin) by Microorganisms: An Overview 193, 3740. The different bas1 mutants showed a significant increase in the production of riboflavin and other growth-related phenotypes (Table 3; Mateos et al., 2006). doi: 10.4028/www.scientific.net/amr.634-638.1031, Lim, S. H., Choi, J. S., and Park, E. Y. The CRISPR/Cas9 strategy comprises expression modules for CAS9 nuclease and complex synthetic guide RNA (sgRNA). 86, 851858. Similarly, excess serine and threonine have the same influence. However, feedback inhibition of important enzymes in their biosynthetic pathways and toxic effects from their excess inhibited cell growth (Lim et al., 2001; Revuelta et al., 2016). (2013) reported that yeasts, Rhodosporidium diobovatum and Trichosporon asahii, are also able to produce riboflavin (Daneshazari et al., 2013). 2000 ). Riboflavin, also known as vitamin B 2, is a water-soluble compound, which can be synthesized by plants and microorganisms, but is essential for animals as they lack an endogenous biosynthetic pathway. Flux limitation towards AMP provides committed substrate GTP for riboflavin overproduction without detrimental effects on biomass formation. Wu et al. an overview. Biotechnol. Bioproc. Small but nutritionally sub- stantial amounts are incorporated in most bread flours and breakfast foods, in some pharmaceuticals, and in nearly all poultry and hog feeds. 2:008. The ADM (United States) improved the production of riboflavin using the yeast C. famata via aerobic fermentation, but later the plant was shut down due to the iron sensitivity of C. famata to the iron/steel equipment, which complicated the process (Abbas and Sibirny, 2011). doi: 10.1007/s002530051649, Sugimoto, T., Morimoto, A., Nariyama, M., Kato, T., and Park, E. Y. doi: 10.1002/bit.25167. The genome of A. gossypii is organized into seven chromosomes and genes responsible for riboflavin biosynthesis, and it is not clustered as in bacteria. and summarize their biosynthetic pathway optimizations through genetic and metabolic engineering, combined with random chemical mutagenesis and rational medium components to increase riboflavin production. Patel, M. V., and Chandra, T. S. (2020). Bioeng. Microbiology 164, 908919. Hohmann, H.-P., Mouncey, N. J., Schlieker, H. W., and Stebbins, J. W. (2010). Microb. 3, 2329. doi: 10.1016/s1389-1723(00)80033-x, Suzuki, G. T., Macedo, J. However, researchers are hopeful about riboflavin biosynthesis in A. gossypii with glucose, fructose, sucrose, starch, maltose, and degraded collagen as carbon sources (Ledesma-Amaro et al., 2015; Aguiar et al., 2017). Species can either have both transporter and biosynthesis genes (L. plantarum, Pediococcus pentosaceus, B. subtilis, Staphylococcus aureus), only one of the two (Lactobacillus johnsonii, Lactobacillus brevis, Lactobacillus delbrueckii, Streptococcus pneumoniae, Enterococcus faecalis), or lack both systems (Listeria monocytogenes, Lactobacillus casei). Fact. 91, 13151326. The CRISPR-assisted simultaneous up- and downregulation of the different genes expression (promoter-based transcription, molecular chaperone-assisted protein folding, protease-mediated degradation) during expression of amylase BLA improve in B. subtilis to 260-fold yield value of the target product BLA in a single cycle (Lu et al., 2019). The pGMBsub03 plasmid (ENA ID: LT622642) included part of the B. subtilis rib operon (ribA, ribH, ribT). Although several mechanisms have been suggested in recent decades based on intense scientific investigations, many key aspects concerning the behavior of riboflavin synthase in catalysis remain largely unknown. 100, 21072119. Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363.

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