Riboflavin Production Research Articles

Manipulation of Purine Metabolic Networks for Riboflavin Production in Bacillus subtilis.

Guanosine monophosphate, the precursor for riboflavin biosynthesis, can be converted to or generated from other purine compounds in purine metabolic networks. In this study, genes in these networks were manipulated in a riboflavin producer, Bacillus subtilis R, to test their contribution to riboflavin biosynthesis. Knocking out adenine phosphoribosyltransferase (apt), xanthine phosphoribosyltransferase (xpt), and adenine deaminase (adeC) increased the riboflavin production by 14.02, 6.78, and 41.50%, respectively, while other deletions in the salvage pathway, interconversion pathway, and nucleoside decomposition genes have no positive effects. The enhancement of riboflavin production in apt and adeC deletion mutants is dependent on the purine biosynthesis regulator PurR. Repression of ribonucleotide reductases (RNRs) led to a 13.12% increase of riboflavin production, which also increased in two RNR regulator mutants PerR and NrdR by 37.52 and 8.09%, respectively. The generation of deoxyribonucleoside competed for precursors with riboflavin biosynthesis, while other pathways do not contribute to the supply of precursors; nevertheless, they have regulatory effects.

Statistical significance in high-dimensional linear mixed models.

This paper concerns the development of an inferential framework for high-dimensional linear mixed effect models. These are suitable models, for instance, when we have n repeated measurements for M subjects. We consider a scenario where the number of fixed effects p is large (and may be larger than M), but the number of random effects q is small. Our framework is inspired by a recent line of work that proposes de-biasing penalized estimators to perform inference for high-dimensional linear models with fixed effects only. In particular, we demonstrate how to correct a 'naive' ridge estimator in extension of work by Bühlmann (2013) to build asymptotically valid confidence intervals for mixed effect models. We validate our theoretical results with numerical experiments, in which we show our method outperforms those that fail to account for correlation induced by the random effects. For a practical demonstration we consider a riboflavin production dataset that exhibits group structure, and show that conclusions drawn using our method are consistent with those obtained on a similar dataset without group structure.

A copper-specific microbial fuel cell biosensor based on riboflavin biosynthesis of engineered Escherichia coli.

Copper pollution poses a serious threat to the aquatic environment; however, in situ analytical methods for copper monitoring are still scarce. In the current study, Escherichia coli Rosetta was genetically modified to express OprF and ribB with promoter Pt7 and PcusC , respectively, which could synthesize porin and senses Cu2+ to produce riboflavin. The cell membrane permeability of this engineered strain was increased and its riboflavin production (1.45-3.56 μM) was positively correlated to Cu2+ (0-0.5 mM). The biosynthetic strain was then employed in microbial fuel cell (MFC) based biosensor. Under optimal operating parameters of pH 7.1 and 37°C, the maximum voltage (248, 295, 333, 352, and 407 mV) of the constructed MFC biosensor showed a linear correlation with Cu2+ concentration (0.1, 0.2, 0.3, 0.4, 0.5 mM, respectively; R2 = 0.977). The continuous mode testing demonstrated that the MFC biosensor specifically senses Cu2+ with calculated detection limit of 28 μM, which conforms to the common Cu2+ safety standard (32 μM). The results obtained with the developed biosensor system were consistent with the existing analytical methods such as colorimetry, flame atomic absorption spectrometry, and inductively coupled plasma optical emission spectrometry. In conclusion, this MFC-based biosensor overcomes the signal conversion and transmission problems of conventional approaches, providing a fast and economic analytical alternative for in situ monitoring of Cu2+ in water.

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.

Neuroprotective effects associated with immune modulation by selected lactic acid bacteria in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative process that affects the motor function and involves an inflammatory response and B vitamin deficiencies. The aim of this study was to evaluate the effect of B-group vitamin-producing and immunomodulatory lactic acid bacteria (LAB) in a murine model of PD. The effect of Lactobacillus plantarum CRL 2130 (a riboflavin producer), Streptococcus thermophilus CRL 807 (an immunomodulatory strain), and Streptococcus thermophilus CRL 808 (a folate producer) were evaluated individually and as a mixture in mice injected with 1-methyl-4-fenil-1,2,3,6-tetrahidropiridina. Motor capacity, tyrosine hydrolase in the brain, and cytokine concentrations in serum and brain tissues were evaluated in 1-methyl-4-fenil-1,2,3,6-tetrahidropiridina-treated mice after bacterial supplementation. The mice receiving the selected LAB showed significantly improved motor skills compared with those that did not receive bacterial supplementation. When given the mixture of all 3 strains together, the animals had higher brain tyrosine hydrolase-positive cell counts, decreased inflammatory cytokines interleukin 6 and tumor necrosis factor alpha in serum, and increased antiinflammatory cytokine interleukin 10 in serum and brain tissues compared with animals that did not receive LAB supplementation. The results showed the potential of a selected LAB mixture to improve motor behavior and neuroinflammation in PD. This probiotic mixture could be used as an adjunct treatment in the control of PD.

Robust high-dimensional semiparametric regression using optimized differencing method applied to the vitamin B2 production data

Background and purpose: By evolving science, knowledge, and technology, we deal with high-dimensional data in which the number of predictors may considerably exceed the sample size. The main problems with high-dimensional data are the estimation of the coefficients and interpretation. For high-dimension problems, classical methods are not reliable because of a large number of predictor variables. In addition, classical methods are affected by the presence of outliers and collinearity.Methods: Nowadays, many real-world data sets carry structures of high-dimensional problems. To handle this problem, we used the least absolute shrinkage and selection operator (LASSO). Also, due to the flexibility and applicability of the semiparametric model in medical data, it can be used for modeling the genomic data. Motivated by these, here an improved robust approach in a high-dimensional data set was developed for the analysis of gene expression and prediction in the presence of outliers.Results: Among the common problems in regression analysis, there was the problem of outliers. In the regression concept, an outlier is a point that fails to follow the main linear pattern of the data. The ordinary least-squares estimator was found potentially sensitive to the outliers; this fact provided necessary motivations to investigate robust estimations. Generally, the robust regression is among the most popular problems in the statistics community. In the present study, the least trimmed squares (LTS) estimation was applied to overcome the outlier problem.Conclusions: We have proposed an optimization approach for semiparametric models to combat outliers in the data set. Especially, based on a penalization LASSO scheme, we have suggested a nonlinear integer programming problem as the semiparametric model which can be effectively solved by any evolutionary algorithm. We have also studied a real-world application related to the riboflavin production. The results showed that the proposed method was reasonably efficient in contrast to the LTS Method.

A novel strain of acetic acid bacteria Gluconobacter oxydans FBFS97 involved in riboflavin production

A novel bacterial strain of acetic acid bacteria capable of producing riboflavin was isolated from the soil sample collected in Wuhan, China. The isolated strain was identified as Gluconobacter oxydans FBFS97 based on several phenotype characteristics, biochemicals tests, and 16S rRNA gene sequence conducted. Furthermore, the complete genome sequencing of the isolated strain has showed that it contains a complete operon for the biosynthesis of riboflavin. In order to obtain the maximum concentration of riboflavin production, Gluconobacter oxydans FBFS97 was optimized in shake flask cultures through response surface methodology employing Plackett–Burman design (PBD), and Central composite design (CCD). The results of the pre-experiments displayed that fructose and tryptone were found to be the most suitable sources of carbon and nitrogen for riboflavin production. Then, PBD was conducted for initial screening of eleven minerals (FeSO4, FeCl3, KH2PO4, K2HPO4, MgSO4, ZnSO4, NaCl, CaCl2, KCl, ZnCl2, and AlCl3.6H2O) for their significances on riboflavin production by Gluconobacter oxydans strain FBFS97. The most significant variables affecting on riboflavin production are K2HPO4 and CaCl2, the interaction affects and levels of these variables were optimized by CCD. After optimization of the medium compositions for riboflavin production were determined as follows: fructose 25 g/L, tryptone 12.5 g/L, K2HPO4 9 g/L, and CaCl2 0.06 g/L with maximum riboflavin production 23.24 mg/L.

Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis.

Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine–Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.

Molecular Elucidation of Riboflavin Production and Regulation in Candida albicans, toward a Novel Antifungal Drug Target.

Candida albicans is a major cause of fungal infections, both superficial and invasive. The economic costs as well as consequences for patient welfare are substantial. Only a few treatment options are available due to the high resemblance between fungal targets and host molecules, as both are eukaryotes. Riboflavin is a yellow pigment, also termed vitamin B2 Unlike animals, fungi can synthesize this essential component themselves, thereby leading us to appreciate that targeting riboflavin production is a promising novel strategy against fungal infections. Here, we report that the GTP cyclohydrolase encoded by C. albicansRIB1 (CaRIB1) is essential and rate-limiting for production of riboflavin in the fungal pathogen. We confirm the high potential of CaRib1 as an antifungal drug target, as its deletion completely impairs in vivo infectibility by C. albicans in model systems. Furthermore, the stimulating effect of iron deprivation and PKA activation on riboflavin production seems to involve CaRib1 and the upstream transcription factor CaSef1. Gathering insights in the synthesis mechanism of riboflavin in pathogenic fungi, like C. albicans, will allow us to design a novel strategy and specifically target this process to combat fungal infections.IMPORTANCECandida albicans is an important fungal pathogen causing common superficial infections as well as invasive diseases with an extremely high morbidity and mortality. Antifungal therapies are limited in efficiency and availability. In this research, we describe the regulation of riboflavin production in C. albicans Since riboflavin biosynthesis is essential to this organism, we can appreciate that targeting it would be a promising new strategy to combat these fungal infections. We provide evidence that one particular enzyme in the production process, CaRib1, would be most promising as an antifungal drug target, as it plays a central role in regulation and proves to be essential in a mouse model of systemic infection.

Effect of Inorganic Nitrogen on Riboflavin Production by Fed-Batch Culture of Bacillus subtilis

Effect of Inorganic Nitrogen on Riboflavin Production by Fed-Batch Culture of Bacillus subtilis

Dataset for supporting a modular autoinduction device for control of gene expression in Bacillus subtilis.

Modular and tuneable genetic tools for Metabolic Engineering fuels the development of chassis for the efficient production of biocompounds at industrial scale. We have constructed an autoinduction device for gene expression in Bacillus subtilis based on the LuxR/I quorum sensing system [1]. Here, we present raw and processed data regarding to B. subtilis growth measured as OD600, performed in three different scales: microcultivation on 96-well plates (200 µL), test tubes (12 mL), and Erlenmeyer flasks (50 mL). We also present raw and processed data on gene expression measured as GFP fluorescence (485/535 nm), luminescence and riboflavin production. Measurements were performed on a microplate reader Tecan 200 PRO (iControl software) and on spectrophotometer (Thermo Fisher Scientific GENESYS 10S UV-Vis). Processed data are presented as product/OD600, maximum and minimum promoter activity, fold of induction, and the induction OD600.

Rational engineering of transcriptional riboswitches leads to enhanced metabolite levels in Bacillus subtilis

Many metabolic pathways in bacteria are regulated by metabolite sensing riboswitches that exert their control at the level of transcription employing a termination-antitermination mechanism. These riboswitches represent engineering targets to modulate expression of genes and operons relevant for the biotechnological production of commercially relevant compounds. We show that removal of the transcriptional riboswitches that control purine biosynthesis and riboflavin biosynthesis in Bacillus subtilis leads to auxotrophic strains. As an alternative, we report a rational approach for engineering transcriptional riboswitches independently from the availability of structural data. This approach consists in the identification and deletion of a key nucleotide sequence exclusively involved in transcription termination without affecting formation of other secondary and tertiary structures, which can be involved in other functions. To demonstrate the efficacy of our approach, we tested it with regard to deregulation of the purine and the riboflavin biosynthetic pathways in B. subtilis. Following validation of the engineered transcriptional riboswitches using specialized reporter strains, our approach was implemented into a B. subtilis wild-type strain employing CRISPR-Cas9 genome editing. The resulting purine and riboflavin production strains were characterized at the level of gene expression, metabolite synthesis and growth, and a substantial enhancement was measured at each level. Moreover, applying our approach to deregulate the purine pathway of an industrial riboflavin overproducing strain with impaired growth led to an increase in biomass by 53%, which resulted in an enhanced total production of riboflavin in the culture.

Genomic analysis of a riboflavin-overproducing Ashbya gossypii mutant isolated by disparity mutagenesis

BackgroundAshbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out.ResultsIn the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process.ConclusionThis study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.

Transforming Candida hispaniensis, a promising oleaginous and flavogenic yeast.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity.

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata.

Riboflavin (vitamin B2 ) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production.

SummaryThe potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l−1). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l−1 after 12‐h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high‐temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

Rediverting Electron Flux with an Engineered CRISPR-ddAsCpf1 System to Enhance the Pollutant Degradation Capacity of Shewanella oneidensis.

Pursuing efficient approaches to promote the extracellular electron transfer (EET) of extracellular respiratory bacteria is essential to their application in environmental remediation and waste treatment. Here, we report a new strategy of tuning electron flux by clustered regularly interspaced short palindromic repeat (CRISPR)-ddAsCpf1-based rediverting (namely STAR) to enhance the EET capacity of Shewanella oneidensis MR-1, a model extracellular respiratory bacterium widely present in the environment. The developed CRISPR-ddAsCpf1 system enabled approximately 100% gene repression with the green fluorescent protein (GFP) as a reporter. Using a WO3 probe, 10 representative genes encoding for putative competitive electron transfer proteins were screened, among which 7 genes were identified as valid targets for EET enhancement. Repressing the valid genes not only increased the transcription level of the l-lactate metabolism genes but also affected the genes involved in direct and indirect EET. Increased riboflavin production was also observed. The feasibility of this strategy to enhance the bioreduction of methyl orange, an organic pollutant, and chromium, a typical heavy metal, was demonstrated. This work implies a great potential of the STAR strategy with the CIRPSR-ddAsCpf1 system for enhancing bacterial EET to favor more efficient environmental remediation applications.

Cell Wall Composition of the Riboflavin Over Producer Eremotheciumashbyii

Eremothecium ashbyii is a filamentous hemiascomycete fungus very closely related to the yeast, Saccharomyces cerevisiae and is a natural overproducer of riboflavin (vitamin B2). An interesting observation is that the riboflavin accumulation is not uniform in all the hyphal cells. Some cells accumulate more than others and some cells do not accumulate riboflavin at all suggesting some kind of regulation at the subcellular level/ developmental level. Riboflavin is not toxic to humans but for fungi and yeast it is toxic, causing cytotoxicity of the cells, damaging DNA, RNA and proteins. Considering the cytotoxic effect, the overproduction of riboflavin by Eremothecium ashbyii raises many questions. The main question is, whether riboflavin overproducers benefit from this property. Cell walls are essential to fungal growth and survival. The rigidity of the fungal cell well determines the shape of the fungal cell and also provides protection from many environmental stresses. Recent studies have revealed that the fungal cell wall plays a regulatory role in response to positive and negative environmental stimuli. In the case of Eremothecium ashbyii significant lacunae exist in our understanding of cellular processes such as vesicle trafficking, cytoskeletal organization and its regulation. These processes are vital with regard to riboflavin production and secretion. Hence the work reported here was undertaken with an aim to gain a preliminary understanding of the cell wall composition of Eremothecium ashbyii and thus gain a better insight into its development and how it affects riboflavin synthesis/storage/ transport.

Screening and Spontaneous Mutation of Pickle-Derived Lactobacillus plantarum with Overproduction of Riboflavin, Related Mechanism, and Food Application.

Riboflavin, also known as vitamin B2, plays an important role in human cell metabolism and participates in various redox reactions and in energy utilization. In this study, 90 riboflavin-producing lactic acid bacteria (LAB) were screened out from pickle juices. The yields of riboflavin in these LAB were about 0.096–0.700 mg/L, and one strain, Lactobacillus plantarum RYG-YYG-9049, was found to produce the highest riboflavin content. Next, roseoflavin was used to induce the spontaneous mutation of RYG-YYG-9049, and selected roseoflavin-resistant colonies generally produced higher riboflavin contents, ranging from 1.013 to 2.332 mg/L. The No. 10 mutant, L. plantarum RYG-YYG-9049-M10, had the highest riboflavin content. Next, the molecular mechanism of enhancing riboflavin production in RYG-YYG-9049-M10 was explored, leading to the finding that roseoflavin treatment did not change the rib operons including the ribA, ribB, ribC, ribH, and ribG genes. Unexpectedly, however, this mechanism did induce an insertion of a 1059-bp DNA fragment in the upstream regulatory region of the rib operon, as compared to the wild-type RYG-YYG-9049. To the best of our knowledge, this is the first report that roseoflavin could induce an insertion of DNA fragment in LAB to increase riboflavin content, representing a new mutation type that is induced by roseoflavin. Finally, in order to fortify riboflavin content in soymilk, RYG-YYG-9049 and RYG-YYG-9049-M10 were used to ferment soymilk, and several fermentation parameters were optimized to obtain the fermented soymilk with riboflavin contents of up to 2.920 mg/L. In general, roseoflavin induction is an economical and feasible biotechnological strategy to induce riboflavin-overproducing LAB, and this strategy can be used to develop LAB-fermented functional foods that are rich in riboflavin.

Mechanistic investigations in ultrasound-induced intensification of fermentative riboflavin production

This paper reports mechanistic studies in ultrasound-assisted riboflavin production by wild strain of D. hansenii var. hansenii. Statistical optimization of fermentation resulted in riboflavin yield of 1.8 mg/g DCW. Supplementation of medium with glycine, Fe3+, cobalt and zinc further enhanced yield to 4.1 mg/g DCW. Intensification of fermentation by sonication (at 10% duty cycle) in four intervals of 12 h each (in total log phase of 48 h) was attempted. Sonication in final 12 h of log phase was most effective with the highest yield of 20 mg/g DCW. Kinetic analysis of the ultrasound-assisted fermentation revealed higher maximum specific growth rate of cells (0.30 h−1) with reduced Monod constant (5.67 g/L), indicating faster cell transport and higher substrate affinity. SDS-PAGE analysis of proteins involved in riboflavin metabolism showed overexpression of all proteins in ultrasound-assisted fermentation, which enhanced the metabolism. These results are attributed to intense microturbulence generated by sonication.