Riboflavin Production Research Articles

Growth stress triggers riboflavin overproduction in Ashbya gossypii

The filamentous fungus Ashbya gossypii is used for riboflavin biosynthesis on an industrial scale, but even the wild type displays overproduction. Because riboflavin overproduction was known to start at the transition between growth and stationary phase, it was suspected that overproduction was induced at low growth rates. However, chemostatic cultivations performed at different growth rates did not result in any detectable riboflavin formation. In this study, we report that it was not the final growth rate that triggered riboflavin overproduction but a decline in growth rate. Therefore, continuous fermenter cultivations with dilution rate shifts were performed. Peaks of riboflavin overproduction were observed in the wild type and in a RIB3placZ reporter strain after downshifts in dilution rate. Accumulation of riboflavin correlated with an increased expression of lacZ reporter activity. The step size of the downshifts corresponded to the peak size of riboflavin formation and reporter activity. Expression of further RIB genes encoding riboflavin biosynthetic enzymes was analyzed by RT-PCR. RIB mRNA levels of the ribulose-5-phosphate branch of the divided riboflavin biosynthesis pathway (RIB3, RIB4, and RIB5) were found to increase in the riboflavin production phase, whereas the RIB2 and RIB7 mRNA levels belonging to the GTP branch remained constant. We propose that a decline in growth rate triggers the increased expression of RIB3, RIB4, and RIB5 resulting in riboflavin overproduction. Because although a reduction in oxygen supply, temperature increase or decrease, or salt stress did affect growth, but neither did lead to riboflavin overproduction nor did induce RIB3 reporter expression, we conclude that declining nutrition must be the stress stimulus. Because about half of the cells in the hyphae of Ashbya gossypii did not accumulate riboflavin, the regulatory response on the cellular level can be estimated to be at least twice as great in comparison to what we detected as overall signals.

Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs

A sequential optimization strategy, based on statistical experimental designs, was used to enhance the production of riboflavin by recombinant Bacillus subtilis RH44. In the first instance, the medium components were optimized in shake flask cultures. After preliminary experiments of nitrogen source selection, the two-level Plackett-Burman (PB) design was implemented to screen medium components that significantly influence riboflavin production. Among the 15 variables tested, glucose, NaNO(3), K(2)HPO(4), ZnSO(4), and MnCl(2) were identified as the most significant factors (confidence levels above 95%) for riboflavin production. The optimal values of these five variables were determined by response surface methodology (RSM) based on the central composite design (CCD). The validity of the model developed was verified, and the optimum medium led to a maximum riboflavin concentration of 6.65 g/l, which was 44.3 and 76.4% higher than the improved medium and the basal medium, respectively. A glucose-limited fed-batch culture profile in a 5-l fermentor was consequently designed according to the above optimum medium in shake flasks. A final riboflavin concentration of 16.36 g/l was obtained in 48 h, which further verified the practicability of this optimum strategy.

Riboflavin production from mutants ofAshbya gossypii utilising orange rind as a substrate

The use of agroindustrial wastes such as orange rind as an alternative in the production of riboflavin was evaluated in this study withAshbya gossypii mutants.Ashbya gossypii mutants were obtained using 300 μg/mL of MNNG after an exposition period of 90 min with 2.3% of survival rate. A total of 11 mutant high yield strains of riboflavin were selected. Of these mutants, the most productive in YM medium were ASHLVII, ASHLX, ASHLXI and ASHLXV. Three additional different mutants were shown to be unsusceptible to inhibition by itaconate in the medium. When we used orange rind at 0.3% in YM medium without malt extract, the rate of riboflavin production was enhanced in the mutant strains producing 223 mg/L of this vitamin, an increase of 184% in the ASHLXI and ASHLXV mutants, as compared with the wild strain.

Isocitrate dehydrogenase and isocitrate lyase are essential enzymes for riboflavin production inAshbya gossypii

For this study, we hypothesized that mitochondrial NAD+-dependent isocitrate dehydrogenase 1 (ICDH1) and isocitrate lyase (ICL1) were important enzymes for riboflavin synthesis in the fungusAshbya gossypii. Here, the genes encoding ICDH1 and ICL1 were disrupted in order to analyze the enzymes' functions on riboflavin production by the fungus. The riboflavin production resulting from these disruptants was markedly decreased compared to the concentration produced by its parental strain when cultured in a rich nutrient medium used to optimize riboflavin production. Furthermore, when comparing the transcription levels of the genes encoding ICDH1 and ICL1, between wild-typeA. gossypii and an itaconate resistant mutant ofA. gossypii obtained by UV irradiation, the mRNA levels in the mutant were 1.8- and 2.0-fold higher than those in the wild-type strain, respectively. These results indicate that ICDH1 and ICL1 are key enzymes for riboflavin synthesis inA. gossypii.

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology

To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.

Enhancement of riboflavin production by overexpression of acetolactate synthase in aptamutant ofBacillus subtilis

Genetic alterations of carbon flux into the acetoin biosynthesis pathway as a possible means to reduce acid accumulation were investigated in the riboflavin-producing Bacillus subtilis during growth on glucose. The lower rates of cell growth and riboflavin production were found in the pta-disrupted mutant while the rate of acetate formation was reduced. In contrast, acid accumulation was significantly reduced, to one-fifth that of the parental strain RH33::[pRB63](n), and a 50% increase in the riboflavin yield was obtained when the expression of the gene encoding acetolactate synthase was increased in the pta-disrupted mutant. Metabolic analysis, together with enzyme activity assays, indicated that the tricarboxylic acid cycle fluxes are significantly increased in response to acetolactate synthase overexpression in pta-disrupted mutant. Moreover, the intracellular ATP-to-ADP ratio also increased 5.8-fold. The high concentration of ATP could explain the increased riboflavin production.

Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis

The establishment and the improvement of industrial bioprocesses calls for the selection of media compositions and process conditions in highly parallel experiments as well as for the intensified screening of new biocatalysts and improved production strains. This work presents for the first time the scale-down and the successful adaptation of an industrial riboflavin fed-batch production process with Bacillus subtilis to a fully automated setup with 48 parallel stirred bioreactors at a milliliter scale (10 mL). The feasibility of an intermittent feeding mode and a discontinuous at-line pH control for parallel cultivations over up to 53 h is demonstrated together with interlaced process analyses at a microliter scale for quasi-simultaneous at-line monitoring of biomass, substrate and product concentration. The discontinuous feeding mode necessitated an increased oxygen input, resulting in lower final biomass concentrations. However, the product yields and volumetric productivities in the milliliter setup were equivalent to the yields and productivities obtained during the reference cultivations at laboratory scale, which allows considering the automated system together with the developed schedule as a screening tool for high-throughput bioprocess design of the described production process.

Intensification of Rates and Selectivity Using Tri-liquid versus Bi-liquid Phase Transfer Catalysis: Insight into Reduction of 4-Nitro-o-xylene with Sodium Sulfide

Reactions in three immiscible liquid phases are attractive, and one of the phases can be the locale of the reaction, which will have a dramatic effect on the product distribution in complex reactions. Thus, converting a bi-liquid (L−L) system into tri-liquid (L−L−L) phases is of considerable scientific and commercial interest. Such systems encounter mass-transfer resistances for transfer across two interfaces and the kinetic analysis becomes difficult. In the case of phase transfer catalysis (PTC), a majority of preparatory and industrial reactions are conducted in two phases. The L−L PTC reactions are conducted under milder conditions, using less-expensive solvents at much faster reaction rates and improved selectivities to desired products. However, the phase transfer catalyst is not recovered but treated as waste, because the quantities are very small and do not contribute much to the expensive product. L−L PTC can be elegantly modified to convert it to L−L−L PTC, to recover and reuse the catalyst and also to enhance selectivity, thereby improving profitability and environmental benefits. 3,4-Dimethyl aniline is a useful starting material for the production of riboflavin (vitamin B2) and also is used as a precursor for many drugs. It can be produced via the reduction of 4-nitro-o-xylene in toluene with aqueous sodium sulfide, using tetrabutylammonium bromide (TBAB) as a catalyst under L−L−L PTC at 80 °C. Comparison between tri- and bi-liquid phases was also conducted, and it was observed that, under L−L−L conditions, the rates of reaction of the formation of 3,4-dimethyl aniline had been dramatically enhanced. The kinetics and mechanism of complex L−L-L PTC processes have been explored in detail. The yield, based on the initial amount of reactant, was >95%, and the selectivity was 100%.

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.

Purine biosynthesis, riboflavin production, and trophic-phase span are controlled by a Myb-related transcription factor in the fungus Ashbya gossypii.

Ashbya gossypii is a natural riboflavin overproducer used in the industrial production of the vitamin. We have isolated an insertional mutant exhibiting higher levels of riboflavin production than the wild type. DNA analysis of the targeted locus in the mutant strain revealed that a syntenic homolog of the Saccharomyces cerevisiae BAS1 gene, a member of the Myb family of transcription factors, was inactivated. Directed gene disruption of AgBAS1 confirmed the phenotype observed for the insertional mutant, and the Deltabas1 mutant also showed auxotrophy for adenine and several growth defects, such as a delay in the germination of the spores and an abnormally prolonged trophic phase. Additionally, we demonstrate that the DNA-binding domain of AgBas1p is able to bind to the Bas1-binding motifs in the AgADE4 promoter; we also show a clear nuclear localization of a green fluorescent protein-Bas1 fusion protein. Real-time quantitative PCR analyses comparing the wild type and the Deltabas1 mutant revealed that AgBAS1 was responsible for the adenine-mediated regulation of the purine and glycine pathways, since the transcription of the ADE4 and SHM2 genes was virtually abolished in the Deltabas1 mutant. Furthermore, the transcription of ADE4 and SHM2 in the Deltabas1 mutant did not diminish during the transition from the trophic to the productive phase did not diminish, in contrast to what occurred in the wild-type strain. A C-terminal deletion in the AgBAS1 gene, comprising a hypothetical regulatory domain, caused constitutive activation of the purine and glycine pathways, enhanced riboflavin overproduction, and prolonged the trophic phase. Taking these results together, we propose that in A. gossypii, AgBAS1 is an important transcription factor that is involved in the regulation of different physiological processes, such as purine and glycine biosynthesis, riboflavin overproduction, and growth.

Knockout of the HprK Gene in Bacillus subtilis CcpA Mutant and Its Influence on Riboflavin Fermentation

In Bacillus subtilis, increasing the carbon catabolite content in vivo would lead to carbon catabolite repression (CCR) and restrain the absorption of glucose. By deleting CcpA, the CCR effect could be alleviated, while the absorption of glucose remains restrained. The phosphoenol-pyruvate (PEP)-sugar phosphotransferase system (PTS) is the main glucose transportation system in B. subtilis. HPr protein together with HprK/P participates in the glucose transportation. The HPr protein is phosphorylated at His-15 resulting in the formation of HPr-His-15-P, and the phosphate group is transferred from HPr to E II. The HprK/P phosphorylates HPr at Ser-46 form HPr-Ser-46-P. HPr-Ser-46-P cannot participate in the transportation of glucose. The knockout of ccpA gene increases the amount of fructose-1, 6-bisphosphate (FBP) in vivo and FBP could activate HPr kinase. So when CcpA is deleted, most part of HPr will be phosphorylated at Ser-46. Therefore, absorption of glucose is blocked. In this study, by disruption of the hprK gene, the obtained B. subtilis ZHc/pMX45 reaches the peak riboflavin production of 4.374 mg/mL at the optimum glucose concentration of 10%, 19.2% higher than that of B. subtilis 24A1/pMX45 at the optimum glucose concentration of 8%.

Biomass recycling from a riboflavin cultivation withB. subtilis: Lysis, extract production and testing as substrate in riboflavin cultivation

Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5-7.5. At a temperature of 40 degrees C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract.

Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid310, lysine333, and alanine417) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

Characterization of d-ribose biosynthesis in Bacillus subtilis JY200 deficient in transketolase gene

d-Ribose is a functional five-carbon sugar, which has been used for the commercial production of riboflavin. Mechanisms of d-ribose biosynthesis from xylose were investigated in the genetically engineered Bacillus subtilis JY200 with a deficiency in transketolase. A transketolase gene ( tkt) disruption cassette in plasmid pUNKC was introduced into the chromosomal tkt gene in the wild type B. subtilis 168. Analysis of culture broth by thin layer chromatography confirmed that the disruption of tkt allowed B. subtilis JY200 to produce d-ribose. In a batch culture of B. subtilis JY200, a loss of cell viability was observed after glucose depletion. Fed-batch cultivation by feeding 400 g l −1 glucose solution as a co-substrate was carried out to supply energy to xylose metabolism and to maintain cell viability throughout cultivation. Fed-batch cultivation of B. subtilis JY200 in a complex medium containing 11 g l −1 xylose and 5 g l −1 glucose initially gave the best result of 10.1 g l −1 d-ribose concentration, 0.24 g g −1 d-ribose yield and 0.29 g l −1 h −1 productivity, corresponding to 40-, 5- and 12-fold increases compared with those in the batch culture. A kinetic study of d-ribose production in fed-batch cultivations of B. subtilis JY200 suggested that xylose uptake might be critical to maximize d-ribose biosynthesis from xylose.

Positive selection of mutants defective in transcriptional repression of riboflavin synthesis by iron in the flavinogenic yeast

It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.

Scale Down of the Industrial Riboflavin Production Process for High‐throughput Bioprocess Design

Scale Down of the Industrial Riboflavin Production Process for High‐throughput Bioprocess Design

Effect of low frequency ultrasonic stimulation on the secretion of siboflavin produced by Ecemothecium Ashbyii

The structure of the cell membrane of Ecemothecium ashbyii is considered to be one of the main obstacles in the improvement of riboflavin productivity, which impedes the release of riboflavin from the cell into the fermentation broth. The results of the present study show that stimulation on growth and riboflavin biosynthesis phase, the content of riboflavin was most obviously enhanced, as compared with the control and the total riboflavin quantity released by ultrasonic treatment. The optimal stimulation time was from 104 to 112 h and ultrasonic should be loaded every 1.5 h.

Transient expression and flux changes during a shift from high to low riboflavin production in continuous cultures ofBacillus subtilis

At the onset of glucose-limited continuous cultures, riboflavin production in recombinant Bacillus subtilis declines significantly within 3 generations. This phenomenon was specific to riboflavin production and was not correlated with any other physiological parameter. Physiological analyses excluded genetic degeneration or co-metabolism of previously generated overflow metabolites as possible causes for the riboflavin transients. By developing a novel method for (13)C-based metabolic flux analysis under non-steady-state conditions, we showed that the pentose precursors of riboflavin were exclusively synthesized via the non-oxidative pentose-phosphate (PP) pathway as long as riboflavin production was high. The complete redirection of carbon flux to the oxidative branch of the PP pathway was achieved at unaltered PP pathway gene expression and correlated with the declining riboflavin production. With the possible exception of a slight down-regulation of the purine biosynthesis pathway, genome-wide expression analysis indicated that transcriptional regulation was not responsible for the production decline.

Utilization of waste activated bleaching earth containing palm oil in riboflavin production by Ashbya gossypii

AbstractWaste activated bleaching earth (ABE) discharged by an oil refinery plant, which contained 40% palm oil, was used for riboflavin production by a culture of the filamentous fungus Ashbya gossypii. When 188 g/L waste ABE containing 75 g/L palm oil was added to the culture, 80% of the palm oil was consumed after 4 d, and the riboflavin concentration reached 2.1 g/L after 10 d of culture. This concentration was almost 1.5 times higher than for cultures grown on pure palm oil, in which case only 68% of the palm oil was consumed. Before the culture was initiated, the surface of the waste ABE was smooth and resembled clay that was covered with palm oil. After the culture, the oil content decreased to 8%. The black color of waste ABE gradually faded and turned yellow, with the ABE finally forming a yellow powder. Eighty percent of the riboflavin produced during the culture period was transferred to oil‐depleted waste ABE and the waste‐oil‐depleted ABE had 14 mg of riboflavin/g of oil‐depleted waste ABE. The waste ABE containing waste palm oil was suitable for use as a raw material for the production of value‐added riboflavin. Thus, this research might provide a good model for the reuse of waste resources containing vegetable oil.

Oxidation of Rapeseed Oil in Waste Activated Bleaching Earth and Its Effect on Riboflavin Production in Culture of Ashbya gossypii

Long-term behavior of rapeseed oil in waste activated bleaching earth (ABE) and the effect of this oil on riboflavin production in the culture of Ashbya gossypii were investigated. Waste ABE with 40% (w/w) rapeseed oil was stored for 80 d, and the extent of oxidation of rapeseed oil was measured by several analytical methods to determine the chemical properties of the oil at different stages of the oil deterioration process:peroxide value, acid value, concentrations of organic acids, acetaldehyde and unsaturated fatty acid, and content of polymerized triglycerides. Peroxide value, acid value, and concentrations of organic acids and acetaldehyde did not affect riboflavin production. However, the content of polymerized triglycerides markedly increased the viscosity of rapeseed oil and was the main reason for the exponential decrease in riboflavin production. A good correlation between the polymerized triglyceride content or viscosity and riboflavin production in the culture of A. gossypii using rapeseed oil as the sole carbon source was found.