Genetic engineering of Bacillus subtilis for the commercial production of riboflavin

Recombinant DNA engineering was combined with mutant selection and fermentation improvement to develop a strain of Bacillus subtilis that produces commercially attractive levels of riboflavin. The B. subtilis riboflavin production strain contains multiple copies of a modified B. subtilis riboflavin biosynthetic operon (rib operon) integrated at two different sites in the B. subtilis chromosome. The modified rib operons are expressed constitutively from strong phage promoters located at the 5′ end and in an internal region of the operon. The engineered strain also contains purine analog-resistant mutations designed to deregulate the purine pathway (GTP is the precursor for riboflavin), and a riboflavin analog-resistant mutation in ribC that deregulates the riboflavin biosynthetic pathway. Received 22 June 1998/ Accepted in revised form 6 November 1998     

Commercial riboflavin production by recombinant Bacillus subtilis: down-stream processing and comparison of the composition of riboflavin produced by fermentation or chemical synthesis

A novel process for riboflavin production using a recombinant Bacillus subtilis strain has been developed. Here we describe a down-stream processing procedure to obtain riboflavin qualities having a minimal content of 96% (‘feed-grade’) and 98% (‘food/pharma-grade’) riboflavin, respectively. Compared to riboflavin produced by chemical synthesis, products with improved chemical purity were obtained. All compounds representing more than 0.1% of the final products were identified. Feed-grade riboflavin material ex fermentation contained small amounts of amino acids and amino sugars and the biosynthetic riboflavin precursor dimethyl-ribityl-lumazine. All other side products found were derived from riboflavin, resulted from the purification procedure and were also found in riboflavin obtained by chemical synthesis. The Bacillus-produced riboflavin does not contain DNA. The data presented here were used to obtain product approval for the commercial application in the USA, Japan and the UK. Received 22 July 1998/ Accepted in revised form 8 November 1998     

Kinetic modeling of riboflavin biosynthesis in Bacillus subtilis under production conditions

To study the network dynamics of the riboflavin biosynthesis pathway and to identify potential bottlenecks in the system, an ordinary differential equation-based model was constructed using available literature data for production strains. The results confirmed that the RibA protein is rate limiting in the pathway. Under the conditions investigated, we determined a potential limiting order of the remaining enzymes under increased RibA concentration (>0.102 mM) and therefore higher riboflavin production (>0.045 mmol g _CDW ^?1 h^?1 and 0.0035 mM s^?1, respectively). The reductase activity of RibG and lumazine synthase (RibH) might be the next most limiting steps. The computational minimization of the enzyme concentrations of the pathway suggested the need for a greater RibH concentration (0.251 mM) compared with the other enzymes (RibG: 0.188 mM, RibB: 0.023 mM).     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production

The 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.     

Isolation of an oxalate-resistant Ashbya gossypii strain and its improved riboflavin production

An oxalate-resistant strain of Ashbya gossypii was naturally isolated from spores grown on an oxalate-containing medium, and its medium was optimized to improve riboflavin production. Riboflavin production by the resistant strain was three-fold higher than that by the wild-type organism when grown in flask cultures. Medium optimization increased the riboflavin production by the resistant strain to 5 g l⁻¹, which was five-fold higher than that obtained by the wild-type strain. The productivity was reproduced in a 3-l bioreactor. During the early growth phase, the specific activity of isocitrate lyase in the oxalate-resistant strain was slightly higher than that in the wild-type strain. Proteomic analysis of the oxalate-resistant strain revealed that the expression of aldose reductase and cobalamin-independent methionine synthase decreased significantly. This is the first report that describes the natural isolation of a riboflavin producer using an antimetabolite-containing medium to enhance the riboflavin production level. This method should also be useful for improving the productivity of other bioproducts since it does not require any mutations or genetic modifications of the microorganism.     

Importance of malate synthase in the glyoxylate cycle of <Emphasis Type="Italic">Ashbya gossypii</Emphasis> for the efficient production of riboflavin

The glyoxylate cycle is an anabolic pathway that is necessary for growth on nonfermentable carbon sources such as vegetable oils and is important for riboflavin production by the filamentous fungus Ashbya gossypii. The aim of this study was to identify malate synthase in the glyoxylate cycle of A. gossypii and to investigate its importance in riboflavin production from rapeseed oil. The ACR268C gene was identified as the malate synthase gene that encoded functional malate synthase in the glyoxylate cycle. The ACR268C gene knockout mutant lost malate synthase activity, and its riboflavin production and oil consumption were 10- and 2-fold lower, respectively, than the values of the wild-type strain. In contrast, the ACR268C gene-overexpressing strain showed a 1.6-fold increase in the malate synthase activity and 1.7-fold higher riboflavin production than the control strain. These results demonstrate that the malate synthase in the glyoxylate cycle has an important role not only in riboflavin production but also in oil consumption.     

Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety

In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of [1′-¹⁴C]riboflavin. The vitamin B₁₂ isolated was ¹⁴C-labeled. It was shown by chemical degradation that the ¹⁴C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety. This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S. typhimurium. Received: 25 August 1998 / Accepted: 27 October 1998     

Metaphosphate-dependent phosphorylation of riboflavin to FMN by Corynebacterium ammoniagenes

Using the bifunctional FAD synthetase from Corynebacterium ammoniagenes, which has the two sequential activities of flavokinase and FMN adenylyl-transferase in FAD biosynthesis, a method of production of the intermediate FMN without any accumulation of FAD was investigated. Various phosphate polymers having no adenylyl moiety were tested for their ability to phosphorylate riboflavin to FMN, using a crude enzyme from C. ammoniagenes/pKH46, which is an FAD-synthetase-gene-dosed strain. Only metaphosphate, other than ATP, could phosphorylate riboflavin to FMN, but FAD did not accumulate at all. The conditions for the conversion of riboflavin to FMN were optimized. The metaphosphate-dependent phosphorylation reaction required Mg²⁺ as the most effective divalent cation. The best concentrations were 10 mM for MgCl₂ and 3mg/ml for metaphosphate. The riboflavin added to the reaction mixture was almost completely converted into FMN after 6 h incubation in the presence of high concentrations of the enzyme preparation.     

A developmental stage of hyphal cells shows riboflavin overproduction instead of sporulation in Ashbya gossypii

The hemiascomycete Ashbya gossypii develops a mycelium. Nutritional stress leads to its differentiation into sporangia. These generate spores. In parallel, the yellow pigment riboflavin is produced. Intracellularly accumulated riboflavin, made visible as a bright green fluorescence, was observed in only 60 % of the hyphal cells. For the remaining 40 %, it was unclear whether these cells simply export riboflavin or its biosynthesis remains down-regulated in contrast to the accumulating cells. The approach followed in this work was to convert the hyphae into protoplasts by enzymatic degradation of the cell wall. Afterwards, the protoplasts were sorted by fluorescence-activated cell sorting on the basis of riboflavin accumulation. When a reporter strain expressing lacZ under the control of the most important riboflavin biosynthesis promoter, RIB3, was used, green protoplasts were found to have more than tenfold greater reporter activity than hyaline protoplasts. This was true on the basis of total protein as well as on the basis of hexokinase specific activity, a marker for constitutive expression. These results allow the conclusion that hyphal cells of A. gossypii differ in phenotype regarding riboflavin overproduction and accumulation.     

The improvement of riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> via disparity mutagenesis and DNA microarray analysis

We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin. By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites. One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon flux from β-oxidation to the riboflavin biosynthetic pathway.     

Growth inhibition ofMicrococcus pyogenes by manganese and riboflavin

Summary Riboflavin, in the presence of manganese, inhibited the growth ofMicrococcus pyogenes var.aureus. The inhibitory action was shown to be dependent upon aerobiosis, and was characterized predominately as a prolongation of the lag phase. Increasing concentrations of either manaanese or riboflavin, in the presence of a constant quantity of the other, enhanced the inhibitory effect. Ultra-violet irradiation of riboflavin reduced its toxic capacity in proportion to the amount of riboflavin degraded. Riboflavin-5-phosphate was equally as inhibitory as riboflavin. Ferrous iron was capable of a non-stoichiometric antagonism at low concentrations of riboflavin and/or manganese. Suported, in part, by a research grant (E-605) from the National Institutes of Health, U.S. Public Health Service. A preliminary report has appeared (Ramsey andWilson, 1956).     

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B₂) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-l-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3–3.5 times higher as compared to the parental strain. It produced 50–70 times more riboflavin in iron sufficient synthetic media relative to the parental wild-type strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.     

Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the Gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity. Received: 19 November 1999 / Accepted: 21 December 1999     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L⁻¹ in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.     

Correlation between riboflavin carrier protein induction and its mRNA activity in estrogen stimulated chicken liver and oviduct

Poly A enriched RNA from either liver or oviduct of estradiol-17β treated immature chicks supported [³H]-leucine incorporation into immunoprecipitable riboflavin carrier protein in a dose-dependent manner when translated in the rabbit reticulocyte lysate system. Primary translation product of riboflavin carrier protein had a molecular weight of 38,000 which on incubation with a stripped hepatic microsomal preparation was processed to a product with a size comparable to native riboflavin carrier protein. Poly A enriched RNA from both the liver and the oviduct of estrogen-treated birds stimulated [³H]-leucine incorporation into riboflavin carrier protein and this was 2–3 fold higher during secondary stimulationvis-a-vis primary stimulation with the steroid. Poly A enriched RNA from the liver of progesteronetreated birds during secondary stimulation did not support riboflavin carrier protein synthesis. In contrast, poly A enriched RNA from the oviduct of the birds treated with progesterone during secondary (but not primary) stimulation did exhibit riboflavin carrier protein-mRNA activity which was comparable to that stimulated by estradiol-17β