Cloning of structural genes involved in riboflavin synthesis of the yeast Candida famata

The riboflavin overproducing mutants of the flavinogenic yeast Candida famata isolated by conventional selection methods are used for the industrial production of vitamin B2. Recently, a transformation system was developed for C. famata using the leu2 mutant as a recipient strain and Saccharomyces cerevislae LEU2 gene as a selective marker. In this paper the cloning of C. famata genes for riboflavin synthesis on the basis of developed transformation system for this yeast species is described. Riboflavin autotrophic mutants were isolated from a previously selected C. famata leu2 strain. C. famata genomic DNA library was constructed and used for cloning of the corresponding structural genes for riboflavin synthesis by complementation of the growth defects on a medium without leucine and riboflavin. As a result, the DNA fragments harboring genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding GTP cyclohydrolase, reductase, dimethylribityllumazine synthase, dihydroxybutanone phosphate synthase and riboflavin synthase, were isolated and subsequently subcloned to the smallest possible fragments. The plasmids with these genes successfully complemented riboflavin auxotrophies of the corresponding mutants of another flavinogenic yeast Pichia guilliermondii. This suggested that C. famata structural genes for riboflavin synthesis and not some of the supressor genes were cloned.     

Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B₂ was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B₂ higher than 1 mg L^?1. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B₂ content.     

Multivitamin production in Lactococcus lactis using metabolic engineering

The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).     

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     

Screening of Bacillus subtilis transposon mutants with altered riboflavin production

To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

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.     

Optimized conditions for high erythritol production by<Emphasis Type="Italic">Penicillium</Emphasis> sp. KJ-UV29, mutant of<Emphasis Type="Italic">Penicillium</Emphasis> sp. KJ81

To improve the erythritol productivity ofPenicillium sp. KJ81, mutants were obtained using UV irradiation and NTG treatment. Among these mutants,Penicillium sp. KJ-UV29 revealed no morphological changes, yet was superior to the wild strain in the following three points: (1)Penicillium sp. KJ-UV29 produced more erythritol than the wild strain under the same conditions, (2) no foam was produced during cultivation, unlike the wild strain, and (3) the mutant produced a significantly lower amount of glycerol.Penicillium sp KJ-UV29 produced as much as 15.1 g/L of erythritol, whereas the wild-typePenicillium sp. KJ-UV29 produced as much as 15.1 g/L of erythritol, whereas the wild-typePenicillium sp. KJ81 only produced 11.7 g/L.Penicillium sp. KJ-UV29 only generated 6.1 g/L of glycerol, compared to 19.4 g/L produced by the wild strain. When investigating the optimal culture conditions for erythritol production by the mutant strainPenicillium sp. KJ-UV29, sucrose was idetified as the most effective carbon source, and the mutant was even able to produce erythritol in a 70% sucrose-containing medium, although a 30% sucrose medium exhibited the highest productivity. The production of erythritol byPenicillium sp. KJ-UV29 was also significantly increased by the addition of ammonium carbonate, potassium nitrate, and sodium nitrate. Accordingly, under optimal conditions,Penicillium sp. KJ-UV29 produced 45.2 g/L of erythritol in a medium containing 30% sucrose, 0.5% yeast extract, 0.5% (NH₄)₂C₂O₄ 0.1% NaNO₃, and 0.01% FeSO₄ with 1 vvm aeration and 200 rpm agitation at 37°C for 7 days in a 5-L jar fermentor.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Improved phloroglucinol production by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.     

Genome shuffling of marine derived bacterium <Emphasis Type="Italic">Nocardia</Emphasis> sp. ALAA 2000 for improved ayamycin production

Genome shuffling is a recent development in microbiology. The advantage of this technique is that genetic changes can be made in a microorganism without knowing its genetic background. Genome shuffling was applied to the marine derived bacterium Nocardia sp. ALAA 2000 to achieve rapid improvement of ayamycin production. The initial mutant population was generated by treatment with ethyl methane sulfonate (EMS) combined with UV irradiation of the spores, resulting in an improved population (AL/11, AL/136, AL/213 and AL/277) producing tenfold (150 μg/ml) more ayamycin than the original strain. These mutants were used as the starting strains for three rounds of genome shuffling and after each round improved strains were screened and selected based on their ayamycin productivity. The population after three rounds of genome shuffling exhibited an improved ayamycin yield. Strain F3/22 yielded 285 μg/ml of ayamycin, which was 19-fold higher than that of the initial strain and 1.9-fold higher than the mutants used as the starting point for genome shuffling. We evaluated the genetic effect of UV + EMS-mutagenesis and three rounds of genome shuffling on the nucleotide sequence by random amplified polymorphic DNA (RAPD) analysis. Many differences were noticed in mutant and recombinant strains compared to the wild type strain. These differences in RAPD profiles confirmed the presence of genetic variations in the Nocardia genome after mutagenesis and genome shuffling.     

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     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process

The aim of the present study was to investigate the production of 1,3-propanediol (PDO) under non-sterile fermentation conditions by employing the strain Clostridium butyricum VPI 1718. A series of batch cultures were performed by utilizing biodiesel-derived crude glycerol feedstocks of different origins as the sole carbon source, in various initial concentrations. The strain presented similarities in terms of PDO production when cultivated on crude glycerol of various origins, with final concentrations ranging between 11.1 and 11.5 g/L. Moreover, PDO fermentation was successfully concluded regardless of the initial crude glycerol concentration imposed (from 20 to 80 g/L), accompanied by sufficient PDO production yields (0.52–0.55 g per gram of glycerol consumed). During fed-batch operation under non-sterile culture conditions, 67.9 g/L of PDO were finally produced, with a yield of 0.55 g/g. Additionally, the sustainability of the bioprocess during a continuous operation was tested; indeed, the system was able to run at steady state for 16 days, during which PDO effluent level was 13.9 g/L. Furthermore, possible existence of a microbial community inside the chemostat was evaluated by operating a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, and DGGE results revealed the presence of only one band corresponding to that of C. butyricum VPI 1718. Finally, non-sterile continuous cultures were carried out at different dilution rates (D), with inlet glycerol concentration at 80 g/L. Maximum PDO production was achieved at low D values (0.02 h⁻¹) corresponding to 30.1 g/L, while the elaboration of kinetic data from continuous cultures revealed the stability of the bioprocess proposed, with global PDO production yield corresponding to 0.52 g/g.     

Improvement of Lactobacillus brevis NM101-1 grown on sugarcane molasses for mannitol,lactic and acetic acid production

The conversion of sugarcane molasses for the production of lactic acid, acetic acid, and mannitol was enhanced by subjecting Lactobacillus brevis NM101-1 wild strain to various doses of gamma irradiation. Four mutants (LM-1-LM-4) obtained at gamma ray doses of 30, 60, 90, and 120 Gy produced higher levels of lactic acid, acetic acid, and mannitol than the wild-type. Among all the mutants tested, LM-3 strain showed the highest mannitol and acetic acid production which reached 198.95 and 96.86 g/l, respectively. On the other hand, mutant LM-1strain exhibited the best performance with respect to lactic acid production (143.73 g/l). Random amplified polymorphic DNA polymerase chain reaction technique (RAPD-PCR) using three primers (RP, R5, and M13) was used in order to detect the variation in DNA profile in response to gamma irradiation treatments. RAPD analysis indicated the appearance and disappearance of DNA polymorphic bands at different gamma ray doses. The results showed the potential of these mutants to be potential candidates for economical production of mannitol, lactic and acetic acids from molasses on a commercial scale.