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.     

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.     

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     

Development of a fermentation process based on a defined medium for the production of pregallidermin, a nontoxic precursor of the lantibiotic gallidermin

In this work, a defined medium was developed and optimized for the mutant strain Staphylococcus gallinarum ΔP, which produces pregallidermin (PGDM), a nontoxic precursor of the lantibiotic gallidermin (GDM). The availability of a defined medium is a prerequisite for a rational process development and the investigation of medium effects on final product concentration, yield, and volumetric productivity. We identified four vitamins and three metal ions as essential for growth and PGDM production with S. gallinarum ΔP. The strain was capable of growing without any added amino acids, but the addition of proline had a strong growth-stimulatory effect. The concentrations of all essential compounds were balanced in a continuous culture using a medium-shift technique. Based on this balanced medium, a fed-batch process was developed in which S. gallinarum ΔP was grown up to a biomass concentration of 67 g l⁻¹ and produced 1.95 g l⁻¹ PGDM, equivalent to 0.57 mM. In the fermentation broth, we identified other GDM precursors in addition to those with a 12 or 14-amino-acid-long leader peptide that had been observed previously. Including those precursors with shorter leader sequences, the final concentration would correspond to 0.69 mM. In molar terms, this represents a roughly fourfold or fivefold increase, respectively, over established, complex medium-based gallidermin production processes (Kempf et al. 2000). With the same medium and feed protocol, the maximum concentration of mature GDM produced by wild-type S. gallinarum Tü 3928 was only 0.08 mM.     

Enhanced hydrogen production from glucose using ldh- and frd-inactivated Escherichia coli strains

We improved the hydrogen yield from glucose using a genetically modified Escherichia coli. E. coli strain SR15 (ΔldhA, ΔfrdBC), in which glucose metabolism was directed to pyruvate formate lyase (PFL), was constructed. The hydrogen yield of wild-type strain of 1.08 mol/mol glucose, was enhanced to 1.82 mol/mol glucose in strain SR15. This figure is greater than 90 % of the theoretical hydrogen yield of facultative anaerobes (2.0 mol/mol glucose). Moreover, the specific hydrogen production rate of strain SR15 (13.4 mmol h⁻¹ g⁻¹ dry cell) was 1.4-fold higher than that of wild-type strain. In addition, the volumetric hydrogen production rate increased using the process where cells behaved as an effective catalyst. At 94.3 g dry cell/l, a productivity of 793 mmol h⁻¹ l⁻¹ (20.2 l h⁻¹ l⁻¹ at 37 °C) was achieved using SR15. The reported productivity substantially surpasses that of conventional biological hydrogen production processes and can be a trigger for practical applications.     

Ferric iron and extracellular electron shuttling increase xylose utilization and butanol production during fermentation with multiple solventogenic bacteria

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild-type cells are goals for biofuel production. Xylose consumption, butanol production, and hydrogen production increased in both Clostridium beijerinckii and a novel solventogenic bacterium (strain DC-1) when anthraquinone-2,6,-disulfonate (AQDS) or riboflavin were used as redox mediators to transfer electrons to poorly crystalline Fe(OH)₃ as an extracellular electron sink. Strain DC-1 was most closely related to Rhizobiales bacterium Mfc52 based on 95% 16S rRNA gene sequence similarity, which demonstrates that this response is not limited to a single genus of xylose-fermenting bacteria. Xylose utilization and butanol production were negligible in control incubations containing cells plus 3% (w/v) xylose alone during a 10-day batch fermentation, for both strains tested (n-butanol titers of 0.05 g L⁻¹). Micromolar concentrations of AQDS and riboflavin were added as electron shuttling compounds with poorly crystalline Fe(OH)₃ as an insoluble electron acceptor, and respective n-butanol titers increased to 6.35 and 7.46 g L⁻¹. Increases in xylose consumption for the iron treatments were relatively high, from less than 0.49 g L⁻¹ (xylose alone, no iron or electron shuttling molecules) to 25.98 and 29.15 g L⁻¹ for the AQDS and riboflavin treatments, respectively. Hydrogen production was also 3.68 times greater for the AQDS treatment and 5.27 greater for the riboflavin treatment relative to controls. Strain DC-1 data were similar, again indicating that the effects are not specific to the genus Clostridium.

     

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α‐linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose‐utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52 g L^?1 ALA, 96.6 mg L^?1 riboflavin, and 2.9 g L^?1 TAL, are obtained from xylose in flask cultures and 1.42 g L^?1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.     

Enantioselective biocatalytic hydrolysis of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-mandelonitrile for production of (<Emphasis Type="Italic">R</Emphasis>)-(−)-mandelic acid by a newly isolated mutant strain

(R)-(−)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl–ultraviolet irradiation and low energy N⁺ ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g⁻¹, in comparison with wild-type strain WT10 of 53.09 U g⁻¹. The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7–8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h⁻¹ g⁻¹. The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.     

Improved productivity of <Emphasis Type="Italic">β</Emphasis>-fructofuranosidase by a derepressed mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> from conventional and non-conventional substrates

Summary Wild-type cultures of Aspergillus niger produced a basal level of β-fructofuranosidase on glucose of 1 IU l⁻¹ h⁻¹. In contrast, a catabolite-derepressed mutant strain of the same organism produced a markedly higher level (25 IU l⁻¹ h⁻¹) of this enzyme when grown on the same carbon source. Wheat bran induced both the wild type (252 IU l⁻¹ h⁻¹) and the mutant strain (516 IU l⁻¹ h⁻¹) to produce 252- to 516-fold higher levels of this enzyme than was observed with the wild-type grown on glucose and was the best carbon source. When corn steep liquor served as a nitrogen source, the wild-type organism showed a higher activity of enzyme on monosaccharides and disaccharides comparable to that produced by corncobs in the basal medium and that mutant was a potentially improved (> 2-fold) organism for the production of β-fructofuranosidase on all carbon sources. Enhanced substrate consumption and product formation kinetic parameters suggest that the mutant organism may be exploited for bulk production of this useful enzyme.     

Study of paclitaxel prodution from an antifungal variant of Taxus callus

The antifungal variant from Taxus callus has been screened. The accumulation of biomass and paclitaxel of resistant and wild-type cells on modified MS medium or treated by fungal-elicitor was compared. The results showed that the production of biomass and paclitaxel of resistant cells was approximate as that of the wild-type cells on normal modified MS medium, but it was 70-fold higher when stimulated by fungal-elicitor. And there were evident difference on the activity of peroxidase (POD), phenylalanine ammonialyase (PAL) and the content of superoxide anion (O₂ ^–) between the antifungal variant and wild-type cells. The results implied that the intensity and velocity of the occurrence of secondary metabolism of antifungal variant were higher than those of the wild-type cells and the selection of resistant variant will be a good new path to improve paclitaxel production.     

Effects of reactive oxygen species on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis

The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular O₂ ^- concentration and α-tocopherol productivity in E. gracilis W₁₄ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast) was higher than that of the W₁₄ZUL strain. In the case of the wild strain, the O₂ ⁻ generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O₂ ⁻ generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O₂ ⁻ generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.     

Genetic transformation of <Emphasis Type="Italic">Solanum chrysotrichum</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and the production of antifungal saponins

Solanum chrysotrichum (Solanaceae) synthesizes a family of six antifungal spirostanol saponins designated as SC-1 to SC-6. The production of saponins by wild-type plants is variable depending on the environmental conditions. In order to develop an in vitro system for the sustained production of these saponins, transformed cell suspension cultures of S. chrysotrichum were established from nodal explants of 3-mo-old plantlets by infecting with the Agrobacterium tumefaciens strain C58/pBI12. From these cultures, kanamycin-resistant and phytohormone-independent cell suspension line C58 5.1.1 was obtained. PCR and Southern blot analyses were used to confirm the integration of the wild-type T-DNA into the plant genome. Batch cultures of the C58 5.1.1 cell line were grown in phytohormone-free MS liquid medium for 25 d. First-order growth kinetics and the production of the antifungal saponins (SC-2, SC-3, and SC-4) were determined by dry weight and quantified by HPLC, respectively, from the cells as well as the culture medium. Based on the cell biomass, the specific growth rate was 0.09 d⁻¹ and the yield of SC-2 reached 5.5% of dry weight, representing 40 times higher amount than that produced in plant leaves. SC-3 was recovered with a maximum yield of 0.9% of dry weight, whereas SC-4 was accumulated at 1.1% of dry weight. Saponins SC-2 and SC-3 were also excreted into the culture medium in low concentrations.     

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.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production

One of the proteins encoded by the riboflavin operon of Bacillus subtilis, RibA, was identified as the rate limiting enzyme in an industrial riboflavin producing strain. An additional single copy of the ribA gene was introduced into the sacB locus of the riboflavin production strain and was expressed constitutively from the medium strength vegI promoter. This led to improved riboflavin titers and yields of riboflavin on glucose of up to 25%. Both enzymatic activities of RibA, the 3,4-dihydroxy-2-butanone 4-phosphate synthase activity located in the N-terminal half of the protein and the GTP cyclohydrolase II activity of the C-terminal domain, are necessary for the improved riboflavin productivity. Received 16 June 1998/ Accepted in revised form 30 October 1998     

Effect of modified glucose catabolism on xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

In this study, the glucose 6-phosphate dehydrogenase gene (XOO2314) was inactivated in order to modulate the intracellular glucose 6-phosphate, and its effects on xanthan production in a wild-type strain of Xanthomonas oryzae were evaluated. The intracellular glucose 6-phosphate was increased from 17.6 to 99.4 μmol g⁻¹ (dry cell weight) in the gene-disrupted mutant strain. The concomitant increase in the glucose 6-phosphate was accompanied by an increase in xanthan production of up to 2.23 g l⁻¹ (culture medium). However, in defined medium supplemented with 0.4% glucose, the growth rate of the mutant strain was reduced to 52.9% of the wild-type level. Subsequently, when a family B ATP-dependent phosphofructokinase from Escherichia coli was overexpressed in the mutant strain, the growth rate was increased to 142.9%, whereas the yields of xanthan per mole of glucose remained approximately the same.     

Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l^–1 h^–1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l^–1 h^–1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.