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.     

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

Enhancement of riboflavin production by overexpression of acetolactate synthase in a pta mutant of Bacillus 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.     

枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响

CcpA蛋白是介导枯草芽孢杆菌碳分解代谢物阻遏(CCR)的全局调控因子,由ccpA基因编码。CCR效应的存在影响B.subtilis对葡萄糖的利用,降低B.subtilis生产发酵产品的效率。采用基因重组技术敲除了核黄素发酵菌株B.subtilis24/pMX45的ccpA基因,构建了CcpA缺陷株B.subtilis24A1/pMX45。发酵结果显示:B.subtilis24A1/pMX45能够在70h内基本耗尽10%的葡萄糖,生物量达到1.5×109个细胞/mL,溢流代谢产物积累量减少,在8%和10%葡萄糖浓度下,B.subtilis24A1/pMX45核黄素产量分别比B.subtilis24/pMX45提高了62%和95%。CcpA的缺陷,可以缓解葡萄糖引起的CCR效应,显著提高菌株的核黄素产量。     

透明颤菌vgb基因在产核黄素B.subtilis中的整合表达研究

目的:将透明颤菌血红蛋白vgb基因应用于核黄素的工业化生产。方法:以枯草芽孢杆菌整合载体pAmyE构建了vgb基因的整合表达载体pAudV,采用化学转化法将vgb基因整合到枯草芽孢杆菌GJ08的染色体上,并通过发酵摇瓶实验检测核黄素的产量。结果:得到产核黄素枯草芽孢杆菌GJ09,摇瓶试验结果表明,在限氧条件下核黄素的产量分别提高了5.23%和3.42%。结论:透明颤菌血红蛋白vgb基因能够促进核黄素产量的提高,可以应用于核黄素的工业化生产中。     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

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.     

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.     

过量无机盐及谷氨酸抑制重组菌核黄素发酵

在重组枯草芽孢杆菌24/pMX45核黄素发酵中,酵母粉促进核黄素合成,酵母抽提物抑制核黄素合成。分析显示,酵母抽提物的无机离子和游离氨基酸含量均高于酵母粉。在酵母粉基础发酵培养基中,添加各种无机离子和游离氨基酸,使其含量与酵母抽提物相同。摇瓶发酵结果表明:过量的无机离子和谷氨酸对核黄素合成有显著的抑制作用。酵母抽提物含有较高浓度的谷氨酸,是其抑制核黄素合成的主要原因。     

The kinetics of riboflavin secretion by Eremothecium ashbyii nrrl 1363

For riboflavin production a highly flavinogenic strain Eremothecium ashbyii is used. Vitamins are classified as chemical substances that control and effect the physiological processes; Riboflavin is one among them. The deficiency of riboflavin in human beings results in the cracking of lips and corners of mouth (cheilosis); nerve tissues are affected. For riboflavin production a highly flavinogenic strain Eremothecium ashbyii NRRL 1363 was used. Investigations were conducted in shake flask using inexpensive and abundantly available raw materials. Among the stimulants, a combination of hog casings and beef extract stimulated the highest and promoted the maximum riboflavin yield followed by the combination of fish meal and beef extract. The fermented broth (an enriched, riboflavin concentrate) can be directly used as a feed grade riboflavin. To upgrade it to pharmaceutical grade further investigations are required.     

Production of fungal cell wall degrading enzymes by a biocontrol strain of Bacillus subtilis AF 1

Fungal cell wall degrading chitinases and glucanases attained significance in agriculture, medicine, and environment management. The present study was conducted to describe the optimum conditions required for the production of beta-1,4-N-acetyl glucosaminidase (NAGase) and beta-1,3-glucanase by a biocontrol strain of Bacillus subtilis AF 1. B. subtilis AF 1 was grown in minimal medium with colloidal chitin (3.0%) and yeast extract (0.3% YE ) and incubated at pH 7.0 and 30 degrees C on constant shaker at 180 rpm for 6 days produced highest amounts of NAGase. Presence of 0.5 mM of phenyl methyl sulfonyl fluoride (PMSF) and 0.04% of Tween 20 further improved the enzyme production. B. subtilis AF 1 grown in minimal medium with laminarin (1%) and yeast extract (0.3%) for 3 days produced maximum amount of beta-1,3-glucanase. These conditions can be further scaled-up for large-scale production of NAGase and beta-1,3-glucanase by B. subtilis AF 1.     

Redirection electron flow to high coupling efficiency of terminal oxidase to enhance riboflavin biosynthesis

The metabolic impact of redirection electron flow to high coupling efficiency of terminal oxidases on riboflavin biosynthetic ability was quantitatively assessed during batch culture in this paper. While disruption of the low coupling bd oxidase of the riboflavin overproducing B. subtilis PK, the apparent phenotype with more rapid specific growth rate and higher biomass yield was achieved. Compared to by-products formation, a discernible shift to less acetate and more acetoin in cyd mutant was observed. As the overflow metabolism was decreased in B. subtilis PK cyd, more carbon source was directed to biomass and riboflavin biosynthetic pathway, which resulted in higher biomass and about 30% improvement of riboflavin biosynthetic ability. The higher product-corrected biomass yield in mutant showed that the efficient energy generation is an important factor for exponential growth of riboflavin overproducing B. subtilis strain in batch culture.     

Effect of glucose and its derivatives on systems of riboflavin uptake and excretion in the yeast Pichia guilliermondii]

Riboflavin uptake by washed cells of riboflavin deficient mutant MS1-3 of Pichia guilliermondii yeast was strongly depressed by D-glucose, L-sorbose, alpha-methyl-D-glucoside, sucrose, trehalose, maltose and salicin but not by D-mannose, D-galactose, D-fructose or ribitol. Glucose decreased also the initial uptake rate of riboflavin analogue, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine; the inhibition having a competitive character (Ki==5,7 mM). Apparently riboflavin permease is able to accept not only riboflavin and its analogues but also glucose and some of glucose derivates. Cells preloaded with riboflavin and transferred into riboflavin-free medium excreted vitamin B2 into the medium. This excretion was strongly stimulated by D-glucose, D-fructose, D-mannose but not by citrate or succinate. In contrast to riboflavin, 8-piperidyl-10-(1'-D-galactityl) isoalloxazine was not excreted into the medium even in the presence of glucose. The rate of riboflavin excretion depended on temperature and pH of incubation medium (pH optimum approximately 7.0) and was decreased in the presence of different inhibitors of energy metabolism. It seems that the exit of riboflavin from the cells is accomplished by energy-dependent specific system of excretion (excretase) which in some properties is different from that of riboflavin permease.     

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 gl(-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 gl(-1) xylose and 5 gl(-1) glucose initially gave the best result of 10.1 gl(-1)D-ribose concentration, 0.24 gg(-1)D-ribose yield and 0.29 gl(-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.     

Formation and Location of 1,4-beta-Glucanases and 1,4-beta-Glucosidases from Penicillium janthinellum

Formation and location of 1,4-beta-glucanases and 1,4-beta-glucosidases were studied in cultures of Penicillium janthinellum grown on Avicel, sodium carboxymethyl cellulose, cellobiose, glucose, mannose, and maltose. Endo-1,4-beta-glucanases were found to be cell free, and their formation was induced by cellobiose. 1,4-beta-Glucosidases, on the other hand, were formed constitutively and were primarily cell free, but with a small amount strongly associated with the cell wall. Low 1,4-beta-glucosidase activities of periplasmic or intracellular origin were also found. A rotational viscosimetric method was developed to measure the total endo-1,4-beta-glucanase activity of the culture (broth and solids). By this method, it was possible to determine the endo-1,4-beta-glucanase activity not only in the supernatant of the culture but also on the surface of the mycelium or absorbed on residual Avicel. During a 70-liter batch cultivation of P. janthinellum, the adsorption of endo-1,4-beta-glucanases by residual and newly added 10% Avicel was measured. The adsorption of soluble protein and endo-1,4-beta-glucanases by Avicel was found to be largely independent of the pH value but dependent on temperature.     

Acrylonitrile induces autolysis Bacillus subtilis

Acrylonitrile (AN) is an industrial chemical used in the manufacture of plastics and other polymers. AN has been reported to be an acute toxin and is a known carcinogen in rodents. When AN was mixed with suspensions of Bacillus subtilis, the bacteria began autolysis. It was determined that AN is partially converted to cyanide, a strong protonophore in B. subtilis. Autolytic enzymes in B. subtilis become active when the protonmotive force is dissipated. The amount of cyanide produced from AN, however, was not enough to promote autolysis in exponential B. subtilis. This is the first report showing that AN may induce autolytic reactions in bacteria. It is suggested the autolysis of B. subtilis may be useful in the environmental monitoring of AN. In addition, the metabolism of AN by bacilli may be useful in bioremediation.     

Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.     

Use of malt extract as a nitrogen source for continuous cultivation of Candida utilis]

The effect of an aqueous extract from malt sprouts on the growth and development of fodder yeast was investigated during their continuous cultivation. The extract can be used as a nitrogen source during continuous cultivation of fodder yeast. It is supposed that the extract contains compounds capable to inhibit yeast growth. The extract should be added to the nutrient medium in the amount of 4 g/l of dry weight.     

Preparation and properties of immobilized riboflavin kinase from Pichia guilliermondii

Riboflavin kinase (ATP: riboflavin-5'-phosphotransferase, EC 2.7.1.26) from n-alkane utilizing Pichia guilliermondii yeast has been immobilized by covalent attachment to CNBr-activated agarose beads. The enzyme activity yield during immobilization reached 71.6%. Immobilized riboflavin kinase showed no significant changes in temperature and pH optima as well as in specificity of the action in relation to synthetic substrate analogues with the substitution of methyl groups at positions 7 and 8 of the isoalloxazine ring. Immobilized riboflavin kinase was stable during FMN synthesis in the continuous-flow packed column enzyme reactor with half-life of 27 days.