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.     

枯草芽孢杆菌基因修饰生产核黄素

【目的】研究枯草芽孢杆菌核黄素合成途径、木糖代谢相关基因修饰对核黄素合成的影响。【方法】单独过表达或共同过表达核黄素操纵子中的基因、过表达木糖代谢相关基因构建相应的重组菌株。通过测定和比较重组菌株摇瓶发酵的核黄素产量和生物量,表征各个基因修饰的效应。采用摇瓶和5 L罐发酵,考察木糖作为主要碳源以及木糖与蔗糖共代谢对核黄素发酵的影响。【结果】ribA基因单独过表达,使核黄素产量提高99%,但生物量降低30%,出现细胞自溶现象。ribA-ribH基因共表达,使核黄素产量提高280%,并且无细胞自溶和生物量下降现象。1.5%蔗糖与6.5%木糖作为碳源,5 L发酵罐发酵70 h,核黄素产量达到3.6 g/L,与8%蔗糖为碳源的发酵相比,核黄素产量提高80%。木糖代谢相关基因过表达,均明显降低核黄素产量。【结论】与ribA基因单独过表达相比,ribA-ribH基因共表达可有效避免细胞自溶现象,并能进一步提高核黄素产量。蔗糖与木糖共代谢,能够改善前体物供给,有利于提高核黄素产量。     

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。     

Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis

We present redirection of electron flow to more efficient proton pumping branches within respiratory chains as a generally applicable metabolic engineering strategy, which tailors microbial metabolism to the specific requirements of high cell density processes by improving product and biomass yields. For the example of riboflavin production by Bacillus subtilis, we reduced the rate of maintenance metabolism by about 40% in a cytochrome bd oxidase knockout mutant. Since the putative Yth and the caa(3) oxidases were of minor importance, the most likely explanation for this improvement is translocation of two protons per transported electron via the remaining cytochrome aa(3) oxidase, instead of only one proton via the bd oxidase. The reduction of maintenance metabolism, in turn, significantly improved the yield of recombinant riboflavin and B. subtilis biomass in fed-batch cultures.     

Estimation of P-to-O ratio in Bacillus subtilis and its influence on maximum riboflavin yield.

Simultaneous growth and riboflavin overproduction were investigated using a previously developed stoichiometric model of Bacillus subtilis metabolism. A fit of model predictions to experimental data was used to obtain estimates of fundamental energetic parameters of B. subtilis. Although multiple solutions describe the experimental data, evidence for a P-to-O ratio of about 1(1/3) mole of ATP produced per atom of oxygen consumed in oxidative phosphorylation was provided by genomic analysis of electron transport components, because no homologue of the proton-translocating NADH dehydrogenase I was found in the B. subtilis genome database. These results allow us to devise a rational metabolic engineering strategy to improve riboflavin production. The potential influence of increased energy coupling in oxidative phosphorylation on riboflavin yield is discussed. Higher coupling is most significant under carbon-limiting conditions in slow-growing cells, that is, in fed-batch processes of industrial interest.     

Sensing of Nitrogen Limitation by Bacillus subtilis: Comparison to Enteric Bacteria

Previous studies showed that Salmonella typhimurium apparently senses external nitrogen limitation as a decrease in the concentration of the internal glutamine pool. To determine whether the inverse relationship observed between doubling time and the glutamine pool size in enteric bacteria was also seen in phylogenetically distant organisms, we studied this correlation in Bacillus subtilis, a gram-positive, sporulating bacterium. We measured the sizes of the glutamine and glutamate pools for cells grown in batch culture on different nitrogen sources that yielded a range of doubling times, for cells grown in ammonia-limited continuous culture, and for mutant strains (glnA) in which the catalytic activity of glutamine synthetase was lowered. Although the glutamine pool size of B. subtilis clearly decreased under certain conditions of nitrogen limitation, particularly in continuous culture, the inverse relationship seen between glutamine pool size and doubling time in enteric bacteria was far less obvious in B. subtilis. To rule out the possibility that differences were due to the fact that B. subtilis has only a single pathway for ammonia assimilation, we disrupted the gene (gdh) that encodes the biosynthetic glutamate dehydrogenase in Salmonella. Studies of the S. typhimurium gdh strain in ammonia-limited continuous culture and of gdh glnA double-mutant strains indicated that decreases in the glutamine pool remained profound in strains with a single pathway for ammonia assimilation. Simple working hypotheses to account for the results with B. subtilis are that this organism refills an initially low glutamine pool by diminishing the utilization of glutamine for biosynthetic reactions and/or replenishes the pool by means of macromolecular degradation.     

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

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.     

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.     

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.     

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.     

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.     

Engineering of a Bacillus subtilis strain with adjustable levels of intracellular biotin for secretory production of functional streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

核黄素基因工程研究进展

核黄素 (维生素B2 )为天然水溶性的B族维生素 ,是维持机体正常代谢所必须的物质 ,具有重要的生理功能。目前核黄素的生产方法主要有化学合成法和微生物发酵法。其中微生物发酵法是后来发展起来的一种十分经济有效的方法 ,并在核黄素主产中开始占据主导地位。为进一步获得核黄素高产菌株 ,人们对核黄素合成基因及其表达调控的机制做了深入细致的研究 ,并以此为依据 ,通过基因工程手段构建出了核黄素高产菌株 ,大大提高了核黄素的产量 ,其中尤以枯草芽孢杆菌最为成功。综述发酵法生产核黄素的现状、核黄素生物合成的分子生物学以及基因工程研究进展 ,讨论了其进一步的发展方向。     

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.     

Monitoring growth of Escherichia coli using a two-channel optical sensor

Summary The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.Correspondence to: W. S. Kunz     

Oversynthesis of riboflavin by yeast Pichia guilliermondii in response to oxidative stress

The effect of oxidative stress on riboflavin (vitamin B2) biosynthesis and iron accumulation in flavinogenic yeast P. guilliermondii was investigated. Treatment of P. guilliermondii cells with superoxidgenerating agent methylviologen leads to elevated production of malondialdyhyd (MDA) which reflects the overall cellular oxidation state. Increased iron content in the cells and enhanced productivity of flavinogenesis under these conditions has been shown too. Significant increasing of MDA and riboflavin production by yeast cells under iron deficiency was observed. Riboflavin overproducing P. guilliermondii mutant strains rib80, rib81 and hit, possess high iron transport and synthesize increased quantity of MDA. The role of riboflavin overproduction and activation of iron assimilation in the P. guilliermondii antioxidant defence is discussed.     

Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse

By treatment of a wild-type strain of Cellulomonas flavigena with N-methyl-N'-nitro-N-nitrosoguanidine at 150 g/ml, mutants PN-7 and PN-10 were obtained, which produce 1.38 and 1.5 times more carboxymethylcellulase than the wild strain when cultured in a batch system with sugar cane bagasse as the sole carbon source. These mutants also exhibited higher specific growth rates compared to the wild strain. From a second mutagenesis of mutant PN-10, mutant PN-120 was obtained in continuous culture. This mutant was able to use a larger portion of sugar cane bagasse than did the wild-type and therefore its biomass yield was also higher. The mutant showed a specific growth rate on sugar cane bagasse threefold higher than the wild strain.     

肌苷产生菌缺失核苷水解酶活性突变株选育及其发酵生产试验

以枯草芽孢杆菌ＪＳＩＭ－１０１９为出发菌株,经物理、化学诱变剂连续处理,获得一株缺失核苷水解酶活性的突变株ＪＳＩＭ－Ｂ－１９８。该突变株不能降解肌苷,应用于发酵生产中,肌苷产量显著增加。在２００００升发酵罐中,连续１０罐批,平均产肌苷８．３８ｇ／Ｌ,对糖转化率２１．９８％,发酵周期５０ｈ￣６０ｈ,该菌株遗传性能稳定,已在工业生产中应用。