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。     

Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.     

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.     

Genetic control of riboflavin synthetase in Bacillus subtilis

Summary The level of riboflavin synthetase in growing cultures of Bacillus subtilis is controlled by repression. The enzyme level is derepressed in flavinogenic mutants of the microorganism. Riboflavin-deficient mutants accumulating 6,7-dimethyl-8-ribityllumazine are devoid of riboflavin synthetase.     

Heavy riboflavin synthase from Bacillus subtilis. Quaternary structure and reaggregation

Heavy riboflavin synthase of Bacillus subtilis was purified by a simplified procedure. The enzyme is a complex protein containing about 3 alpha-subunits (23.5 X 10(3) Mr) and 60 beta-subunits (16 X 10(3) Mr). The 10(6) Mr protein dissociates upon exposure to pH values above neutrality. Phosphate ions increase the stability at neutral pH. The dissociation induced by exposure of the enzyme to elevated pH is reversible in phosphate buffer at neutral pH. The stability of the enzyme at elevated pH values is greatly enhanced by the substrate analogue, 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione. Electron micrographs of negatively stained enzyme specimens show spherical particles with a diameter of 15.6 nm. Various immunochemical methods show that the alpha-subunits are not accessible to antibodies in the native molecule. The native enzyme is not precipitated by anti-alpha-subunit serum, and riboflavin synthase activity is not inhibited by the serum. However, these tests become positive at pH values that lead to dissociation of the enzyme. Subsequent to dissociation of the native enzyme at elevated pH values, the beta-subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 S and 70 S. The average molecular weight was approximately 5.6 X 10(6). Homogeneous preparations have not been obtained. Electron micrographs show hollow, spherical vesicles with diameters of about 29 nm. The substrate analogue 5-nitroso-6-ribitylamino-2,4(1H, 3H)-pyrimidinedione can induce the reaggregation of isolated beta-subunits with formation of smaller molecules, which are structurally similar to native riboflavin synthase. A homogeneous preparation of reaggregated molecules was obtained by renaturation of beta-subunits from 6.4 M-urea in the presence of the ligand. The sedimentation velocity of this aggregate is about 7% smaller than that of the native enzyme. The molecular weight is 96 X 10(4). Electron micrographs show spherical particles with a diameter of about 17.4 nm. Inspection of the micrographs tentatively suggests the presence of a central cavity. It appears likely that these molecules, which are devoid of alpha-subunits, have the same number and spatial arrangement of beta-subunits as the native enzyme. All data are consistent with the hypothesis that the native enzyme consists of a central core of alpha-subunits surrounded by a capsid-like arrangement of beta-subunits. The number of beta-subunits and the shape of the protein suggest a capsid-like arrangement of beta-subunits.(ABSTRACT TRUNCATED AT 400 WORDS)     

The bounds of the riboflavin operon in Bacillus subtilis

All the structural genes of riboflavin biosynthesis are shown to be located on the 2.8 MD DNA fragment, using the collection of plasmids, carrying the Bacillus subtilis riboflavin operon fragments and Bacillus subtilis strains, containing various deletions of rib-operon for analysis. The proximal Bgl II site is shown to be located between promoter P1 and the first structural gene ribG. The distal Hind III site of fragment C is the left bound of the rib-operon.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Enhancement of riboflavin production by deregulating gluconeogenesis in Bacillus subtilis

The regulation of metabolic flux through glycolytic versus the gluconeogenic pathway plays an important role in central carbon metabolism. In this study, we made an attempt to enhance riboflavin production by deregulating gluconeogenesis in Bacillus subtilis. To this end, gapB (code for NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase), fbp (code for fructose-1,6-bisphosphatase) and pckA (code for phosphoenolpyruvate carboxykinase) were overexpressed in parental strain B. subtilis RH33. Compared with RH33, overexpression of fbp and gapB resulted in approximately 18.0 and 14.2 % increased riboflavin production, respectively, while overexpression of pckA obtained the opposite result. Significant enhancement of riboflavin titers up to 4.89 g/l was obtained in shake flask cultures when gapB and fbp were co-overexpressed, nevertheless the specific growth rate decreased slightly and the specific glucose uptake rate remained almost unchanged. An improvement by 21.9 and 27.8 % of the riboflavin production was achieved by co-overexpression of gapB and fbp in shake flask and fed-batch fermentation, respectively. These results imply that deregulation of gluconeogenesis is an effective strategy for production of metabolites directly stemming from the pentose phosphate pathway as well as other NADPH-demanding compounds with glucose as carbon source in B. subtilis.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

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).     

Nitrogen assimilation enzymes in Bacillus subtilis mutants with hyperproduction of riboflavin

Three groups of the nitrogen assimilation cycle enzymes (glutamate synthases (GTS), glutamine synthases (GS), and glutamate dehydrogenases (GD)) were studied in Bacillus subtilis strains with hyperproduction of riboflavin (vitamin B2). It was found that in all strains tested activity of GS was virtually the same, activity of GD was absent, and activity of GTS was reduced. In strains 41 and 24, riboflavin producers, activity of GTS was 30-60% the enzyme activity in the original strain (wild-type RosR). The most pronounced decrease in the activity of GTS (0-12% relative to RosR) was observed in the strain AS5, which had the highest level of biosynthetic activity relative to the other strains. According to the results of determination of the sensitivity of induction of beta-xylosidase to glucose- and fructose-induced catabolic repression, none of the strains studied was characterized by disorders in the protein CcpA, a global regulator of the catabolic repression in gram-positive bacteria, which is required for reducing amination and resulting activation of biosynthesis of glutamic acid in cell. It was suggested that mutations responsible for partial or complete inhibition of GTS biosynthesis caused an increase in the intracellular pool of glutamine. The intracellular pool of glutamine is a nitrogen source for riboflavin in cell. It follows from the results of this work that there is a trend toward an increase in the rate of biosynthesis of vitamin B2 in mutants with inhibited GTS activity. However, the complexity of the processes of regulation of nitrogen assimilation enzymes makes it difficult to find a distinct correlation between GTS activity and riboflavin biosynthesis in these strains.     

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.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine by heavy riboflavin synthase from Bacillus subtilis

The beta subunits of heavy riboflavin synthase catalyze the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and a carbohydrate phosphate, Compound X. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate is not a substrate for the enzyme, although it is an established intermediate in the biosynthesis of riboflavin. It follows that this pyrimidine phosphate must be dephosphorylated prior to the formation of 6,7-dimethyl-8-ribityllumazine.     

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.     

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     

Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis

The establishment and the improvement of industrial bioprocesses calls for the selection of media compositions and process conditions in highly parallel experiments as well as for the intensified screening of new biocatalysts and improved production strains. This work presents for the first time the scale-down and the successful adaptation of an industrial riboflavin fed-batch production process with Bacillus subtilis to a fully automated setup with 48 parallel stirred bioreactors at a milliliter scale (10 mL). The feasibility of an intermittent feeding mode and a discontinuous at-line pH control for parallel cultivations over up to 53 h is demonstrated together with interlaced process analyses at a microliter scale for quasi-simultaneous at-line monitoring of biomass, substrate and product concentration. The discontinuous feeding mode necessitated an increased oxygen input, resulting in lower final biomass concentrations. However, the product yields and volumetric productivities in the milliliter setup were equivalent to the yields and productivities obtained during the reference cultivations at laboratory scale, which allows considering the automated system together with the developed schedule as a screening tool for high-throughput bioprocess design of the described production process.     

Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis

Carbon flow in Bacillus subtilis through the pentose phosphate (PP) pathway was modulated by overexpression of glucose-6-phosphate dehydrogenase (G6PDH) under the control of the inducible Pxyl promoter in B. subtilis PY. Alteration of carbon flow into the PP pathway will affect the availability of ribulose-5-phosphate (Ru5P) and the riboflavin yield. Overexpression of G6PDH resulted in the glucose consumption rate increasing slightly, while the specific growth rate was unchanged. An improvement by 25% ± 2 of the riboflavin production was obtained. Compared to by-products formation in flask culture, low acid production (acetate and pyruvate) and more acetoin were observed. Metabolic analysis, together with carbon flux redistribution, indicated that the PP pathway fluxes are increased in response to overexpression of G6PDH. Moreover, increased flux of the PP pathway is associated with an increased intracellular pool of Ru5P, which is a precursor for riboflavin biosynthesis. The high concentrations of Ru5P could explain the increased riboflavin production.