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1.
A genome-scale metabolic model of the Gram-positive bacteria Corynebacterium glutamicum ATCC 13032 was constructed comprising 446 reactions and 411 metabolites, based on the annotated genome and available biochemical information. The network was analyzed using constraint based methods. The model was extensively validated against published flux data, and flux distribution values were found to correlate well between simulations and experiments. The split pathway of the lysine synthesis pathway of C. glutamicum was investigated, and it was found that the direct dehydrogenase variant gave a higher lysine yield than the alternative succinyl pathway at high lysine production rates. The NADPH demand of the network was not found to be critical for lysine production until lysine yields exceeded 55% (mmol lysine (mmol glucose)(-1)). The model was validated during growth on the organic acids acetate and lactate. Comparable flux values between in silico model and experimental values were seen, although some differences in the phenotypic behavior between the model and the experimental data were observed. 相似文献
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Lars Regestein Thomas Maskow Andreas Tack Ingo Knabben Martin Wunderlich Johannes Lerchner Jochen Büchs 《Biotechnology and bioengineering》2013,110(5):1386-1395
Non‐invasive methods for online monitoring of biotechnological processes without compromising the integrity of the reactor system are very important to generate continuous data. Even though calorimetry has been used in conventional biochemical analysis for decades, it has not yet been specifically applied for online detection of product formation at technical scale. Thus, this article demonstrates a calorespirometric method for online detection of microbial lysine formation in stirred tank bioreactors. The respective heat generation of two bacterial strains, Corynebacterium glutamicum ATCC 13032 (wild‐type) and C. glutamicum DM1730 (lysine producer), was compared with the O2‐consumption in order to determine whether lysine was formed. As validation of the proposed calorespirometric method, the online results agreed well with the offline measured data. This study has proven that calorespirometry is a viable non‐invasive technique to detect product formation at any time point. Biotechnol. Bioeng. 2013; 110: 1386–1395. © 2012 Wiley Periodicals, Inc. 相似文献
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Jian‐Zhong Xu Han‐Kun Yang Li‐Ming Liu Ying‐Yu Wang Wei‐Guo Zhang 《Biotechnology and bioengineering》2018,115(7):1764-1777
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Andreas Tauch Thomas Hermann Andreas Burkovski Reinhard Krämer Alfred Pühler J. Kalinowski 《Archives of microbiology》1998,169(4):303-312
By complementation analysis of an isoleucine-uptake-deficient Escherichia coli strain, it was shown that a 1.6-kb HindIII-StuI fragment of Corynebacterium glutamicum ATCC 13032, located downstream of the aecD gene, encodes an isoleucine uptake system. Sequence analysis revealed that the complementing fragment carried an open reading
frame, termed brnQ, that encodes a protein with sequence similarities to branched-chain amino acid carriers of gram-positive and gram-negative
bacteria. The brnQ gene specifies a predominantly hydrophobic protein of 426 amino acid residues with a calculated molecular mass of 44.9 kDa.
A topology prediction by neural network computer analysis suggests the existence of 12 hydrophobic segments that most probably
form transmembrane α-helices. A C. glutamicum mutant strain harboring a defined deletion of brnQ in the chromosome showed a considerably lower isoleucine uptake rate of 0.04 nmol min–1 mg (dry mass)–1 as compared to the wild-type strain rate of 1.2 nmol min–1 mg (dry mass)–1. Overexpression of brnQ by means of a tac promotor resulted in an elevated uptake rate for isoleucine of 11.3 nmol min–1 mg (dry mass)–1. Evidently, the brnQ gene encodes the only transport system in C. glutamicum directing isoleucine uptake.
Received: 16 October 1997 / Accepted: 27 November 1997 相似文献
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Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants. 相似文献
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萜类化合物具有可观的商业价值,但生产过程复杂,产量低,利用微生物异源合成萜类化合物已成为热点。谷氨酸棒状杆菌内含合成萜类色素的途径,具有异源合成萜类化合物的天然优势和研究前景。首次对谷氨酸棒状杆菌合成萜类化合物进行了综述,从萜类合成途径、关键酶和全局调控机制三个方面进行了途经介绍。概述了谷氨酸棒状杆菌中单萜、倍半萜、四萜类化合物的异源合成,并对利用谷氨酸棒状杆菌高效合成萜类化合物所需解决的问题进行讨论,为谷氨酸棒状杆菌高效合成萜类化合物提供建议。 相似文献
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Hermann Sahm Lothar Eggeling Bernd Eikmanns Reinhard Krämer 《FEMS microbiology reviews》1995,16(2-3):243-252
The Gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of l-glutamate and l-lysine. In the last 10 years, genetic engineering and amplification of relevant structural genes have become fascinating methods for the construction of strains with desired genotypes. By cloning and expressing the various genes of the l-lysine pathway in C. glutamicum we could demonstrate that an increase of the flux of l-aspartate semialdehyde to l-lysine could be obtained in strains with increased dehydrodipicolinate synthase activity. By combined overexpression of deregulated aspartate kinase and dihydrodipicolinate synthase, the l-lysine secretion could be increased (10–20%). Recently we detected that in C. glutamicum two pathways exist for the synthesis of dl-diaminopimelate and l-lysine. Mutants defective in one pathway are still able to synthesize enough l-lysine for growth, but the l-lysine secretion is reduced to 50–70%. Using NMR spectroscopy, we could calculate how much of the l-lysine secreted into the medium is synthesized via each pathway. Amplification of the feedback inhibition-insensitive homoserine dehydrogenase and homoserine kinase in a high l-lysine overproducing strain enabled channelling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of l-threonine. For a further flux from l-threonine to l-isoleucine the allosteric control of threonine dehydratase must be eliminated. In addition to all steps considered so far to be important for amino acid overproduction, the secretion into the culture medium also has to be noted. Recently it could be demonstrated that l-glutamate, l-lysine and l-isoleucine are not secreted via passive diffusion but via specific active carrier systems. Analysis of lysine-overproducing C. glutamicum strains indicates that this secretion carrier has a strong influence on the overproduction of this amino acid. Thus, for the construction of strong amino acid overproducing strains by using the gene cloning techniques, the overexpression of the genes for the export systems also seems necessary. 相似文献
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氨基酸是一类在食品、医药及化工等领域具有广泛应用的重要化合物。谷氨酸棒杆菌Corynebacterium glutamicum是生物合成氨基酸最重要的微生物菌株,其年产各类氨基酸超过百万吨。谷氨酸棒杆菌高产氨基酸除具有强大的合成代谢能力外,高效的分泌转运能力也是不可忽略的分子基础。文中综述了近年来谷氨酸棒杆菌中氨基酸分泌转运蛋白及其代谢改造的研究进展,并展望了未来发展方向,为进一步改造提升其发酵生产氨基酸的能力提供了可资借鉴的资料。 相似文献
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Fangyu Cheng Qianying Gong Huimin Yu Gregory Stephanopoulos 《Biotechnology journal》2016,11(4):574-584
Hyaluronic acid (HA) plays important roles in human tissue system, thus it is highly desirable for various applications, such as in medical, clinic and cosmetic fields. The wild microbial producer of HA, streptococcus, was restricted by its potential pathogens, hence different recombinant hosts are being explored. In this work, we engineered Corynebacterium glutamicum, a GRAS (Generally Recognized as Safe) organism free of exotoxins and endotoxins to produce HA with high titer and satisfied Mw. The ssehasA gene encoding hyaluronan synthase (HasA) was artificially synthesized with codon preference of C. glutamicum. Other genes involved in the HA synthetic pathway were directly cloned from the C. glutamicum genome. The operon structures and constitutive or inducible promoters were particularly compared and the preferred environmental conditions were also optimized. Using glucose and corn syrup powder as carbon and nitrogen sources, batch cultures of the engineered C.glutamicum with operon ssehasA‐hasB driven by Ptac promoter were performed in a 5 L fermentor. The maximal HA titer, productivity and yield reached 8.3 g/L, 0.24 g/L/h and 0.22 gHA/gGlucose, respectively; meanwhile the maximal Mw was 1.30 MDa. This work provides a safe and efficient novel producer of HA with huge industrial prospects. 相似文献
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Kataoka M Hashimoto KI Yoshida M Nakamatsu T Horinouchi S Kawasaki H 《Letters in applied microbiology》2006,42(5):471-476
AIM: The ultimate aim is to elucidate the molecular mechanisms for glutamate overproduction by Corynebacterium glutamicum. METHODS AND RESULTS: Gene expression in response to the conditions inducing glutamate overproduction was investigated by using a DNA microarray technique. Most genes involved in the EMP pathway, the PPP, and the TCA cycle were downregulated, while five genes that were highly upregulated (NCgl0917, NCgl2944, NCgl2945, NCgl2946, and NCgl2975) were identified under all the three conditions for overproduction that are studied here. Gene products of NCgl2944, NCgl2945, and NCgl2946 were highly homologous to each other, did not resemble any other protein, and have remained uncharacterized thus far. The product of NCgl0917 showed a similarity to a few hypothetical and uncharacterized proteins. NCgl2975 was homologous to metal-binding proteins. CONCLUSIONS: The decrease in the activity of 2-oxoglutarate dehydrogenase complex, a key enzyme that is downregulated during glutamate overproduction, can be mainly attributed to the downregulation of odhA and sucB. Five highly upregulated genes were also identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Although fermentative production of glutamate has been carried out for more than 45 years, information on the molecular mechanisms of glutamate overproduction is still limited. This study further elucidates these mechanisms. 相似文献
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为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。 相似文献
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Julian A. da Luz Enrico Hans Doinita Frank An‐Ping Zeng 《Engineering in Life Science》2017,17(5):512-522
Engineering of enzymes and pathways is generally required for the development of efficient strains for bioproduction processes. To this end, quantitative and reliable data of intracellular metabolites are highly desired, but often not available, especially for conditions more close to industrial applications, i.e. at high cell density and product concentration. Here, we investigated the intracellular metabolite profiles of an engineered l ‐lysine‐producing Corynebacterium glutamicum strain and the corresponding wild‐type strain to assess the impacts of deregulation of product inhibition of the key enzymes aspartate kinase and phosphoenolpyruvate carboxylase and to identify potentials for their further improvement. A bioreactor system with automated fast‐sampling, filtration and on‐filter quenching of the metabolism was used for a more reliable determination of intracellular metabolites in batch cultures with optical cell density (OD660) up to 40. The l ‐lysine‐producing strain showed substantially different metabolite profiles in the amino acid metabolism, including increased intracellular pool sizes in the l ‐lysine‐, l ‐homoserine‐ and l ‐threonine pathways and decreased intracellular pool sizes for all other determined amino acids. By comparing data of in vitro inhibition of the engineered enzymes and determined intracellular concentrations of the inhibitors it was found that the inferred in vivo activities of these enzymes are still significantly below their in vitro maximums. This work demonstrates the usefulness of metabolic analysis for assessing the impact of engineered enzymes and identifying targets for further strain development. 相似文献
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Isolation and characterization of a gene encoding meso-diaminopimelate dehydrogenase fromGlycine max
A detailed characterization of the lysine biosynthetic pathway in plants is yet to be completed. It is, however, assumed that
the diaminopimelic acid pathway exists in the plant kingdom, as commonly described forEscherichia coli.
Modification and refinement of lytic complementation, a technique previously utilized in bacterial systems, facilitated the
isolation of a functional Diaminopimelate Dehydrogenase gene from aGlycine max nuclear gene library. The isolated gene codes for the enzyme meso-diaminopimelate dehydrogenase. The coding capacity for
the enzyme was originally contained on a 6.6kb fragment in a Charon 4a soybean gene bank. Subcloning of the 6.6kb fragment
resulted in the recombinant plasmid pMW75. Subsequent subcloning resulted in a 4.05 kb fragment contained in pLW14. One region
of homology was observed upon hybridization to EcoR1 digested soybean DNA. Homologous sequences were also observed in Triticum
DNA.
Meso-diaminopimelate dehydrogenase activity was demonstrated inGlycine max embryos. Maximum enzymatic activity of the cloned enzyme was observed at a pH of 8.0. The enzyme encoded by the soybean gene
has an apparent molecular weight of 67 000. 相似文献
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Takeo Tomita 《Bioscience, biotechnology, and biochemistry》2017,81(11):2050-2061
Amino acids are essential components in all organisms because they are building blocks of proteins. They are also produced industrially and used for various purposes. For example, L-glutamate is used as the component of “umami” taste and lysine has been used as livestock feed. Recently, many kinds of amino acids have attracted attention as biological regulators and are used for a healthy life. Thus, to clarify the mechanism of how amino acids are biosynthesized and how they work as biological regulators will lead to further effective utilization of them. Here, I review the leucine-induced-allosteric activation of glutamate dehydrogenase (GDH) from Thermus thermophilus and the relationship with the allosteric regulation of GDH from mammals. Next, I describe structural insights into the efficient production of L-glutamate by GDH from an excellent L-glutamate producer, Corynebacterium glutamicum. Finally, I review the structural biology of lysine biosynthesis of thermophilic bacterium and archaea. 相似文献
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Francesco Falcioni Bruno Bühler Andreas Schmid 《Biotechnology and bioengineering》2015,112(2):322-330
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