The Corynebacterium glutamicum aecD gene encodes a C-S lyase with alpha, beta-elimination activity that degrades aminoethylcysteine.

S-(beta-Aminoethyl)-cysteine (AEC) resistance was achieved in Corynebacterium glutamicum by cloning a chromosomal 1.5-kb EcoRV-BglII DNA fragment on a multicopy plasmid. DNA sequence analysis of the 1.5-kb DNA fragment revealed an open reading frame (ORF326) which represents the AEC resistance gene, designated aecD. The aecD gene directs the synthesis of a 36-kDa protein which was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The aecD gene is a nonessential gene and mediates AEC resistance only in an amplified state. C. glutamicum strains harboring an amplified aecD gene can utilize AEC as an alternative nitrogen source, indicating that the AEC resistance mechanism is due to AEC degradation. Since the AEC degradation products analyzed by high-pressure liquid chromatography were found to be pyruvate and aminoethanethiol (cysteamine), it was concluded that the aecD gene encodes a C-S lyase with alpha, beta-elimination activity. Besides AEC, the C-S lyase was also able to use cysteine, cystine, and cystathionine as substrates.     

The tetAB genes of the Corynebacterium striatum R-plasmid pTP10 encode an ABC transporter and confer tetracycline, oxytetracycline and oxacillin resistance in Corynebacterium glutamicum

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI-Sa/I DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated beta-lactam antibiotic oxacillin.     

谷氨酸棒杆菌10147基因组中启动子活性片段的克隆与分析

摘要：【目的】获得谷氨酸棒杆菌10147基因组中具有启动子活性片段的结构序列,为构建表达载体做准备。【方法】利用启动子探测载体pAKC6,采用鸟枪法克隆经过限制性内切酶Sau3A I完全酶切的谷氨酸棒杆菌10147染色体DNA片段,并测定pAKC6上报告基因编码的氯霉素乙酰转移酶（CAT）的比活力,以筛选有启动子功能的片段。【结果】共克隆到30个具有启动子功能的片段。其中有三个插入片段起动的氯霉素乙酰转移酶比活力大于24 U/mg,插入片段F57起动的CAT比活力为32.50 U/mg;而插入有启动子Ptrc的阳性对照的CAT比活力为26.33 U/mg。【结论】获得三个DNA插入片段具有与已知启动子Ptrc相当的启动活性,这些片段可以用于构建谷氨酸棒杆菌表达载体。     

Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.     

Cloning of the pyruvate kinase gene (pyk) of Corynebacterium glutamicum and site-specific inactivation of pyk in a lysine-producing Corynebacterium lactofermentum strain.

The pyruvate kinase gene pyk from Corynebacterium glutamicum was cloned by applying a combination of PCR, site-specific mutagenesis, and complementation. A 126-bp DNA fragment central to the C. glutamicum pyk gene was amplified from genomic DNA by PCR with degenerate oligonucleotides as primers. The cloned DNA fragment was used to inactivate the pyk gene in C. glutamicum by marker rescue mutagenesis via homologous recombination. The C. glutamicum pyk mutant obtained was unable to grow on minimal medium containing ribose as the sole carbon source. Complementation of this phenotype by a gene library resulted in the isolation of a 2.8-kb PstI-BamHI genomic DNA fragment harboring the C. glutamicum pyk gene. Multiple copies of plasmid-borne pyk caused a 20-fold increase of pyruvate kinase activity in C. glutamicum cell extracts. By using large internal fragments of the cloned C. glutamicum gene, pyk mutant derivatives of the lysine production strain Corynebacterium lactofermentum 21799 were generated by marker rescue mutagenesis. As determined in shake flask fermentations, lysine production in pyk mutants was 40% lower than that in the pyk+ parent strain, indicating that pyruvate kinase is essential for high-level lysine production. This finding questions an earlier hypothesis postulating that redirection of carbon flow at the phosphoenol pyruvate branch point of glycolysis through elimination of pyruvate kinase activity results in an increase of lysine production in C. glutamicum and its close relatives.     

Cloning of the trp gene cluster from a tryptophan-hyperproducing strain of Corynebacterium glutamicum: identification of a mutation in the trp leader sequence.

Corynebacterium glutamicum ATCC 21850 produces up to 5 g of extracellular L-tryptophan per liter in broth culture and displays resistance to several synthetic analogs of aromatic amino acids. Here we report the cloning of the tryptophan biosynthesis (trp) gene cluster of this strain on a 14.5-kb BamHI fragment. Subcloning and complementation of Escherichia coli trp auxotrophs revealed that as in Brevibacterium lactofermentum, the C. glutamicum trp genes are clustered in an operon in the order trpE, trpD, trpC, trpB, trpA. The cloned fragment also confers increased resistance to the analogs 5-methyltryptophan and 6-fluorotryptophan on E. coli. The sequence of the ATCC 21850 trpE gene revealed no significant changes when compared to the trpE sequence of a wild-type strain reported previously. However, analysis of the promoter-regulatory region revealed a nonsense (TGG-to-TGA) mutation in the third of three tandem Trp codons present within a trp leader gene. Polymerase chain reaction amplification and sequencing of the corresponding region confirmed the absence of this mutation in the wild-type strain. RNA secondary-structure predictions and sequence similarities to the E. coli trp attenuator suggest that this mutation results in a constitutive antitermination response.     

Cloning and study of Corynebacterium glutamicum genes complementing ilvA and thrA2 mutations in Escherichia coli

Molecular cloning and expression of Corynebacterium glutamicum genes complementing Escherichia coli mutations thrA2 and ilvA was performed. It was demonstrated that the thrA2 gene of C. glutamicum is located close to thrB on EcoRI DNA fragment 4.1 kb long. The fragment was cloned in pUC18 vector. The thrA2 gene is expressed in the recombinant plasmid pOBT3 under control of the vector pUC18 Plac promoter. In E. coli minicells, the genes thrA2 and thrB determined synthesis of proteins of Mr 43kD and 25 kD, respectively. A gene complementing ilvA mutation of E. coli was identified in a library of EcoRI C. glutamicum DNA fragments. This library was constructed using plasmid vector. It was shown that the ilvA gene of C. glutamicum is located inside the 3.6 kb EcoRI fragment and is expressed using its own promoter.     

Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.

Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.     

Structural organization of the Corynebacterium glutamicum plasmid pCG100.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1.9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetR or lacZ alpha genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.     

Cloning and structural-functional analysis in Escherichia coli of genes of glutamate-producing corynebacteria controlling biosynthesis of amino acids of aspartic acid series

A library of EcoRI DNA fragments from Brevibacterium flavum was constructed using plasmid vector. The genes complementing ThrA2 and ThrB mutations in Escherichia coli were identified in the library. The gene thrA2 of B. flavum codes for mutant enzyme homoserine dehydrogenase insensitive to inhibition by threonine. The genes thrA2 and thrB are localized wihtin the EcoRI fragment 4.1 kb long and are expressed under the control of their own promoters in E. coli cells. Structural and functional analysis of cloned C. glutamicum gene ilvA was performed. The gene of C. glutamicum complemented ilvA mutation in E. coli and appeared to be localized within the EcoRI--SacI DNA fragment 1.6 kb in size. Using E. coli minicells we have demonstrated that the gene ilvA of C. glutamicum controls the synthesis of polypeptide of relative molecular mass 50 kD.     

一步法生产1,5-戊二胺谷氨酸棒杆菌基因工程菌的构建

1,5-戊二胺是一种重要的化工原料,发酵法生产1,5-戊二胺是一条新颖且具有潜在竞争力的生产途径。以蜂房哈夫尼菌(Hafnia alvei)AS1.1009基因组为模板,通过PCR扩增,得到大小约为2.2kb的赖氨酸脱羧酶基因ldc。以大肠杆菌(Escherichia coli)/谷氨酸棒杆菌(Corynebacterium glutamicum)穿梭质粒pXMJl9为载体,将扩增得到的目的基因片段克隆至谷氨酸棒杆菌C.glutamicum TK260512,获得重组菌株C.glutamicum TK260512/pXMJl9-ldc.在摇瓶发酵水平上,通过IPTG诱导ldc基因的表达,并采用反相高效液相色谱方法测定了发酵液中1,5-戊二胺的含量,结果显示,经36h发酵,工程菌C.glutamicum TK260512/pXMJ19-ldc的1,5-戊二胺产量为0.96g/L。     

Isolation of ftsI and murE genes involved in peptidoglycan synthesis from Corynebacterium glutamicum

Corynebacterium glutamicum is known to excrete large amounts of L-glutamic acid upon treatment by penicillin. However, the mechanism of L-glutamate overproduction by penicillin treatment is still unknown. A 5.3-kb HindIII fragment was isolated by directly introducing the C. glutamicum (Brevibacterium lactofermentum) ATCC 13869 gene library into the temperature-sensitive Escherichia coli murE mutant and selecting temperature resistant clones. Two open reading frames (ORFs) were found in this fragment: (1) murE, encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-diaminopimelate ligase, and (2)ftsI, encoding septum-peptidoglycan synthetase, one of the targets of penicillin (penicillin-binding protein 3). Both ORFs were involved in peptidoglycan synthesis. Proteins were synthesized from the C. glutamicum murE and ftsI genes, 55 kDa and 73 kDa respectively, in an in vitro protein synthesis system, using E. coli S30 extracts.     

Cloning vector system for Corynebacterium glutamicum.

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.     

Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics

Endogenous plasmids and selectable resistance markers are a fundamental prerequisite for the development of efficient recombinant DNA techniques in industrial microorganisms. In this article, we therefore summarize the current knowledge about endogenous plasmids in amino acid-producing Corynebacterium glutamicum isolates. Screening studies identified a total of 24 different plasmids ranging in size from 2.4 to 95 kb. Although most of the C. glutamicum plasmids were cryptic, four plasmids carried resistance determinants against the antibiotics chloramphenicol, tetracycline, streptomycin-spectinomycin, and sulfonamides. Considerable information is now available on the molecular genetic organization of 12 completely sequenced plasmid genomes from C. glutamicum. The deduced mechanism of plasmid DNA replication and the degree of amino acid sequence similarity among replication initiator proteins was the basis for performing a classification of the plasmids into four distinct C. glutamicum plasmid families.     

Characterization of the cryptic plasmid pCC1 from Corynebacterium callunae and its use for vector construction

The complete nucleotide sequence of the cryptic plasmid pCC1 from Corynebacterium callunae (4109 bp) was determined. DNA sequence analysis revealed five open reading frames longer than 200 bp. One of the deduced polypeptides showed homology with the Rep proteins encoded by plasmids of the pIJ101/pJV1 family of plasmids replicating by the rolling-circle (RC) mechanism. Within this plasmid family, the Rep protein of pCC1 showed the highest degree of similarity to the Rep proteins of corynebacterial plasmids pAG3 and pBL1. These data suggest that the plasmid pCC1 replicates by the RC mechanism. The Escherichia coli/Corynebacterium glutamicum shuttle cloning vector pSCCD1, carrying the pCC1 rep gene on the 2.1-kb DNA fragment and the streptomycin/spectinomycin resistance determinant, was constructed. This vector is stably maintained in population of C. glutamicum cells grown in the absence of selection pressure and it is compatible with plasmid vectors based on corynebacterial plasmids pBL1 and pSR1.     

Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.     

Cloning of a DNA fragment fromCorynebacterium glutamicum conferring aminoethyl cysteine resistance and feedback resistance to aspartokinase

Summary TheCorynebacterium glutamicum/Escherichia coli shuttle vector plasmid pZ1 was used to clone the S-(2-aminoethyl)-d,l-cysteine (AEC)-resistance gene from a lysine-excreting, AEC-resistant strain ofC. glutamicum, the aspartokinase activity of which was released from feedback inhibition by mixtures of lysine and threonine or AEC and threonine respectively. A recombinant plasmid designated pCS2 carrying a 9.9-kb chromosomal insert that conferred AEC resistance and the ability to excrete lysine to its host was isolated. The aspartokinase activity of the pCS2-carrying strain was resistant towards inhibition by mixtures of lysine and threonine or AEC and threonine respectively. By deletion analysis the DNA region conferring AEC resistance to the host and feedback resistance to its aspartokinase activity could be confined to a 1.2-kb DNA fragment.