Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

Threonine Locus of Escherichia coli K-12: Genetic Structure and Evidence for an Operon

Three genes, thrA, thrB, and thrC, were previously defined and localized in the threonine locus of Escherichia coli K-12. thrA, thrB, and thrC specify the enzymes aspartokinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase, respectively. A complementation analysis of the threonine cluster using derivatives of a lambda phage carrying the threonine genes (lambdadthr(c)) demonstrates that: (i) thrB and thrC each consist of a single cistron; and (ii) thrA is composed of two cistrons, thrA(1) and thrA(2), although it specifies a single polypeptide chain. thrA(1) and thrA(2) correspond to aspartokinase I and homoserine dehydrogenase I, respectively. Their relative order is established. The demonstration of polar effects of mutations (nonsense or induced by phage Mu) in thrA and thrB is taken as evidence for the existence of a thrA thrB thrC operon, transcribed in this order.     

Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrc genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed-field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.     

Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions.

We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction. The intergenic sequence thrA and thrB is only one base pair. The coding region for homoserine kinase is 927 base pairs long. It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB. The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes. The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I.     

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.     

含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响

摘要：【目的】通过分子生物学手段构建重组质粒,将其转入野生型大肠杆菌W3110,分析含苏氨酸操纵子基因的质粒及质粒定点突变解除反馈抑制时,对L-苏氨酸积累的影响。【方法】以W3110染色体DNA为模板,PCR扩增苏氨酸操纵子基因,即启动子THrLp、编码前导肽基因thrL以及thrA、thrB、thrC基因,通过重叠延伸PCR的方法对thrA基因定点突变,解除苏氨酸对它的反馈抑制,构建出重组表达质粒WYE112和WYE134,5 L发酵实验测定L-苏氨酸的产量。【结果】经5 L发酵罐发酵产酸实验,W3110的L-苏氨酸产量为0.036 ± 0.004 g/L,携带含苏氨酸操纵子质粒的W3110菌株L-苏氨酸产量为2.590 ± 0.115 g/L,质粒上thrA解除反馈抑制后,L-苏氨酸的产量增加到9.223 ± 1.279 g/L。【结论】过表达苏氨酸操纵子基因可以使L-苏氨酸积累,进一步解除thrA基因的反馈抑制,可以增强L-苏氨酸积累的效果,为L-苏氨酸工程菌改造的进一步研究奠定了基础。     

Cloning and characterization of the mutated threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens

The entire threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens TLr156, which lacks threonine-mediated feedback inhibition of both aspartokinase I (AK I) and homoserine dehydrogenase I (HD I), was cloned on a multicopy plasmid pLG339. Hybrid plasmid pSK301 carried a 6.5-kb chromosomal DNA. Several derivatives of pSK301 with Tn1000 insertions were obtained. By examining the phenotypes and the physical maps of these plasmids, we could define the loci of the thrA(1)5A(2)5, thrB, and thrC genes. The thrA(1)5A(2)5 and thrC gene products were identified by the maxicell method as proteins with Mrs of 85,000 and 43,000, respectively. The thrA(1)5A(2)5 genes encode a single polypeptide similar to the thrA1A2 genes of Escherichia coli. Plasmid pSK301 was introduced into S. marcescens T-1112, in which both AK I and HD I are produced constitutively. The resulting transformant carried five to six copies of pSK301 per chromosome and produced the AK I and HD I enzymes at three to four times higher level than control strain T-1112[pLG339]. Strain T-1112[pSK301] produced four times higher levels of threonine than strain T-1112[pLG339], yielding about 35 mg of threonine per ml of a medium containing sucrose and urea.     

Analysis of genomic sequences by Chaos Game Representation

MOTIVATION: Chaos Game Representation (CGR) is an iterative mapping technique that processes sequences of units, such as nucleotides in a DNA sequence or amino acids in a protein, in order to find the coordinates for their position in a continuous space. This distribution of positions has two properties: it is unique, and the source sequence can be recovered from the coordinates such that distance between positions measures similarity between the corresponding sequences. The possibility of using the latter property to identify succession schemes have been entirely overlooked in previous studies which raises the possibility that CGR may be upgraded from a mere representation technique to a sequence modeling tool. RESULTS: The distribution of positions in the CGR plane were shown to be a generalization of Markov chain probability tables that accommodates non-integer orders. Therefore, Markov models are particular cases of CGR models rather than the reverse, as currently accepted. In addition, the CGR generalization has both practical (computational efficiency) and fundamental (scale independence) advantages. These results are illustrated by using Escherichia coli K-12 as a test data-set, in particular, the genes thrA, thrB and thrC of the threonine operon.     

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.     

Nucleotide sequence and fine structural analysis of the Corynebacterium glutamicum hom-thrB operon

The complete nucleotide sequence of the Corynebacterium glutamicum hom-thrB operon has been determined and the structural genes and promoter region mapped. A polypeptide of Mr 46,136 is encoded by hom and a polypeptide of Mr 32,618 is encoded by thrB. Both predicted protein sequences show amino acid sequence homology to their counterparts in Escherichia coli and Bacillus subtilis. The promoter region has been mapped by S1-nuclease and deletion analysis. Located between -88, RNA start site and -219 (smallest deletion clone with complete activity) are sequence elements similar to those found in E. coli and B. subtilis promoters. Although there are no obvious attenuator-like structures in the 5'-untranslated region, there is a dyad-symmetry element, which may act as an operator.     

A probabilistic measure for alignment-free sequence comparison

MOTIVATION: Alignment-free sequence comparison methods are still in the early stages of development compared to those of alignment-based sequence analysis. In this paper, we introduce a probabilistic measure of similarity between two biological sequences without alignment. The method is based on the concept of comparing the similarity/dissimilarity between two constructed Markov models. RESULTS: The method was tested against six DNA sequences, which are the thrA, thrB and thrC genes of the threonine operons from Escherichia coli K-12 and from Shigella flexneri; and one random sequence having the same base composition as thrA from E.coli. These results were compared with those obtained from CLUSTAL W algorithm (alignment-based) and the chaos game representation (alignment-free). The method was further tested against a more complex set of 40 DNA sequences and compared with other existing sequence similarity measures (alignment-free). AVAILABILITY: All datasets and computer codes written in MATLAB are available upon request from the first author.     

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.     

Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli

Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.