Production of threonine byBrevibacterium flavum containing threonine biosynthesis genes fromEscherichia coli

Genes of the threonine operon ofEscherichia coli were used for the construction of aBrevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Km^r/Nm^r), various plasmids carryingE. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment ofB. flavum carrying these recombinant plasmids. A mutant ofB. flavum CCM 351 carrying the cloned genesthrA andthrB accumulated 12 g/L of threonine after 48 h of cultivation.     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Fine structure analysis of the threonine operon in Escherichia coli K-12.

Summary A fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.     

A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin

The DNA sequence changes of 31 mutations altering the attenuation control mechanism of the histidine operon are presented. These mutations are discussed in terms of a model for operon regulation that involves a his leader peptide gene whose translation regulates formation of alternative stem-loop structures in the his leader messenger RNA. Three suppressible mutations generate nonsense codons (ochre and UGA) in the his leader peptide gene, demonstrating that translation of this gene is essential for operon expression. Eight mutations presumably reduce the efficiency of translation initiation of the his leader peptide gene, causing reduced levels of operon expression. Five of these mutations directly alter the leader peptide gene initiator codon (AUG). Three mutations alter sequences just in front of the initiator codon and presumably alter the ribosome recognition site. Fourteen mutations reduce the stability of the his leader mRNA stem-loop structures that are alternatives to the attenuator stem. The properties of these mutations provide support for the role of these stem-loop structures in preventing formation of the attenuator stem. Finally, we show that mutations that alter the attenuator stem suppress hisO mutations. This lends support to the proposal that these hisO mutations cause reduced levels of operon expression due to excessive attenuator stem formation. The properties of these 31 mutations provide substantial support for the model of his operon regulation described in this paper.     

Increased stability of recombinant plasmids by Tn1000 insertion in chemostat cultures of recombinantEscherichia coli GT123

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.     

Expression plasmid vectors containing Escherichia coli tryptophan promoter transcriptional units lacking the attenuator

Two DNA fragments which contain the Escherichia coli tryptophan promoter-operator region but lack the attenuator have been used in the construction of a series of pAT153 based plasmids suitable for the regulated expression of foreign genes in E. coli. The first, a 139-bp HhaI fragment includes 59 bp of the trp leader sequence, ending within the “attenuator peptide” coding sequence, eleven codons from the N-terminus. A fusion-type expression plasmid incorporating this fragment has been constructed. The second, a 99-bp HaeIII-TaqI fragment contains no coding sequence but includes the “attenuator peptide” SD site situated 4 bp upstream of the TaqI site. This fragment has been incorporated in expression vectors which result in the direct expression of cloned gene sequences. To further maximise expression, plasmids with directly repeating trp promoter HaeIII-TaqI units have been constructed.     

Expression of leucine genes from an extremely thermophilic bacterium in Escherichia coli

Summary The organisation of the leucine genes in Thermus thermophilus HB8 was analysed by examining the ability of recombinant DNAs to complement Escherichia coli mutations. The arrangement of the genes is different from that in the mesophilic bacteria E. coli and Salmonella typhimurium. The promoter responsible for the expression of the leuB, leuC and leuD genes of Thermus HB8 in E. coli was identified. The sequence of Thermus DNA containing this promoter revealed structural similarities to the promoter and attenuator regions of the E. coli leucine operon.     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown function) and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO ‘operator’ in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase III_S.t. and RNase III_E.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.     

Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12

Summary The nucleotide sequence of the Escherichia coli K12 -methylgalactoside transport operon, mgl, was determined. Primer extension analysis indicated that the synthesis of mRNA initiates at guanine residue 145 of the determined sequence. The operon contains three open reading frames (ORF). The operator proximal ORF, mglB, encodes the galactose binding protein, a periplasmic protein of 332 amino acids including the 23 residue amino-terminal signal peptide. Following a 62 nucleotide spacer, the second ORF, mglA, is capable of encoding a protein of 506 amino acids. The amino-terminal and carboxyl-terminal halves of this protein are homologous to each other and each half contains a putative nucleotide binding site. The third ORF, mglC, is capable of encoding a hydrophobic protein of 336 amino acids which is thought to generate the transmembrane pore. The overall organization of the mglBAC operon and its potential to encode three proteins is similar to that of the ara FGH high affinity transport operon, located approximately 1 min away on the E. coli K12 chromosome.