大肠杆菌色氨酸生物合成途径关键酶的调控

为了通过基因工程手段提高大肠杆菌色氨酸产量, 对色氨酸生物合成途径中的关键基因trpR、tnaA、aroG和trpED进行了改造。首先通过敲除trpR基因解除了基因组上色氨酸合成和转运关键酶受到的反馈阻遏调控, 进而又敲除了tnaA基因, 阻断了色氨酸的分解代谢。然后, 将色氨酸合成途径的关键酶aroGfbr和trpEDfbr基因串联表达, 以去除色氨酸生物合成途径的瓶颈。与对照MG1655相比, trpR基因单敲菌色氨酸浓度提高了10倍, 双敲菌色氨酸浓度提高了约20倍。pZE12-trpEDfbr转入双敲菌后色氨酸浓度提高到168 mg/L, 而将aroGfbr和trpEDfbr转入双敲菌后, 色氨酸浓度提高到820 mg/L。为构建色氨酸高产菌奠定了基础。     

大肠杆菌色氨酸生物合成途径关键酶的调控

为了通过基因工程手段提高大肠杆菌色氨酸产量, 对色氨酸生物合成途径中的关键基因trpR、tnaA、aroG和trpED进行了改造.首先通过敲除trpR基因解除了基因组上色氨酸合成和转运关键酶受到的反馈阻遏调控, 进而又敲除了tnaA基因, 阻断了色氨酸的分解代谢.然后, 将色氨酸合成途径的关键酶aroGfbr和trpEDfbr基因串联表达, 以去除色氨酸生物合成途径的瓶颈.与对照MG1655相比, trpR基因单敲菌色氨酸浓度提高了10倍, 双敲菌色氨酸浓度提高了约20倍.pZE12-trpEDfbr转入双敲菌后色氨酸浓度提高到168 mg/L, 而将aroGfbr和trpEDfbr转入双敲菌后, 色氨酸浓度提高到820 mg/L.为构建色氨酸高产菌奠定了基础.     

Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.     

Genetic Analysis of Regulation of the RecF Pathway of Recombination in Escherichia coli K-12

Genetic evidence is provided supporting the hypothesis that one or more genes of the RecF pathway of recombination other than recA are controlled by the lexA repressor. Using lexA, recA, and recA operator mutations, we also analyze the role of recA and sbcB in regulating the RecF pathway.     

Degradation of mRNA in Escherichia coli

Degradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression. One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli (E. coli). In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E. coli. The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described. At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.     

Ribosome Degradation and the Degradation Products in Starved Escherichia coli

The properties of the abnormal ribonucleoprotein particles produced by Escherichia coli Q-13 starved for glucose were studied. Smaller species of these partially deproteinized particles separable to six distinct sizes contained partially degraded ribonucleic acids. The mode of ribosome degradation under this condition is discussed in terms of differential appearance of these intermediate particles.     

Accumulation of Intermediates of the Carbon-Phosphorus Lyase Pathway for Phosphonate Degradation in phn Mutants of Escherichia coli

The catabolism of phosphonic acids occurs in Escherichia coli by the carbon-phosphorus lyase pathway, which is governed by the 14-cistron phn operon. Here, several compounds are shown to accumulate in strains of E. coli with genetic blocks in various phn cistrons when the strains are fed with phosphonate.Phosphonates (Pn), which contain the carbon-phosphorus bond, are quite abundant in nature, primarily as components of phosphonolipids, where 2-aminoethyl phosphonate (AEPn) is analogous to ethanolamine phosphate as the constituent of phospholipids. In addition, Pn are constituents of polysaccharides, glycoproteins, glycolipids, and several antibiotics (25). Furthermore, large amounts of manmade Pn enter the environment (24). Pn utilization polypeptides in Escherichia coli are specified by the phnCDEFGHIJKLMNOP operon (15). Of the 14 cistrons, one (phnF) encodes a repressor protein, as inferred from sequence alignment (6); three (phnCDE) encode an ABC transport system for Pn (22); seven (phnGHIJKLM) have been postulated to encode the carbon-phosphorus (CP) lyase activity (26); and three (phnNOP) have been postulated to encode “auxiliary enzymes.” Among the latter genes, the phnO gene has been shown to specify an enzyme with aminoalkylphosphonate N-acetyltransferase activity (5). We previously identified the product of the phnN gene as an enzyme capable of catalyzing the phosphorylation of ribose 1,5-bisphosphate to 5-phosphoribosyl α-d-1-diphosphate (ribose 1,5-bisphosphate phosphokinase; EC2.7.4.23) (11). The relation of these two compounds to Pn catabolism remains to be established. Since both the substrate and the product of ribose 1,5-bisphosphate phosphokinase are phosphorylated compounds, we inferred that at least some of the other intermediates of the pathway might be also phosphate esters. It might therefore be possible to identify these intermediates by labeling cells with radioactive phosphate ion in the presence of Pn followed by visualization by appropriate thin-layer chromatography (TLC) procedures. In addition, we employed mutants defective in individual genes of the pathway, and we looked for the accumulation of radiolabeled compounds as candidates for members of the phn pathway. Finally, the mutant strains used harbored the ΔpstS605 allele to render the expression of the phn operon constitutive and, thus, independent of the phosphate supply.     

芳香烃龙胆酸降解途径蛋白质组学的研究

芳香烃是一类重要的环境污染物,微生物降解是其主要的处理方法。研究显示降解过程中产生保守型和诱导型的各一组同工酶。目前,仅有保守型的龙胆酸加双氧酶（GDOI）及其下游片段被克隆。产碱假单胞菌NCIB9867（P25X）的突变株-SNZ28 GDOI被打断,在龙胆酸诱导的情况下,该突变株仍能检测到龙胆酸加双氧酶活性。采用二维蛋白电泳分析突变株SNZ28在有和没有龙胆酸诱导条件下的蛋白质表达差异。电泳结果显示了两者存在有15个蛋白点的差异。通过MALDI-TOF和Q—TOF分析,其中的12个蛋白质点与数据库中已知多肽片段有同源性。其中,P4点与青枯菌（Ralstonia species）龙胆酸1,2加双氧酶同源。该结果在蛋白质组学上证实了GDOII的存在。     

Genetic Characterization of the Phenylacetyl-Coenzyme A Oxygenase from the Aerobic Phenylacetic Acid Degradation Pathway of Escherichia coli

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.     

Biodegradation of Aromatic Compounds by Escherichia coli

Although Escherichia coli has long been recognized as the best-understood living organism, little was known about its abilities to use aromatic compounds as sole carbon and energy sources. This review gives an extensive overview of the current knowledge of the catabolism of aromatic compounds by E. coli. After giving a general overview of the aromatic compounds that E. coli strains encounter and mineralize in the different habitats that they colonize, we provide an up-to-date status report on the genes and proteins involved in the catabolism of such compounds, namely, several aromatic acids (phenylacetic acid, 3- and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3-hydroxyphenylpropionic acid, and 3-hydroxycinnamic acid) and amines (phenylethylamine, tyramine, and dopamine). Other enzymatic activities acting on aromatic compounds in E. coli are also reviewed and evaluated. The review also reflects the present impact of genomic research and how the analysis of the whole E. coli genome reveals novel aromatic catabolic functions. Moreover, evolutionary considerations derived from sequence comparisons between the aromatic catabolic clusters of E. coli and homologous clusters from an increasing number of bacteria are also discussed. The recent progress in the understanding of the fundamentals that govern the degradation of aromatic compounds in E. coli makes this bacterium a very useful model system to decipher biochemical, genetic, evolutionary, and ecological aspects of the catabolism of such compounds. In the last part of the review, we discuss strategies and concepts to metabolically engineer E. coli to suit specific needs for biodegradation and biotransformation of aromatics and we provide several examples based on selected studies. Finally, conclusions derived from this review may serve as a lead for future research and applications.     

Degradation of Lindane by Escherichia coli

Lindane was degraded by Escherichia coli isolated from rat feces. About 10% of the added lindane was metabolized by the bacterium in Trypticase soy broth containing the pesticide. A single metabolite, 2,3,4,5,6-pentachloro-1-cyclohexene, was detected and identified by gas chromatography and mass spectrometry.     

Tuned Escherichia coli as a host for the expression of disulfide-rich proteins

Disulfide-bond formation is a major post-translational modification and is essential for protein folding, stability, and function. This is especially true for secreted proteins, many of which possess great potential for biotechnological applications. Focusing on the use of Escherichia coli for the production of this class of proteins, we describe the mechanisms that maintain redox compartmentalization in the cell, with an emphasis on those that promote the formation and isomerization of disulfide bonds in the bacterial periplasm, while presenting parallel pathways in the eukaryotic endoplasmic reticulum. Based on these concepts, we review the use of E. coli as a cell factory for the production of heterologous disulfide-containing proteins using either peri- or cytoplasmic expression and, in particular, how these compartments can be tuned to improve the yield of correctly folded recombinant proteins. Finally, we describe a few examples of the production of small disulfide-rich proteins (protease inhibitors) to illustrate how soluble, active, and fully oxidized recombinants may be successfully obtained upon peri- or cytoplasmic expression in E. coli.     

Regulation of the methionine regulon in Escherichia coli

The genes involved in methionine biosynthesis are scattered throughout the Escherichia coli chromosome and are controlled in a similar but not coordinated manner. The product of the metJ gene and S-adenosylmethionine are involved in the repression of this ‘regulon’.     

Pathway Choice in Glutamate Synthesis in Escherichia coli

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.     

Regulation of the Pathway for the Degradation of Anthranilate in Aspergillus niger

Studies were carried out to determine the factors governing the induction of anthranilate hydroxylase and other enzymes in the pathway for the dissimilation of anthranilate by Aspergillus niger (UBC 814). The enzyme was induced by growth in the presence of tryptophan, kynurenine, anthranilate, and, surprisingly, by 3-hydroxyanthranilate, which was not an intermediate in the conversion of anthranilate to 2,3-dihydroxybenzoate. There was an initial lag in the synthesis of anthranilate hydroxylase when induced by tryptophan, anthranilate, and 3-hydroxyanthranilate. Cycloheximide inhibited the enzyme induction. Comparative studies on anthranilate hydroxylase, 2,3-dihydroxybenzoate carboxy-lyase, and catechol 1:2-oxygenase revealed that these enzymes were not coordinately induced by either anthranilate or 3-hydroxyanthranilate. Structural requirements for the induction of anthranilate hydroxylase were determined by using various analogues of anthranilate. The activity of the constitutive catechol oxygenase was increased threefold by exposure to anthranilate, 2,3-dihydroxybenzoate, or catechol. 3-Hydroxyanthranilate did not enhance the levels of catechol oxygenase activity.