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。     

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.     

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.     

MAL1基因启动子区的克隆及其在大肠杆菌中的启动功能分析

目的：MAL1基因启动子区在原核生物大肠杆菌中是否有双向启动外源基因的功能。方法：利用PCR扩增MAL1基因启动子区,将不同方向的MAL1基因启动子区后接上Zeocin基因,构建成重组报告质粒,转化大肠杆菌DH5α,验证该启动子区是否双向都具有启动外源基因的功能。结果：将含不同方向启动子区的重组质粒PUCMZ1和PUCMZ2,转化大肠杆菌,转化子均有Zeoein抗性。结论：证明了MAL1结构基因上游启动子区在原核生物大肠杆菌中具有双向启动的功能,且3′-5′方向的启动能力强于5′-3′方向。     

Arginine Catabolism and the Arginine Succinyltransferase Pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.     

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’.     

Promoter recognition and promoter strength in the Escherichia coli system.

The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude. Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E. coli. The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25. Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found. This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.