【導(dǎo)讀】化學(xué)工程系，加利福尼亞大學(xué)，洛杉磯，CA90034.*作者().摘要代謝工程在復(fù)雜多樣的宿主內(nèi)產(chǎn)生外源代謝物方面取得了可喜的成果。代謝失衡和不理想的產(chǎn)物出現(xiàn)。我們已經(jīng)闡述了代謝工程的另一種進(jìn)程，通過設(shè)計調(diào)?？鼗芈肥拐{(diào)控子根據(jù)細(xì)胞內(nèi)代謝的狀態(tài)來調(diào)節(jié)基因表達(dá)。尤其在大腸桿菌中恢復(fù)和改。變調(diào)整回路體系中的一個Ntr調(diào)控子來控制番茄紅素的生物合成途徑。這樣當(dāng)減少因代謝失衡引起的消極影響時，細(xì)胞內(nèi)操控系統(tǒng)就會明顯提高。番茄紅素的產(chǎn)量。雖然我們論證這種可以提高代謝產(chǎn)量的方法，但是它也可被延伸到。其他領(lǐng)域，而在這些領(lǐng)域中基因表達(dá)必須受細(xì)胞生理學(xué)嚴(yán)格的約束，如基因治療。生物資料和功能基因的學(xué)科進(jìn)軍作為進(jìn)步的結(jié)果。除了要構(gòu)造代謝途徑的遺傳組成成分。動力控制重組細(xì)胞路徑可以潛在地導(dǎo)致產(chǎn)量提高、將生長阻礙減小到最小、減少。有毒副產(chǎn)品形成。在NRII缺失時，NRI才能響應(yīng)ACP水平。能夠?qū)^量的碳流量做出響應(yīng)，而過量的碳流量可以被醋酸鹽的排泄物反映出來。

　　

【正文】 opene. Combined, the p2IDI strain produced acetate 3 times less than pTacIDI, which indicates to the acetate salt of the carbon flow to be diverted to the lycopene, as shown in (Fig. 5A). the pTacIDI strains produce similar fermentation the sample as BW18302 host control, it implies that the expression of lycopene in this strain has not changed metabolic flux (Fig. 5 D). These results are summarized in Table 1. From the p2IDI strain pyruvate excreta still very high, even in the process of most of the time it is less than pTacIDI amount of production (Figure 5 C). Since the intracellular metabolite excretion of pyruvate Be one of the pioneers in the path of the isoprenoid pointed out that carbon is still not effectively transferred to lycopene. Nevertheless, PPS overexpression in the sugar deposition conditions can also cause growth inhibition (Figure 2B). So in order to avoid metabolic imbalance PPS overexpression the final lycopene production strain contains a control idi and pps (glnAp2idi + glnAp2pps) artificial regulator, the remainder of the path of lycopene (DXS, GPS, crtBI) under the control of the Ptac. Lycopene concentration increased by 50% to cause production increased three times from (mg / ) to (mg / ) shown in Figure 5A. This strain does not appear to growth delay phenomenon is shown in Figure 5D illustrates this contrast, strain and contains pTacIDI and pPS184 (Ptacidi + Ptacpps). , GlnAp2 adjust the PPS in the subexpression by increasing the utilization of pyruvate to create the conditions for lycopene yield (Figure 5C and Table 1 above) Figure 5 through the ACP and glnAp2 dynamics control to improve the yield of lycopene from E. coli. (A) containing % of glucose YE medium lycopene production. (B) Titanium secretions. (C) pyruvate secretions. (D) growth activity These results indicate that the kiics provided by the the glnAp2 artificial regulon rate control allows to control the expression of the enzyme, these enzymes are used to help lycopene biosynthesis. In addition, these results propose an effective strategy for metabolic engineering. And not to the past, trying to delete the adjustment loop, or attempt by overexpression of key enzymes to suppress them. We believe that, then under certain circumstances, the metabolic imbalance can be adjusted by a reasonable manner the control handle. This method can be optimized metabolic pathways important for cell viability Biotechnology can also modify. For example, the rules of the cell cycle, protein replacement and high levels of protein products. In addition, he may bee an important means of gene therapy, but reasonable control circuit is designed to expand the path of the dual application will bee an effective design of industrial applications of microorganisms. Experimental program Materials All chemicals from Sigma (St. Louis, MO). Modification and restriction enzyme from the life process. All PCRrelated materials from Promega (Madison, WI), oligonucleotides from Genosys (The Woodlands, TX). Bacterial strains and plasmids E. coli strain BW13711 (lacX74) and BW18302 (lacX74glnL20 01) by Alex Ninfa friendly, the University of Michigan. Strains VJS632 (K12 prototroph) provided by the University of California, Davis. Strains JCL1595 and JCL1596 dissolved glnAP2lacZ together built, used to build the strains JCL1595 and JCL1596 of plasmid PSR551 and λRS45 of antibiotics generously donated by the University of California, Los Angeles, Bob Simon. The plasmid pRW5tkt33 and pJF118EH23 respectively, by the the Palo Alt and Michael Bagdasarian, Michigan State University for the generous gift. Plasmids PAROG by cloning contains pRW5tkt aroGfbr PCR fragment inserted into the EcoRIBamHI sites pJF118EH constructed plasmid pTacIDI clone contains idi PCR fragment from the E. coli chromosome is constructed to The pJF118EH bit point EcoRIPstI plasmid PPS706 has been described. Contains the promoter and NRI binding sites glnAp2 promoter region is from the use of the front guide 5 CAGCTGCAAAGGTCATTGCAC CAAC and the reverse primer 5 GGTACCAGTACGTGTTCAGCGGACATAC coli chromosome PCR amplification, two primers amplify a region of DNA sequence position between 93 and 343. The glnAp2 promoter PCR fragment was cloned into EcoRV the plasmid pAROG, pPS706 and pTacIDI EcoRI sites to the the plasmid p2AROG3, pPSG706 and p2IDI. The plasmid pPS184 and pPSG184 by cutting off from pPS706 the Ptacpps pPSG706 the, glnAp2pps fragments, using EcoRV and BamHI and inserted into the corresponding sites of pACYC184 built. The glnAp2 PCR fragments were cloned into pRS5 The 51 sites EcoRI This produce containing glnAp2 p2GFPuv. the glnAp2lacZ region be transferred to the same restructuring RS45 the antibacterial body to produce l p2GFPuv. Growth conditions all E. coli strains were grown in a fixed medium shake flask culture was placed in a growth medium containing % glucose M9 the fixed salts or containing % glucose, YE fixed salt medium. YE the fixed salt thereof by 14 g / l K2HPO4, 16 g / l KH2PO4, 5 g / l (NH4) 2SO4, 1 g MgSO4, and 1 mg / l thiamine. The cell mixture was stirred at the wavelength of 550nm is detected. SDSPAGE analysis and enzyme Laemmli SDSPAGE describe Bill betanougat activity measurement is acplished by Miller.