重组大肠杆菌生产谷胱甘肽发酵条件的研究

研究了重组Ｅ．Ｃｏｌｉ产ＧＳＨ的发酵条件,重点考察了添加酵母膏、前体氨基酸和ＡＴＰ的影响。结果发现,前体氨基酸和ＡＴＰ均能促进胞内ＧＳＨ的积累,若在发酵０ｈ和１２ｈ分别加入２０ｇ／ＬＡＴＰ和９ｍｍｏｌ／Ｌ前体氨基酸,则细胞干重和胞内ＧＳＨ含量可分别比对照提高２４％和１４倍。应用正交试验得出的针对细胞干重和ＧＳＨ总量的最佳组合,最大细胞干重和ＧＳＨ总量比原试验中的最好结果分别提高了１０％和２６％。在分析了该菌对葡萄糖利用情况的基础上,对该菌进行了指数流加培养,２５ｈ细胞干重与发酵液内ＧＳＨ总量分别达到８０ｇ／Ｌ和８８０ｍｇ／Ｌ,比摇瓶最好结果分别提高了８３和４６倍。     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.     

大肠杆菌原核增强子样序列的克隆及其结构与功能的研究

利用氯霉素乙酰转移酶(cat)以及β-半乳糖苷酶(lacZ)基因作为报告基因,从大肠杆菌MC1061株染色体基因组中克隆到3个原核增强子样序列——MC2,MC8,MC9,这3个片段均具有正反向增强活性,对β-半乳糖苷酶基因的增强活性(正向)在2～5.5倍之间。采用体内转录,RNA Dot blot杂交的方法对MC8的功能进行了研究,结果表明,MC8片段对于基因表达的调控发生在转录水平上。用核酸外切酶III末端缺失的方法对MC8的功能区进行了定位。结果显示,MC8的功能区位于距其正向克隆5′端450～950bp长约500bp的区段内。在450～600bp以及840～950bp区段内至少分别含有一个功能位点。序列分析的结果表明,MC8功能区有3个AT丰富区,其中2个分别位于450～600bp以及840～950bp区段内。     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.     

Butanol production from glycerol by recombinant Escherichia coli

Escherichia coli MG1655 (DE3) with the ability to synthesize butanol from glycerol was constructed by metabolic engineering. The genes thil, adhe2, bcs operon (crt, bcd, etfB, etfA, and hbd) were cloned into the plasmid vectors, pETDuet-1 and pACYCDuet-1, then the two resulting plasmids, pACYC-thl-bcs and pET-adhe2, were transferred to E. coli, and the recombinant strain was able to synthesize up to 18.5 mg/L butanol on a glycerol-containing medium. After the glycerol transport protein gene GlpF was expressed, the butanol production was improved to 22.7 mg/L. The competing pathway of byproducts, such as ethanol, succinate, and lactate, was subsequently deleted to improve the 1-butanol production to 97.9 mg/L. Moreover, a NADH regeneration system was introduced into the E. coli, and finally a 154.0 mg/L butanol titer was achieved in a laboratory-scale shake-flask experiment.     

Improved conditions for production of recombinant plant sesquiterpene synthases in Escherichia coli

Amorpha-4,11-diene synthase (ADS) from Artemisia annua and (+)-germacrene synthase (GDS) from Zingiber officinale were expressed in Escherichia coli under different conditions to optimize the yield of active soluble protein. The cDNAs of these enzymes were inserted into the pET28 vector (Novagen) and expressed in four different bacterial strains; BL21 (DE3), BL21 (DE3) Tuner, BL21 (DE3) pLysS and BL21 (DE3) pLysS Tuner using different inducing agents (IPTG, The Inducer). The effects of induction under osmotic stress in the presence of glycine betaine and sorbitol were investigated. Although background expression for ADS was reduced when using pLysS strains, no significant difference was noted for ADS activity in soluble whole cell lysates after induction with either IPTG or The Inducer. For GDS, on the other hand, the change between BL21 (DE3) cells and BL21 (DE3) Tuner, induced with IPTG, leads to a twofold increase in enzyme activity in the soluble fraction while a reduction in activity is observed when using the pLysS strains. The same doubling of activity is observed for GDS when the commonly used BL.21 (DE3) is induced with The Inducer. Addition of 2.5 mM glycine betaine and 660 mM sorbitol to the bacterial growth media resulted in reduction of growth rate and biomass yield but under these conditions the best overall protein production, for both enzymes, was obtained. Compared to the standard conditions previously used in our laboratory the yield of soluble active protein was increased 7- and 2.5-fold for ADS and GDS, using BL21 (DE3) pLysS Tuner and BL21 (DE3), respectively.     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Assessments of growth conditions on the production of cyanophycin by recombinant Escherichia coli strains expressing cyanophycin synthetase gene

The synthesis of cyanophycin, a biodegradable polymer, is directed by cyanophycin synthetase. Polymerase chain reaction (PCR) cloned the gene cphA coding for cyanophycin synthetase from Synechocystis sp. PCC 6803 into pET-21b followed by transformation into two Escherichia coli hosts. The culture conditions for cyanophycin production were investigated, and the molecular weight and compositions of purified cyanophycin were analyzed. The results showed that E. coli BL21-CodonPlus(DE3)-RIL could produce 120 mg cyanophycin per gram dry cell weight in terrific medium. The purified cyanophycin consisted of insoluble and soluble forms at pH 7. The insoluble form had a higher molecular weight (20-32 kDa) than the soluble form (14-25 kDa). Both forms are composed of three major amino acids, aspartic acid, arginine, and lysine, and the insoluble form showed a higher arginine/lysine molar ratio (4.61 ± 0.31) than the soluble form (0.89 ± 0.05). In addition, the nitrogen sources could affect the yields of insoluble and soluble forms of cyanophycin. The medium containing additional lysine could enhance the proportion of the soluble form, but had little effect on the lysine and arginine percentages of both soluble and insoluble forms. The medium containing additional arginine slightly decreased the proportion of soluble form and altered its amino acid composition, with a minimal effect on the lysine and arginine percentages in the insoluble form.     

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830: pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage p_R promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30°C; the temperature shift to induce production of fusion protein was varied from 36 to 42°C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38°C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/l per hour). By applying a fedbatch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.     

Microcalorimetric studies on the polyhydroxyalkanoates production of recombinant Escherichia coli

The thermogenic curves of metabolism of two strains of Escherichia coli pUC19cab/XL-IBlue and XL-IBlue have been determined by using a LKB-2277 bioActivity Monitor and ampoule method at 37 degrees C. pUC19cab/XL-IBlue was a recombinant E. coli strain bearing a foreign plasmid pUC19cab which brought the polyhydroxyalkanoates (PHAs) production. XL-IBlue was a host bacterium without any foreign DNA. Our studies reveal that the PHA production of recombinant E. coli has an apparent influence on their thermogenic curves of metabolism and therefore the initial time of PHAs production can be determined from these thermogenic curves.     

重组大肠杆菌产尿素酶B高密度发酵条件的优化

探究重组大肠杆菌产尿素酶B（urease B subunit, UreB）的高密度发酵条件。通过实验室摇瓶和30 L发酵罐对UreB基因工程菌的发酵条件进行优化。结果表明：30 L发酵罐中以TB培养基为发酵培养基,接种量为5%,发酵温度为37 ℃,pH为6.8,溶氧量为30%左右,培养至2 h开始恒速流加50%甘油,4 h流加50%酵母提取物和50%胰蛋白胨,并加入终浓度为0.5 mmol/L的异丙基β-D-硫代半乳糖苷（isopropyl β-D-thiogalactoside,IPTG),诱导表达4 h,结束发酵,所得菌体干物质约为25.7 g/L,UreB表达量为31.4%。此工艺可以提高UreB的产量。     

Effects of environmental conditions on xylose fermentation by recombinant Escherichia coli.

In batch fermentations, optimal conversion of xylose to ethanol by recombinant Escherichia coli was obtained under the following conditions: 30 to 37 degrees C, pH 6.4 to 6.8, 0.1 to 0.2 M potassium phosphate buffer, and xylose concentrations of 8% or less. A yield of 39.2 g of ethanol per liter (4.9% ethanol by volume) was observed with 80 g of xylose per liter, equivalent to 96% of the maximum theoretical yield. Maximal volumetric productivity was 0.7 g of ethanol per liter per h in batch fermentations and 30 g of ethanol per liter per h in concentrated cell suspensions (analogous to cell recycling).     

Kinetics of ethanol production by recombinant Escherichia coli KO11

The fermentation kinetics for separate as well as simultaneous glucose and xylose fermentation with recombinant ethanologenic Escherichia coli KO11 are presented. Glucose and xylose were consumed simultaneously and exhibited mutual inhibition. The glucose exhibited 15 times stronger inhibition in xyclose fermentation than vice versa. The fermentation of condensate from steampretreated willow (Salix) was investigated. The kinetics were studied in detoxified as well as in nondetoxified condensate. The fermentation of the condensate followed two phases: First the glucose and some of the pentoses (xylose in addition to small amounts of arabinose) were fermented simultaneously, and then the remaining part of the pentoses were fermented. The rate of the first phase was independent of the detoxification method used, whereas the rate of the second phase was found to be strongly dependent. When the condensate was detoxified with overliming in combination with sulfite, which was the best detoxification method investigated, the sugars in the condensate, 9 g/L, were fermented in 11 h. The same fermentation took 150 h in nondetoxified condensate. The experimental data were used to develop an empirical model, describing the batch fermentation of recombinant E. coli KO11 in the condensate. The model is based on Monod kinetics including substrate and product inhibition and the sum of the inhibition exerted by the rest of the inhibitors, lumped together. (c) 1995 John Wiley & Sons, Inc.     

Extracellular production of phospholipase A2 from Streptomyces violaceoruber by recombinant Escherichia coli

Phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber was successfully produced extracellularly in an active form by using a recombinant strain of Escherichia coli. The PLA(2) gene, which was artificially synthesized with optimized codons for E. coli and fused with pelB signal sequence, was expressed in E. coli using pET system. Most of the enzyme activity was detected in the culture supernatant with negligible activity in the cells. The recombinant enzyme was purified to homogeneity from the culture supernatant simply by ammonium sulfate precipitation and an anion exchange chromatography. The purified enzyme showed a specific activity comparable to that of the authentic enzyme. The recombinant enzyme had the same N-terminal amino acid sequence to that of the mature protein, indicating the correct removal of the signal peptide. An inactive PLA(2) with a mutation at the catalytic center was also secreted to the culture medium, suggesting that the observed secretion was not dependent on enzymatic activity. A simple screening method for the PLA(2)-producing colonies was established by detecting clear zone formation around the colonies on agar media containing lecithin. This is the first example of direct extracellular production of active PLA(2) by recombinant E. coli.     

Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy

Halohydrin dehalogenases are attractive biocatalysts in producing a series of important chiral building blocks. Recombinant expression of halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) in Escherichia coli using T7 promoter-based pGEF(+) system revealed much lower expression level than that of the well-studied halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). In this study, we changed the codon usage in the 5′-end of hheA gene to improve the expression yield of HheA. Our results showed that the expression of HheA could be largely improved by the replacement of G-rich +2 codon (adjacent to the start codon) with less G-containing codons. The expression of one of the resulting mutants HheA-D1 (replaced +2 codon GTG with CCA) was about 4-fold higher and purified yields about 8-fold greater than that of the wild-type HheA. Moreover, the expression level of the resulting HheA variants correlated well with the minimal folding free energy (ΔG) of the mRNA secondary structure surrounding the 5′-end region of the genes. These findings suggested that the G-rich +2 codon of hheA gene might be the main suppressive factor for limiting the recombinant expression of HheA and that +2 codon optimization strategy could be used as a general tool in modulating recombinant protein production in E. coli.     

Beta-lactamase production by Escherichia coli under different growth conditions

Facultative anaerobic bacteria, such as Escherichia coli, are more resistant to cephalosporin antibiotics during anaerobic growth. Strict anaerobic ambience reduces beta-lactamase production or the enzyme affinities for their substrates. A different balance between DNA gyrase and topoisomerase I activity, during aerobic and anaerobic growth condition, could be related to the bacteria behavior.     

The production of polyhydroxyalkanoates in recombinant Escherichia coli

Polyhydroxyalkanoates, the natural polyester that many microorganisms accumulate to store carbon and reducing equivalents, have been considered as a future alternative of traditional plastic due to their special properties. In Escherichia coli, a previous non-polyhydroxyalkanoates producer, pathway engineering has been developed as a very powerful approach to set up microbial production process through the introduction of direct genetic changes by recombinant DNA technology. Various metabolic pathways leading to the polyhydroxyalkanoates accumulation with desirable properties at low-cost and high-productivity have been developed. At the same time, high density fermentation technology of E. coli provides an efficient polyhydroxyalkanoates production strategy. This review focused on metabolic engineering, fermentation and downstream process aiming to polyhydroxyalkanoates production in E. coli.