共查询到20条相似文献,搜索用时 15 毫秒
1.
Keming Liang Claire R. Shen 《Journal of industrial microbiology & biotechnology》2017,44(12):1605-1612
Enzymatic reduction of acetoin into 2,3-butanediol (2,3-BD) typically requires the reduced nicotinamide adenine dinucleotide (NADH) or its phosphate form (NADPH) as electron donor. Efficiency of 2,3-BD biosynthesis, therefore, is heavily influenced by the enzyme specificity and the cofactor availability which varies dynamically. This work describes the engineering of cofactor flexibility for 2,3-BD production by simultaneous overexpression of an NADH-dependent 2,3-BD dehydrogenase from Klebsiella pneumoniae (KpBudC) and an NADPH-specific 2,3-BD dehydrogenase from Clostridium beijerinckii (CbAdh). Co-expression of KpBudC and CbAdh not only enabled condition versatility for 2,3-BD synthesis via flexible utilization of cofactors, but also improved production stereo-specificity of 2,3-BD without accumulation of acetoin. With optimization of medium and fermentation condition, the co-expression strain produced 92 g/L of 2,3-BD in 56 h with 90% stereo-purity for (R,R)-isoform and 85% of maximum theoretical yield. Incorporating cofactor flexibility into the design principle should benefit production of bio-based chemical involving redox reactions. 相似文献
2.
Engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> for efficient cellobiose utilization
Escherichia coli normally cannot utilize the β-glucoside sugar cellobiose as a carbon and energy source unless a stringent selection pressure
for survival is present. The cellobiose-utilization phenotype can be conferred by mutations in the two cryptic operons, chb and asc. In this study, the cellobiose-utilization phenotype was conferred to E. coli by replacing the cryptic promoters of these endogenous operons with a constitutive promoter. Evolutionary adaptation of the
engineered strain CP12CHBASC by repeated subculture in cellobiose-containing minimal medium led to an increase in the rate
of cellobiose uptake and cell growth on cellobiose. An efficient cellobiose-metabolizing E. coli strain would be of great importance over glucose-metabolizing E. coli for a simultaneous saccharification and fermentation process, as the cost of the process would be reduced by eliminating
one of the three enzymes needed to hydrolyze cellulose into simple sugars. 相似文献
3.
Chou CP 《Applied microbiology and biotechnology》2007,76(3):521-532
The advent of recombinant DNA technology has revolutionized the strategies for protein production. Due to the well-characterized
genome and a variety of mature tools available for genetic manipulation, Escherichia coli is still the most common workhorse for recombinant protein production. However, the culture for industrial applications often
presents E. coli cells with a growth condition that is significantly different from their natural inhabiting environment in the gastrointestinal
tract, resulting in deterioration in cell physiology and limitation in cell’s productivity. It has been recognized that innovative
design of genetically engineered strains can highly increase the bioprocess yield with minimum investment on the capital and
operating costs. Nevertheless, most of these genetic manipulations, by which traits are implanted into the workhorse through
recombinant DNA technology, for enhancing recombinant protein productivity often translate into the challenges that deteriorate
cell physiology or even jeopardize cell survival. An in-depth understanding of these challenges and their corresponding cellular
response at the molecular level becomes crucial for developing superior strains that are more physiologically adaptive to
the production environment to improve culture productivity. With the accumulated knowledge in cell physiology, whose importance
to gene overexpression was to some extent undervalued previously, this review is intended to focus on the recent biotechnological
advancement in engineering cell physiology to enhance recombinant protein production in E. coli. 相似文献
4.
Nguyen Huy Thuan Amit Kumar Chaudhary Duong Van Cuong Nguyen Xuan Cuong 《Journal of industrial microbiology & biotechnology》2018,45(3):175-185
Microbial cells have extensively been utilized to produce value-added bioactive compounds. Based on advancement in protein engineering, DNA recombinant technology, genome engineering, and metabolic remodeling, the microbes can be re-engineered to produce industrially and medicinally important platform chemicals. The emergence of co-culture system which reduces the metabolic burden and allows parallel optimization of the engineered pathway in a modular fashion restricting the formation of undesired byproducts has become an alternative way to synthesize and produce bioactive compounds. In this study, we present genetically engineered E. coli-based co-culture system to the de novo synthesis of apigetrin (APG), an apigenin-7-O-β-d-glucopyranoside of apigenin. The culture system consists of an upstream module including 4-coumarate: CoA ligase (4CL), chalcone synthase, chalcone flavanone isomerase (CHS, CHI), and flavone synthase I (FNSI) to synthesize apigenin (API) from p-coumaric acid (PCA). Whereas, the downstream system contains a metabolizing module to enhance the production of UDP-glucose and expression of glycosyltransferase (PaGT3) to convert API into APG. To accomplish this improvement in titer, the initial inoculum ratio of strains for making the co-culture system, temperature, and media component was optimized. Following large-scale production, a yield of 38.5 µM (16.6 mg/L) of APG was achieved. In overall, this study provided an efficient tool to synthesize bioactive compounds in microbial cells. 相似文献
5.
The excretion of the aromatic amino acid l-tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was
eliminated by deleting the tyrR gene. The generation of this l-tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no
structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor
substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing l-tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that l-tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential
growth phase. The final l-tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of l-tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively. 相似文献
6.
Background
Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.Results
The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD600nm of 80 and a product titer of 722.1 mg TAG L-1 at the end of the process.Conclusions
This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.7.
PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA Cma , 1179 bp), acetoacetyl-CoA reductase (phaB Cma , 738 bp), and PHA synthase (phaC Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD600, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa. 相似文献
8.
Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present
work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression
of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g
dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of
four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original
strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture
12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol
production to date and showed promising to make the bioprocess economically feasible. 相似文献
9.
Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of
enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality.
Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers
such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory
strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular
production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies
have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached.
This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that
significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials
and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy. 相似文献
10.
Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH4)2SO4, NH4Cl, (NH4)2HPO4 and KNO3, (NH4)2HPO4 showed the best effect on improving the titer of indigoidine. Different concentrations of (NH4)2HPO4 were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH4)2HPO4. This work provides two efficient methods for the production of this promising blue pigment in E. coli. 相似文献
11.
V. B. Panichkin V. A. Livshits I. V. Biryukova S. V. Mashko 《Applied Biochemistry and Microbiology》2016,52(9):783-809
The review summarizes the main approaches applied during the creation of L-tryptophan producing strains based on Escherichia coli for the industrial production of this amino acid. In addition, some prospects for the further improvement of tryptophan producers to increase their productivity and improve their technological characteristics based on systems metabolic engineering approaches are outlined in the review. These approaches can be used to obtain the producers of other aromatic amino acids and tryptophan precursors or derivatives. 相似文献
12.
Su Jin Lee Geun-Young Sim Youngshim Lee Bong-Gyu Kim Joong-Hoon Ahn 《Journal of industrial microbiology & biotechnology》2017,44(11):1551-1560
Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized. 相似文献
13.
Function of the <Emphasis Type="Italic">rel</Emphasis> Gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> 总被引:6,自引:0,他引:6
Sokawa et al. suggest that rel- strains of Escherichia coli possess abnormal protein synthesizing machinery, which cannot carry out normal protein synthesis when the supply of amino-acids is limited. 相似文献
14.
V. N. Verbenko L. V. Kuznetsova E. P. Krupyan V. I. Shalguev 《Russian Journal of Genetics》2009,45(10):1192-1199
Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed
1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA
+ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein. 相似文献
15.
O. D. Hendrickson N. I. Smirnova A. V. Zherdev V. K. Gasparyan B. B. Dzantiev 《Applied Biochemistry and Microbiology》2017,53(1):107-113
In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 106 and 5 × 105 cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens. 相似文献
16.
Na-Rae Lee Ji-Yeong Yun Sun-Mee Lee Jin-Byung Park 《Biotechnology and Bioprocess Engineering》2015,20(6):1088-1098
Solvent stress occurs during whole-cell biocatalysis of organic chemicals. Organic substrates and/or products may accumulate in the cellular membranes of whole cells, causing structural destabilization of the membranes, which leads to disturbances in cellular carbon and energy metabolism. Here, we investigate the effect of cyclohexanone on carbon metabolism in Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032. Adding cyclohexanone to the culture medium (i.e., glucose mineral medium) resulted in a decreased specific growth rate and increased cellular maintenance energy in both strains of bacteria. Notably, carbon metabolism, which is mainly involved to increase cellular maintenance energy, was very different between the bacteria. Carbon flux into the acetic acid fermentation pathway was dominantly enhanced in E. coli, whereas the TCA cycle appeared to be activated in C. glutamicum. In fact, carbon flux into the TCA cycle in E. coli appeared to be reduced with increasing amounts of cyclohexanone in the culture medium. Metabolic engineering of E. coli cells to maintain or improve TCA cycle activity and, presumably, that of the electron transport chain, which are involved in regeneration of cofactors (e.g., NAD(P)H and ATP) and formation of toxic metabolites (e.g., acetic acid), may be useful in increasing solvent tolerance and biotransformation of organic chemicals (e.g., cyclohexanone). 相似文献
17.
18.
Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase
mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen
as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction
on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the
peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H2O2 concentration is linear up to the concentration of 8 mM. At higher concentrations of H2O2, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The
peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling.
Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence
on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center
of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b
558, and H2O2 is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological
role for humans and animals. 相似文献
19.
Coupling lactate fermentation with cell growth was investigated in shake-flask and bioreactor cultivation systems by increasing
aeration to improve lactate productivity in Escherichia coli CICIM B0013-070 (ackA
pta
pps
pflB
dld
poxB
adhE
frdA). In shake-flasks, cells reached 1 g dry wt/l then, cultivated at 100 rpm and 42°C, achieved a twofold higher productivity
of lactic acid compared to aerobic and O2-limited two-phase fermentation. The cells in the bioreactor yielded an overall volumetric productivity of 5.5 g/l h and a
yield of 86 g lactic acid/100 g glucose which were 66% higher and the same level compared to that of the aerobic and O2-limited two-phase fermentation, respectively, using scaled-up conditions optimized from shake-flask experiments. These results
have revealed an approach for improving production of fermentative products in E. coli. 相似文献
20.
Bacterial lipoproteins comprise a subset of membrane proteins that are covalently modified with lipids at the amino-terminal Cys. Lipoproteins are involved in a wide variety of functions in bacterial envelopes. Escherichia coli has more than 90 species of lipoproteins, most of which are located on the periplasmic surface of the outer membrane, while others are located on that of the inner membrane. In order to elucidate the mechanisms by which outer-membrane-specific lipoproteins are sorted to the outer membrane, biochemical, molecular biological and crystallographic approaches have been taken. Localization of lipoproteins on the outer membrane was found to require a lipoprotein-specific sorting machinery, the Lol system, which is composed of five proteins (LolABCDE). The crystal structures of LolA and LolB, the periplasmic chaperone and outer-membrane receptor for lipoproteins, respectively, were determined. On the basis of the data, we discuss here the mechanism underlying lipoprotein transfer from the inner to the outer membrane through Lol proteins. We also discuss why inner membrane-specific lipoproteins remain on the inner membrane. 相似文献