Production of succinate and polyhydroxyalkanoate from substrate mixture by metabolically engineered Escherichia coli

The strategic design of this study aimed at producing succinate and polyhydroxyalkanoate (PHA) from substrate mixture of glycerol/glucose and fatty acid in Escherichia coli. To accomplish this, an E. coli KNSP1 strain derived from E. coli LR1110 was constructed by deletions of ptsG, sdhA and pta genes and overexpression of phaC1 from Pseudomonas aeruginosa. Cultivation of E. coli KNSP1 showed that this strain was able to produce 21.07 g/L succinate and 0.54 g/L PHA (5.62 wt.% of cell dry weight) from glycerol and fatty acid mixture. The generated PHA composed of 58.7 mol% 3-hydroxyoctanoate (3HO) and 41.3 mol% 3-hydroxydecanoate (3HD). This strain would be useful for complete utilization of byproducts glycerol and fatty acid of biodiesel production process.     

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli

Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51 g/L (productivity equal to 1.05 g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47 g/L were produced in 10 L bioreactor within 48 h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62 g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56 g/L in 10 L bioreactor within 72 h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production

In the application of engineered Escherichia coli in industrial polyhydroxybutyrate production process, one of the major concerns is the induction of the metabolic pathway. In this study, we developed a stress-induced system by which the PHB biosynthesis pathways can be induced under stress conditions. Fermentation results showed that recombinant E. coli DH5α (pQKZ103) harboring this system was able to accumulate polyhydroxybutyrate up to 85.8% of cell dry weight in minimal glucose medium without adding any inducer. Growth experiment with GFP as a reporter indicated that the induction of this system happened at the late exponential phase and was sensitive to stressed environment. This system can also be applied in many other biotechnological processes.     

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.     

Extracellular production of active vibriolysin engineered by random mutagenesis in Escherichia coli

Vibriolysin, an extracellular protease of Vibrio proteolyticus, is synthesized as a preproenzyme. The N-terminal propeptide functions as an intramolecular chaperone and an inhibitor of the mature enzyme. Extracellular production of recombinant vibriolysin has been achieved in Bacillus subtilis, but not in Escherichia coli, which is widely used as a host for the production of recombinant proteins. Vibriolysin is expressed as an inactive form in E. coli possibly due to the inhibitory effect of the N-terminal propeptide. In this study, we isolated the novel vibriolysin engineered by in vivo random mutagenesis, which is expressed as active mature vibriolysin in E. coli. The Western blot analysis showed that the N-terminal propeptide of the engineered enzyme was processed and degraded, confirming that the propeptide inhibits the mature enzyme. Two mutations located within the engineered vibriolysin resulted in the substitution of stop codon for Trp at position 11 in the signal peptide and of Val for Ala at position 183 in the N-terminal propeptide (where position 1 is defined as the first methionine). It was found that the individual mutations are related to the enzyme activity. The novel vibriolysin was extracellularly overproduced in BL21(DE3) and purified from the culture supernatant by ion-exchange chromatography followed by hydrophobic-interaction chromatography, resulting in an overall yield of 2.2 mg/L of purified protein. This suggests that the novel engineered vibriolysin is useful for overproduction in an E. coli expression system.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods

We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella enterica prpBCDE promoter (P_prpB) and compared it to that from the strongest IPTG-inducible promoter, P_T7. In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from P_prpB was 2- to 4-fold higher than that from P_T7. Unlike P_T7, expression from P_prpB did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins.     

High-level expression and purification of Escherichia coli oligopeptidase B

Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.     

Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli

In the context of recombinant DNA technology, the development of feasible and high-yielding plasmid DNA production processes has regained attention as more evidence for its efficacy as vectors for gene therapy and DNA vaccination arise. When producing plasmid DNA in Escherichia coli, a number of biological restraints, triggered by plasmid maintenance and replication as well as culture conditions are responsible for limiting final biomass and product yields. This termed "metabolic burden" can also cause detrimental effects on plasmid stability and quality, since the cell machinery is no longer capable of maintaining an active metabolism towards plasmid synthesis and the stress responses elicited by plasmid maintenance can also cause increased plasmid instability. The optimization of plasmid DNA production bioprocesses is still hindered by the lack of information on the host metabolic responses as well as information on plasmid instability. Therefore, systematic and on-line approaches are required not only to characterise this "metabolic burden" and plasmid stability but also for the design of appropriate metabolic engineering and culture strategies. The monitoring tools described to date rapidly evolve from laborious, off-line and at-line monitoring to online monitoring, at a time-scale that enables researchers to solve these bioprocessing problems as they occur. This review highlights major E. coli biological alterations caused by plasmid maintenance and replication, possible causes for plasmid instability and discusses the ability of currently employed bioprocess monitoring techniques to provide information in order to circumvent metabolic burden and plasmid instability, pointing out the possible evolution of these methods towards online bioprocess monitoring.     

Expression of a biologically-active conotoxin PrIIIE in Escherichia coli

Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5 mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure-function studies.     

Binding of NorR to three DNA sites is essential for promoter activation of the flavorubredoxin gene, the nitric oxide reductase of Escherichia coli

NorR is a nitric oxide sensor that in Escherichia coli regulates the gene encoding for flavorubredoxin, an enzyme involved in nitrosative detoxification. The present work shows that although purified NorR can bind independently to each of three binding sites in the flavorubredoxin gene promoter, the presence of all sites is required for in vivo nitric oxide-dependent induction of the flavorubredoxin gene. Furthermore, trimerization of NorR upon binding to the three sites was observed by protein cross-linking experiments. These results reveal the importance of the multiple DNA binding sites present on NorR-dependent promoters and suggest that the functional form of NorR is a trimer.