Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.     

Microbial production of medium-chain-length 3-hydroxyalkanoic acids by recombinant Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.     

Motility influences biofilm architecture in Escherichia coli

Eight Escherichia coli strains were studied in minimal medium with a continuous flow system using confocal microscopy. K12 wild-type strains ATCC 25404 and MG1655 formed the best biofilms (∼43 μm thick, 21 to 34% surface coverage). JM109, DH5α, and MG1655 motA formed intermediate biofilms (∼13 μm thick, 41 to 58% surface coverage). BW25113, MG1655 qseB, and MG1655 fliA had poor biofilms (surface coverage less than 5%). The best biofilm-formers, ATCC 25404 and MG1655, displayed the highest motility, whereas the worst biofilm former, BW25113, was motility-impaired. The differences in motility were due to differences in expression of the motility loci qseB, flhD, fliA, fliC, and motA (e.g., qseB expression in MG1655 was 139-fold higher than BW25113 and 209-fold higher than JM109). Motility affected the biofilm architecture as those strains which had poor motility (E. coli JM109, E. coli MG1655 motA, and DH5α) formed flatter microcolonies compared with MG1655 and ATCC 25404, which had more dramatic vertical structures as a result of their enhanced motility. The presence of flagella was also found to be important as qseB and fliA mutants (which lack flagella) had less biofilm than the isogenic paralyzed motA strain (threefold less thickness and 15-fold less surface coverage).     

Expression of Pyruvate Carboxylase Enhances Succinate Production in Escherichia coli without Affecting Glucose Uptake

Plasmids carrying the pyc gene from Rhizobium etli were used to express pyruvate carboxylase in Escherichia coli. Results of batch fermentations of a wild-type E. coli (MG1655), this wild-type with the pUC18 cloning/expression vector (MG1655/pUC18) and this wild-type carrying the pyc gene (MG1655/pUC18-pyc) were compared in glucose-limited medium. The results indicate that the final succinate concentration upon complete glucose utilization was increased from 1.18 g/L to 1.77 g/L by the expression of pyc, while the final succinate concentration in MG1655/pUC18 was slightly lower than in the parent strain. This increased succinate concentration came at the expense of lactate synthesis, whose final concentration decreased from 2.33 g/L to 1.88 g/L. The expression of pyc did not affect the maximum glucose uptake (2.17 g/Lh for MG1655 versus 2.47 g/Lh for MG1655/pUC18-pyc), but did decrease the maximum rate of cell mass production (0.213 g/Lh for MG1655, 0.169 g/Lh for MG1655/pUC18 and 0.199 g/Lh for MG1655/pUC18-pyc).     

2D [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C] NMR study of carbon fluxes during glucose utilization by <Emphasis Type="Italic">Escherichia coli</Emphasis> MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

2D [1H,13C] NMR study of carbon fluxes during glucose utilization by Escherichia coli MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells--glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)--were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655(edd-eda), MG1655(zwf, edd-eda), and MG1655(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

弗氏志贺菌毒力大质粒导入大肠杆菌MG1655

目的:将弗氏2a志贺菌2457T的毒力大质粒pSF导入大肠杆菌MG1655。方法:通过诱动转移技术,将弗氏2a志贺菌2457T的毒力大质粒导入大肠杆菌MG1655。结果:构建了MG1655/pSF:pXL275-virG的毒力大质粒导入突变株,双向电泳初步比较分析表明在重组MG1655中有志贺菌毒力的表达。结论:成功地将弗氏2a志贺菌2457T毒力大质粒pSF导入了大肠杆菌MG1655。     

De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.     

Construction of the new <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12 MG 1655 novel strain with improved growth characteristics for application in metabolic engineering

MG1655 of Escherichia coli K-12 is frequently used in metabolic engineering as the wild-type strain. However, its two mutations, ilvG and rph-1 provide a negative effect on culture growth. The “polar effect” of rph-1 decreases the level of pyrE expression, causing partial auxotrophy for pyrimidines. Mutation ilvG leading to the appearance of Val^S phenotype causes retardation of cell growth rate on media containing amino acids. In this work, the substitution of two loci in the genome of MG1655 with the recovery of the wild-type phenotype was accomplished. Gene rph ^wt from the chromosome of E. coli TG1 was marked via Red-dependent integration of DNA fragment carrying λattL-Cm^R-λattR and transduced with phage P1 into MG1655; later, the Cm^R marker was removed with the use of λXis/Int recombinase. Parallel to this procedure, a spontaneous Val^R mutant of E. coli MG1655 yielding colonies of maximal size on M9 medium with glucose in the presence of L-Val (50 μg/ml) was isolated. It was shown that a nucleotide deletion in the isolated Val^R strain had been generated in the region of the identified ilvG mutation, which led to the recovery of the reading frame and active protein synthesis. This mutation named ilvG-15, which is the only reason for the Val^R phenotype in the obtained strain, was transferred to MG1655-rph ^wt using cotransduction, by analogy to the transfer of rph ^wt. Evaluation of rates of aerobically growing cells (μ, hour-1) on M9 medium with glucose produced the following values: 0.56, 0.69, and 0.73 for strains MG1655,MG1655-rph ^wt, and MG1655-(rph ^wt, ilvG-15), respectively.     

操纵子前导区的修饰及PRPP合成途径的加强对大肠杆菌积累L-组氨酸的影响

目的:基于转酮酶基因缺失菌株MG1655-ΔtktA,研究启动子替换L-组氨酸操纵子前导区及6-磷酸葡萄糖脱氢酶基因zwf、6-磷酸葡萄糖酸脱氢酶基因gnd、PRPP合成酶基因prs的过表达对大肠杆菌产L-组氨酸的影响。方法:通过Red重组系统用T5启动子替换L-组氨酸操纵子前导区;构建gnd和zwf串联表达载体gnd-zwf-pSTV28,prs表达载体prs-pQE30。通过摇瓶发酵,考察上述改造对大肠杆菌积累L-组氨酸的影响。结果:测定结果显示,改造菌株的发酵液中均能实现L-组氨酸积累,平均分别为MG1655-ΔtktA-PT5,60.12 mg/L;MG1655-ΔtktA-PT5(prs-pQE30),66.47mg/L;MG1655-ΔtktA-PT5(zwf-gnd-pSTV28),89.69 mg/L;MG1655-ΔtktA-PT5(prs-pQE30,zwf-gnd-pSTV28),111.56 mg/L。结论:L-组氨酸操纵子前导区的修饰使菌株合成L-组氨酸的能力大大增强,而氧化戊糖磷酸途径的加强和PRPP合成酶活性的提高能够进一步提高产量。     

Physiological studies of Escherichia coli strain MG1655: growth defects and apparent cross-regulation of gene expression

Escherichia coli strain MG1655 was chosen for sequencing because the few mutations it carries (ilvG rfb-50 rph-1) were considered innocuous. However, it has a number of growth defects. Internal pyrimidine starvation due to polarity of the rph-1 allele on pyrE was problematic in continuous culture. Moreover, the isolate of MG1655 obtained from the E. coli Genetic Stock Center also carries a large deletion around the fnr (fumarate-nitrate respiration) regulatory gene. Although studies on DNA microarrays revealed apparent cross-regulation of gene expression between galactose and lactose metabolism in the Stock Center isolate of MG1655, this was due to the occurrence of mutations that increased lacY expression and suppressed slow growth on galactose. The explanation for apparent cross-regulation between galactose and N-acetylglucosamine metabolism was similar. By contrast, cross-regulation between lactose and maltose metabolism appeared to be due to generation of internal maltosaccharides in lactose-grown cells and may be physiologically significant. Lactose is of restricted distribution: it is normally found together with maltosaccharides, which are starch degradation products, in the mammalian intestine. Strains designated MG1655 and obtained from other sources differed from the Stock Center isolate and each other in several respects. We confirmed that use of other E. coli strains with MG1655-based DNA microarrays works well, and hence these arrays can be used to study any strain of interest. The responses to nitrogen limitation of two urinary tract isolates and an intestinal commensal strain isolated recently from humans were remarkably similar to those of MG1655.     

Induction of oxidative stress by high hydrostatic pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Evidence for an evolutionary antagonism between Mrr and Type III modification systems

The Mrr protein of Escherichia coli is a laterally acquired Type IV restriction endonuclease with specificity for methylated DNA. While Mrr nuclease activity can be elicited by high-pressure stress in E. coli MG1655, its (over)expression per se does not confer any obvious toxicity. In this study, however, we discovered that Mrr of E. coli MG1655 causes distinct genotoxicity when expressed in Salmonella typhimurium LT2. Genetic screening enabled us to contribute this toxicity entirely to the presence of the endogenous Type III restriction modification system (StyLTI) of S. typhimurium LT2. The StyLTI system consists of the Mod DNA methyltransferase and the Res restriction endonuclease, and we revealed that expression of the LT2 mod gene was sufficient to trigger Mrr activity in E. coli MG1655. Moreover, we could demonstrate that horizontal acquisition of the MG1655 mrr locus can drive the loss of endogenous Mod functionality present in S. typhimurium LT2 and E. coli ED1a, and observed a strong anti-correlation between close homologues of MG1655 mrr and LT2 mod in the genome database. This apparent evolutionary antagonism is further discussed in the light of a possible role for Mrr as defense mechanism against the establishment of epigenetic regulation by foreign DNA methyltransferases.     

丁醇合成途径关键酶基因在大肠杆菌中的克隆和表达

【目的】克隆丙酮丁醇梭状芽胞杆菌(Clostridium acetobutylicum)ATCC824丁醇合成途径关键酶基因,构建产丁醇的工程大肠杆菌。【方法】以C.acetobutylicum ATCC824基因组为模板,分别扩增丁醇合成途径关键酶基因thil,adhE2和BCS operon(crt-bcd-etfB-etfA-hbd)基因序列,构建BCS operon-adhE2-thil/pTrc99a/MG1655(pBAT)。重组菌E.coli pBAT采用0.1 mmol异丙基-β-硫代半乳糖苷(IPTG)诱导5 h,测定乙酰基转移酶(THL)、3-羟基丁酰辅酶A脱氢酶(HBD)、3-羟基丁酰辅酶A脱水酶(CRT)、丁酰辅酶A脱氢酶(BCD)、醛醇脱氢酶(BYDH/BDH)的酶活。并以该基因工程菌作为发酵菌种,采用好氧、厌氧和微好氧三种培养方式,检测丁醇产量。【结果】酶活测定结果显示:THL酶活达到0.160 U/mg protein,酶活力提高了近30倍;HBD酶活力提高了近5倍;CRT酶活达到1.53 U/mg protein,野生菌株无此酶活;BCD酶活力提高了32倍;BYDH/BDH酶活力无显著提高。3种发酵培养结果显示在微好氧和厌氧条件下,均有丁醇产生,且丁醇的最大产量约为84 mg/L。【结论】本实验通过构建产丁醇基因工程大肠杆菌,实现了丁醇关键酶基因在大肠杆菌中的活性表达以及发酵产丁醇,为发酵法生产丁醇开辟了一条新的途径。     

敲除aceE基因对大肠杆菌生长和丙酮酸代谢的影响

大肠杆菌aceE基因是编码丙酮酸脱氢酶多酶复合体PdhR的关键酶之一。利用Red重组系统敲除大肠杆菌MG1655的aceE基因后,阻断了丙酮酸流向TCA循环,导致丙酮酸的累积,也使菌体生长受到影响,在培养基中补加5 g/L KAc后可以在一定程度上弥补菌株在生长上的缺陷。摇瓶发酵36 h,MG1655没有积累丙酮酸,MG1655ΔaceE∷cat菌株可以积累26.77 g/L丙酮酸,为利用大肠杆菌发酵生产丙酮酸奠定了基础。     

Characterization of the extracellular polymeric substances produced by Escherichia coli using infrared spectroscopic, proteomic, and aggregation studies

The aim of this study was twofold: first, to characterize the free extracellular polymeric substances (EPS) and bound EPS produced by Escherichia coli during different growth phases in different media, and then to investigate the role of the free EPS in promoting aggregation. EPS was extracted from a population of E. coli MG1655 cells grown in different media composition (Luria-Bertani (LB) and Luria-Bertani with the addition of 0.5 w/v% glucose at the beginning of the growth phase (LBG)) and at different growth phases (6 and 24 h). The extracted EPS was characterized using Fourier transform infrared spectroscopy and further identified using one-dimensional gel-based electrophoresis and tandem mass spectrometry. E. coli MG1655 was found to produce significantly lower amounts of bound EPS compared to free EPS under all conditions. The protein content of free EPS increased as the cells progressed from the exponential to stationary phase when grown in LB or LBG, while the carbohydrate content only increased across the growth phases for cells grown in LBG. FTIR revealed a variation in the different functional groups such as amines, carboxyl, and phosphoryl groups for free EPS extracted at the different growth conditions. Over 500 proteins were identified in the free EPS, with 40 proteins common in all growth conditions. Proteins with functionality related to amino acid and carbohydrate metabolism, as well as cell wall and membrane biogenesis were among the highest proteins identified in the free EPS extracted from E. coli MG1655 under all growth and media conditions. The role of bound and free EPS was investigated using a standardized aggregation assay. Bound EPS did not contribute to aggregation of E. coli MG1655. The readdition of free EPS to E. coli MG1655 resulted in aggregation of the cells in all growth conditions. Free EPS extracted from the 24 h E. coli MG1655 cultures grown in LB had the greatest effect on aggregation of cells grow in LBG, with a 30% increase in aggregation observed.     

sucAB and sucCD are mutually essential genes in Escherichia coli

sucAB and sucCD of Escherichia coli encode enzymes that generate succinyl-CoA from 2-oxoglutarate and succinate, respectively. Their mutual essentiality was studied. sucAB and sucCD could be deleted individually, but not simultaneously. The mutual essentiality of sucAB and sucCD was further confirmed by the conditional expression of sucABCD, sucAB, and sucCD under the control of a P(BAD) in E. coli MG1655, E. coli MG1655 (DeltasucCD), and E. coli MG1655 (DeltasucAB), respectively. These strains grew well in Luria-Bertani medium containing 0.1% arabinose, but not in the absence of arabinose unless the medium was supplemented with succinyl-CoA. Our results indicate that either sucAB or sucCD is enough to produce succinyl-CoA that is essential for cell viability.