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1.
Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli. 总被引:1,自引:0,他引:1
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A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose. E. coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate. When pPC37 and pPC58 were transformed into E. coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate. Crude extracts of E. coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP). E. coli FMJ39 had no detectable LDH activity, and E. coli LDH from a wild-type strain was not activated by FDP. Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C. acetobutylicum DNA encodes the FDP-activated LDH. 相似文献
2.
Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization 总被引:2,自引:0,他引:2
The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. coli cell extracts and its kinetic properties were determined. Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP). For pyruvate, partially purified LDH had Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded. The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence. The enzyme did not lose activity when incubated at 65 degrees C for 5 min. 相似文献
3.
Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase. 总被引:16,自引:6,他引:10
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Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion. 相似文献
4.
Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities. 总被引:3,自引:0,他引:3
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In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2. 相似文献
5.
Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc(+)), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc(+)) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc(+) strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc(+) strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation. 相似文献
6.
Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes
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An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum. 相似文献
7.
Regulation of lactate dehydrogenase and change of fermentation products in streptococci. 总被引:41,自引:13,他引:41
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Streptococcus mutans JC 2 produced mainly lactate as a fermentation product when grown in nitrogen-limited continuous culture in the presence of an excess of glucose and produced formate, acetate, and ethanol, but no lactate, under glucose-limited conditions. The levels of lactate dehydrogenase (LDH) in these cultures were of the same order of magnitude, and the activity of LDH was completely dependent on fructose-1,6-diphosphate (FDP). The intracellular level of FDP was high and the level of phosphoenolpyruvate (PEP) was low under the glucose-excess conditions. In the glucose-limited cultures, all glycolytic intermediates studied, except PEP, were low. S. mutans FIL, which had an FDP-independent LDH and similar levels of glycolytic intermediates as S. mutans JC2, produced mainly lactate under glucose-excess or under glucose-limited conditions. LDH of Streptococcus bovis ATCC 9809 was dependent on FDP for activity at a low concentration of pyruvate but had a significant activity without FDP at a high concentration of pyruvate. This strain also produced mainly lactate both under glucose-excess and glucose-limited conditions. The levels and characteristics of these LDHs were not changed by the culture conditions. These results indicate that changes in the intracellular level of FDP regulate LDH activity, which in turn influences the type of fermentation products produced by streptococci. PEP, adenosine 5'-monophosphate, adenosine 5'-diphosphate, and inorganic phosphate significantly inhibited LDH activity from S. mutans JC 2 and may also participate in the regulation of LDH activity in other streptococci. 相似文献
8.
Thermosensitive H1 plasmids determining citrate utilization. 总被引:6,自引:0,他引:6
Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains). 相似文献
9.
E Garza J Zhao Y Wang J Wang A Iverson R Manow C Finan S Zhou 《Journal of industrial microbiology & biotechnology》2012,39(8):1101-1107
A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition. 相似文献
10.
When 50 microM carbonyl cyanide-m-chlorophenyl hydrazone (CCCP), a protonophore, was added to growth medium containing glucose at pH 7.5, Escherichia coli TK1001 (trkD1 kdpABC5) started exponential growth after 30 min; the generation time was 70 min at 37 degrees C. Strain AS1 (acrA), another strain derived from E. coli K-12, also grew in the presence of 50 microM CCCP under the same conditions, except that the lag period was ca. 3 h. When this strain was grown in the presence of 50 microM CCCP and then transferred to fresh medium containing 50 microM CCCP, cells grew without any lag. Neither a membrane potential nor a pH gradient was detected in strain AS1 cells growing in the presence of CCCP. When either succinate or lactate was substituted for glucose, these strains did not grow in the presence of 50 microM CCCP. Thus, it is suggested that E. coli can grow in the absence of a proton motive force when glucose is used as an energy source at pH 7.5. 相似文献
11.
Regulation of Staphylococcus aureus Lactate Dehydrogenase 总被引:8,自引:4,他引:4
The effect of growth conditions on the specific activity of nicotinamide adenine dinucleotide (NAD)-linked lactate dehydrogenase (LDH) in extracts of Staphylococcus aureus strain SG 511A was examined. Kinetic and electrophoretic experiments, with extracts prepared from aerobically and anaerobically grown cells, provided evidence for only one physiologically significant enzyme. The aerobic level of NAD-linked LDH of S. aureus remained constant and was independent of the carbon source. In contrast, the level of LDH produced in an anaerobic environment was variable and was dependent on the carbon source. Growth anaerobically on pyruvate, as the sole fermentable carbon source, resulted in a maximal level of LDH activity, a value about eightfold greater than the aerobic level. Anaerobic growth either on pyruvate plus glucose or on glucose alone, however, resulted in approximately a threefold decrease in this maximum. Experiments with a heme-requiring auxotroph derived from S. aureus demonstrated that the aerobic level of LDH activity was dependent on a functional respiratory chain. 相似文献
12.
Metabolic Analysis of Escherichia coli in the Presence and Absence of the Carboxylating Enzymes Phosphoenolpyruvate Carboxylase and Pyruvate Carboxylase 总被引:1,自引:0,他引:1
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Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc+), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc+) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc+ strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc+ strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation. 相似文献
13.
A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH). 相似文献
14.
Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism 总被引:1,自引:0,他引:1
Romero S Merino E Bolívar F Gosset G Martinez A 《Applied and environmental microbiology》2007,73(16):5190-5198
Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain. 相似文献
15.
Influence of substrate carbon on the metabolism of Clostridium thermohydrosulfuricum 总被引:1,自引:0,他引:1
Luiz Donaduzzi Pierre Germain Fatiou Toukourou Sein Myint 《FEMS microbiology letters》1989,57(2):213-217
The concentration of carbon sources has a significant influence on the growth, carbohydrate uptake and metabolite distribution in Clostridium thermohydrosulfuricum. The growing concentrations of glucose or starch increase the production of ethanol and lactate, the intracellular fructose-1,6-diphosphate (FDP) and the specific activity of lactate dehydrogenase (LDH), but decrease the ethanol/lactate ratio. 相似文献
16.
In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824. 总被引:5,自引:1,他引:4
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The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824. 相似文献
17.
A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogenlyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23 were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa. 相似文献
18.
Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1). 相似文献
19.
Construction and expression of human aldolase A and B expression plasmids in Escherichia coli host 总被引:2,自引:0,他引:2
M Sakakibara I Takahashi Y Takasaki T Mukai K Hori 《Biochimica et biophysica acta》1989,1007(3):334-342
E. coli expression plasmids for human aldolases A and B (EC 4.1.2.13) have been constructed from the pIN-III expression vector and their cDNAs, and expressed in E. coli strain JM83. Enzymatically active forms of human aldolase have been generated in the cells when transfected with either pHAA47, a human aldolase A expression plasmid, or pHAB 141, a human aldolase B expression plasmid. These enzymes are indistinguishable from authentic enzymes with respect to molecular size, amino acid sequences at the NH2- and COOH-terminal regions, the Km for substrate, fructose 1,6-bisphosphate and the activity ratio of fructose 1,6-bisphosphate/fructose 1-phosphate (FDP/F1P), although net electric charge and the Km for FDP of synthetic aldolase B differed from those for a previously reported human liver aldolase B. In addition, both the expressed aldolases A and B complement the temperature-sensitive phenotype of the aldolase mutant of E. coli h8. These data argue that the expressed aldolases are structurally and functionally similar to the authentic human aldolases, and would provide a system for analysis of the structure-function relationship of human aldolases A and B. 相似文献
20.
【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。 相似文献