Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter⁻¹ of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter⁻¹). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.Thymidine, which is composed of 2-deoxyribose and a thymine base, is a commercially useful precursor in the chemical synthesis of various antiviral drugs, including stavudine and zidovudine (azidothymidine), the active ingredient in a formulation for the treatment of AIDS (18, 19). Because thymidine is required only in DNA synthesis, intracellular thymidine levels are very low and are tightly controlled (40). For the production of precursors for antiviral drugs, thymidine is either biologically produced in a low yield by a few modified microorganisms or chemically synthesized through a very costly process (17, 33, 48, 49). Thus, there is a need for developing a more efficient strain for thymidine production on a large scale.In nature, there are two distinct pathways for dTTP synthesis, the salvage and de novo pathways. The salvage pathway enables the cells to utilize preformed nucleobases and nucleosides for nucleotide synthesis, using thymidine phosphorylase (deoA), uridine phosphorylase (udp), and thymidine kinase (tdk) (Fig. ​(Fig.1)1) (40).Open in a separate windowFIG. 1.Thymidine biosynthetic pathway. The steps engineered in this study are indicated by the bold arrows and lines. Components of the catabolism are as follows: pyrA, carbamoylphosphate synthase; pyrBI, aspartate-carbamoyl transferase; pyrC, dihydroorotase; pyrD, dihydroorotate oxidase; pyrE, orotate phosphoribosyltransferase; pyrF, OMP decarboxylase; pyrG, CTP synthetase; pyrH, UMP kinase; TMPase, TMP phosphohydrolase; nrd, nucleotide diphosphate reductase; tdΔI, T4 thymidylate synthase (intron deleted); thyA, thymidylate synthase; dcd, dCTP deaminase; udk, uridine kinase; deoA, thymidine phosphorylase; tdk, thymidine kinase; udp, uridine phosphorylase; dut, deoxyribonucleotide triphosphatase; ndk, nucleotide diphosphate kinase; tmk, TMP kinase; ung, uracil-DNA N-glycosylase; upp, uracil phosphoribosyl-transferase; cdd, cytidine deaminase; codA, cytosine deaminase.As the name indicates, the de novo pathway enables the cells to synthesize nucleobases de novo. The de novo pathway leading to thymidine biosynthesis starts with the condensation of aspartate and carbamoylphosphate, synthesized by carbamoylphosphate synthase (pyrA) (41). This condensation reaction is catalyzed by aspartate-carbamoyl transferase (pyrBI) to produce carbamoyl aspartate, which undergoes several reactions to produce UMP, the common precursor for the synthesis of the pyrimidine ribonucleoside and deoxynucleosides (Fig. ​(Fig.1)1) (39-41). For thymidine biosynthesis, UMP is converted to UDP in a reaction catalyzed by UMP kinase (pyrH), and UDP is converted to dUDP by ribonucleoside diphosphate reductase (nrdAB), which is regulated by NTP effectors through binding to specific allosteric sites on ribonucleotide diphosphate reductase (nrdA). Escherichia coli can synthesize dUMP from both dCDP and dUDP. The major pathway involves phosphorylation of dCDP to dCTP, deamination of dCTP to dUTP, and hydrolysis of dUTP to dUMP. Only 20 to 30% of the cellular dUMP is supplied by hydrolysis of dUTP (29, 37). The deamination of dCTP (dcd) is located at a branch point in the pyrimidine metabolic pathway. Because of its importance, dcd is regulated by a positive homotropic cooperativity toward dCTP and by a feedback inhibition by dTTP (29, 31, 40).Deoxyuridine triphosphatase (dUTPase [dut]) is a pyrophosphatase that contains zinc ions (42). dUTPase catalyzes the hydrolysis of dUTP to PP_i and dUMP, a substrate for thymidylate synthase (thyA). Generally, the intracellular concentration of dUTP is <10 nmol per 1 g dry cell weight (DCW), and that of dTTP exceeds 500 nmol per 1 g DCW (5, 39, 52). The intracellular dUTP-to-dTTP ratio is increased in dut-deficient mutants, leading to an increased frequency of misincorporation of uracil for thymine in DNA (34). This incorporation is transient only because uracil is removed from DNA via a subsequent excision repair initiated by uracil-DNA N-glycosylase, which is encoded by ung (15, 50). Attempted repair of deoxyuridine residues from DNA without adequate dTTP available to complete the repair reaction can result in multiple single-strand breaks, eventually leading to double-strand breaks (15). Indeed, single- and double-strand breaks accumulate in thymidine-deprived cells (16). In such cells, the loss of uracil glycosylase activity should decrease DNA breaks arising from attempted repair and thereby decrease the toxicity of thymidine depletion.The synthesis of dTMP from dUMP involves the transfer of a methylene group and two reducing equivalents from 5,10-methylenetetrahydrofolate to dUMP, catalyzed by the dimeric enzyme thymidylate synthase (thyA). Even though ThyA catalyzes the committed step for de novo synthesis of dTTP, neither the activity of the enzyme nor the expression of the thyA gene seems to be regulated (2, 3).The general strategy used for the development of a thymidine-overproducing strain involves the alleviation of control mechanisms in key pathways. Several different microorganisms have been modified for thymidine production, including E. coli, Brevibacterium helvolum, and Corynebacterium ammoniagenes, by classical mutagenesis methods, and they were selected based on their capacity to grow on toxic thymidine analogues (30, 33, 48, 49). In these studies, feedback inhibition-resistant variants of thymidine biosynthetic enzymes were obtained by random mutation, and high-producing variants were selected. The most optimum B. helvolum strain obtained by this procedure produced 500 mg liter⁻¹ of thymidine by batch fermentation (33). However, engineered B. helvolum and E. coli mutants also produced thymine, deoxyuridine, and uracil, which are unfavorable for thymidine production since it increases costs during the purification process (30, 33, 48, 49). Furthermore, these thymidine-producing strains have residual thymidine degradation activities, resulting in decreased productivities.Thus, we tried to develop a more efficient thymidine-producing strain by enhancing the de novo pathway leading to thymidine biosynthesis and by disrupting the thymidine salvage pathway. The strategy reported here is based on disrupting genes which encode enzymes involved in thymidine degradation and on expressing foreign genes in the de novo pathway leading to thymidine biosynthesis which encode enzymes that are expected to be less sensitive to feedback inhibition by thymidine than the original enzymes in the host strain. The T4 ribonucleotide diphosphate reductase (nrdAB) operon, T4 thioredoxin (nrdC), T4 thymidylate synthase (td), and PBS2 TMP phosphohydrolase (TMPase) were expressed in an E. coli mutant strain which was modified to block the salvage pathway (deoA, tdk, and udp). In order to increase the influx of dUMP, E. coli dCTP deaminase (dcd), deoxyuridine triphosphatase (dut), and uridine kinase (udk) were expressed with phage-derived genes. We found that the dUTP/dTTP ratio was increased by increasing the level of dUTP in our mutant, leading to the frequent misincorporation of dUTP in DNA. In order to prevent frequent temporary DNA breaks and gaps by excision repair caused by the increased intracellular dUTP/dTTP ratio, uracil-DNA N-glycosylase (ung) was additionally disrupted.     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h⁻¹. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h⁻¹ achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.     

Effect of Oxygen on Several Enzymes Involved in the Aerobic and Anaerobic Utilization of Glucose in Escherichia coli

By using the continuous culture technique, the transition from aerobiosis to anaerobiosis and its effect on a number of enzymes has been investigated in Escherichia coli K-12. A decrease in the oxygen partial pressure below 28.0 mm of Hg resulted firstly in an increase of the respiratory enzymes (reduced nicotinamide adenine dinucleotide [NADH] oxidase, 2.53-fold; succinic dehydrogenase, 1.4-fold; cytochrome b(1), 3.91-fold; and cytochrome a(2), 2.45-fold) before the electron transport system gradually collapsed as cytochrome a(2), followed by cytochrome b(1), succinic dehydrogenase, and finally NADH oxidase decreased in activity. The change from respiration to fermentation was initiated well before the oxygen tension reached zero by the increase in levels of fructose diphosphate-aldolase, glucose 6-phosphate, and 6-phosphogluconate dehydrogenases and a decrease in 2-oxoglutarate dehydrogenase. Whem the dissolved oxygen tension reached zero, dry weight and CO(2) formation together with isocitrate dehydrogenase decreased, whereas acid production and phosphofructokinase synthesis started to increase. Enzymatic investigations revealed that the kinetics of the enzyme phosphofructokinase from strict aerobic cultures (6.9 ppm oxygen in solution) was adenosine triphosphate (ATP)-insensitive, whereas the same enzyme from anaerobic cultures was ATP-sensitive. A mechanism is proposed for the change from aerobiosis to anaerobiosis together with the occurring change in glucose regulation.     

Production of mevalonate by a metabolically-engineered Escherichia coli

Mevalonate is biosynthesized from acetyl-CoA and metabolized to isoprenoid compounds in a wide variety of organisms although certain types of prokaryotes employ another route for isoprenoid biosynthesis (the non-mevalonate pathway). To establish a fermentative process for mevalonate production, enzymes for mevalonate synthesis from Enterococcus faecalis were expressed in Escherichia coli, a non-mevalonate pathway bacterium. Mevalonate was accumulated, indicating a redirection of acetate metabolism by the expressed enzyme. The recombinant E. coli produced 47 g mevalonate l(-1) in 50 h of fed-batch cultivation in a 2 l jar fermenter; this is the highest titer ever reported demonstrating the superiority of E. coli in its ability of acetyl-CoA supply and its inability is degrade mevalonate.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Effect of Dihydrostreptomycin on Protein Synthesis in Whole Cells and in Cell-free Extracts of a Streptomycin-dependent Strain of Escherichia coli B

The effect of dihydrostreptomycin on the incorporation of amino acids into protein in antibiotic-deprived cells of a streptomycin-dependent strain of Escherichia coli B has been compared with its effect on protein synthesis in extracts from cells of the same strain. Stimulation of phenylalanine incorporation into protein in whole cells occurred within 5 min of addition of dihydrostreptomycin to a deprived culture and was maximal at an antibiotic concentration of 20 mug/ml. Stimulation of protein synthesis in cell-free extracts from antibiotic-deprived cells was maximal at a dihydrostreptomycin concentration of 10 mug/ml in systems programmed with f2-ribonucleic acid and poly AGU, whereas extracts from cells grown on nonlimiting concentrations of dihydrostreptomycin were unaffected by the addition of antibiotic. These results indicate that protein synthesis is an antibiotic-requiring process in streptomycin-dependent E. coli B.     

Low salt medium for lactate and ethanol production by recombinant Escherichia coli B

Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient l(+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l⁻¹). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18–19 mmol l⁻¹ h⁻¹ for both (initial 48 h of fermentation).     

Function of Ubiquinone in Escherichia coli: a Mutant Strain Forming a Low Level of Ubiquinone

A ubiquinone-deficient mutant of Escherichia coli K-12 forming 20% of the normal amount of ubiquinone was compared with a normal strain. This lowered concentration of ubiquinone is still four times the concentration of cytochrome b(1). The mutant strain grew more slowly than the normal strain on a minimal medium with glucose as sole source of carbon and gave a lower aerobic growth yield than the normal strain. The reduced nicotinamide adenine dinucleotide (NADH) oxidase rate in membranes from the mutant strain was 40% of the oxidase rate in membranes from the normal strain, and the percentage reduction of cytochrome b(1) in the aerobic steady state, with NADH as substrate, was increased in membranes from the mutant strain. It is concluded that ubiquinone is required for maximum oxidase activity at the relatively high concentration (27 times that of cytochrome b(1)) found in normal cells. The results are discussed in relation to a scheme previously advanced for ubiquinone function in E. coli.     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

产姜黄素大肠杆菌工程菌的构建

姜黄素是姜科植物的特征性成分,具有重要的药理活性.文中利用姜黄素生物合成关键酶β-酮酰辅酶A合酶(Diketide-CoA synthase,DCS)基因和姜黄素合酶(Curcumin synthase,CURS)基因构建非天然融合基因DCS::CURS,并将其与4-香豆酰辅酶A连接酶(4-coumarate coen...     

Production of lycopene by metabolically-engineered Escherichia coli

Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5 mg lycopene/l (6.5 mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76 % increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32 %. The optimal strain LYC010 produced 3.52 g lycopene/l (50.6 mg/g DCW) in fed-batch fermentation.     

Fed‐Batch Process for Pyruvate Production by Recombinant Escherichia coli YYC202 Strain

Fed‐batch fermentation was applied to the production of pyruvate by using a recombinant Escherichia coli YYC202 strain. This strain is completely blocked in its ability to convert pyruvate into acetyl‐CoA or acetate, resulting in acetate auxotrophy during growth in glucose minimal medium. By controlling acetate and glucose feed rate, a series of lab‐scale fed‐batch experiments were performed at pH 7 and 37 °C. CO₂ production rate (CTR) was used for on‐line regulation of the acetate feed rate. The correlation between CTR and acetate consumption rate (ACR) was determined experimentally. At optimal process conditions a final pyruvate concentration higher than 62 g/L, a space‐time yield of up to 42 g/L/d and pyruvate/glucose molar yield of 1.11 mol/mol were achieved. Experimental evidence was gathered that pyruvate export is active.     

在大肠杆菌中合成的萤火虫荧光素酶的分离和性质

用生物工程技术将萤火虫荧光素酶基因转移到大肠杆菌,在大肠杆菌中合成荧光素酶。这种工程菌已可通过发酵大量培养,并从菌体分离得到接近纯化的荧光素酶。这种酶的分子量是103kD;巯基试剂5,5’-巯基-2(2-硝基苯甲酸)“DTNB”能抑制酶的活性;对于底物荧光素的K_m为1.2μmol/L;酶反应最适pH为7.77;酶催化的生物发光峰在560nm。     

L-asparaginase synthesis by Escherichia coli B

We have studied the influence of strain of organism, temperature, and medium on the production of the antileukemic intracellular enzyme L-asparaginase by E. coli B grown in shaken flasks. Five strains of E. coli B exhibited wide differences in their capacities to synthesize the EC-2 form of L-asparaginase active against leukemia. For the most productive strain, when grown in a casein hydrolysate medium, maximal production of L-asparaginase occurred at 25°C. At this temperature, the organism required glycerol, glucose, or other mono-saccharides to synthesize L-asparaginase. Synthesis was stimulated when glycerol was used in place of glucose, but not in its presence. The effect of glycerol on L-asparaginase synthesis was most evident when the cells were grown at 37°C, rather than at 25°C. With 0.25% glucose, cells had a specific activity of 409 I.U./g; with glycerol cells had a specific activity of 553 I.U./g. At 25°C, both cell and L-asparaginase synthesis were increased by the use of 0.25% glycerol resulting in only a slight increase in specific activity of the cells. The addition of zinc, copper, manganese, iron, L-asparagine, L-glutamine, or L-aspartic acid had no effect on L-asparaginase synthesis in the casein hydrolysate medium. L-aspartic acid (10^?2 M) enhanced L-asparaginase synthesis in a synthetic medium that lacked these metals or L-asparagine, L-glutamine, or L-aspartic acid; cells grown under these conditions had a specific activity of 90 I.U./g. In the casein hydrolysate medium, cell morphology was correlated with temperature of incubation.     

Restoration by Chloramphenicol of Bacteriophage Production in Escherichia coli B Infected with a Ligase-Deficient Amber Mutant

The addition of chloramphenicol (CM) 5 min after infection of the nonpermissive host Escherichia coli B with the ligase-negative T4 amber, T4 AmH39X, allowed replication of parental deoxyribonucleic acid (DNA) and the production of high-molecular-weight progeny DNA, composed mostly of subunits with a D₂/D₁ of 0.6. When CM was removed after the accumulation of a large pool of this DNA, most of the infected bacteria were able to produce viable progeny phage, with an average yield of approximately 15 bacteriophage per bacterium. This phenomenon is called CM rescue of the ligase-negative T4 Am. CsCl and sucrose gradient analyses showed both the resulting phage and DNA extracted from them to be similar to the phage and DNA produced on the permissive host. The total transfer of the parental label to progeny phages was as high as 20%. In contrast, in bacteria not treated with CM or in bacteria to which CM was added after phage-coded nucleases had already been synthesized, both parental and progeny (newly synthesized) DNA was composed of very short fragments. Phage which are produced under conditions other than those of CM rescue are dead, light in CsCl, and contain only very short fragments of DNA. Parent-to-progeny transfer in this case is below 1%. When light radio-active parental DNA was used to infect heavy bacteria, DNA replicating in the CM rescue conditions assumed only a hybrid density. After removal of CM and maturation, the parental DNA was incorporated into progeny molecules in fragments constituting approximately 7 to 10% of its mass. This pattern of distribution is essentially what is observed in similar experiments in the permissive host. The role of ligase as an enzyme which compensates for the lethal action of phage-coded nuclease and which is stringently required for the repair of single-stranded nicks is emphasized. The possibility of specific sites for a unique cutting enzyme is discussed in connection with the hypothesis of a circularly permuted assembly of sets.     

Production of phloroglucinol by Escherichia coli using a stationary-phase promoter

Escherichia coli was metabolically engineered using a new host-vector system to produce phloroglucinol. The key biosynthetic gene phlD (encoding a type III polyketide synthase) from Pseudomonas fluorescens was expressed in E. coli using the stationary-phase promoter of the fic gene and a high-copy plasmid. In shake-flasks, the engineered strain produced phloroglucinol up to 0.28 g/l with a productivity of 0.014 g/l h. About 9.2% of the glucose consumed was converted to phloroglucinol after 20 h. Compared with the widely used inducible T7 promoter system, this strain did not require IPTG induction and the final titer of phloroglucinol was 22% higher.