Production of Aromatic Compounds by Metabolically Engineered Escherichia coli with an Expanded Shikimate Pathway

Escherichia coli was metabolically engineered by expanding the shikimate pathway to generate strains capable of producing six kinds of aromatic compounds, phenyllactic acid, 4-hydroxyphenyllactic acid, phenylacetic acid, 4-hydroxyphenylacetic acid, 2-phenylethanol, and 2-(4-hydroxyphenyl)ethanol, which are used in several fields of industries including pharmaceutical, agrochemical, antibiotic, flavor industries, etc. To generate strains that produce phenyllactic acid and 4-hydroxyphenyllactic acid, the lactate dehydrogenase gene (ldhA) from Cupriavidus necator was introduced into the chromosomes of phenylalanine and tyrosine overproducers, respectively. Both the phenylpyruvate decarboxylase gene (ipdC) from Azospirillum brasilense and the phenylacetaldehyde dehydrogenase gene (feaB) from E. coli were introduced into the chromosomes of phenylalanine and tyrosine overproducers to generate phenylacetic acid and 4-hydroxyphenylacetic acid producers, respectively, whereas ipdC and the alcohol dehydrogenase gene (adhC) from Lactobacillus brevis were introduced to generate 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, respectively. Expression of the respective introduced genes was controlled by the T7 promoter. While generating the 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol producers, we found that produced phenylacetaldehyde and 4-hydroxyphenylacetaldehyde were automatically reduced to 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol by endogenous aldehyde reductases in E. coli encoded by the yqhD, yjgB, and yahK genes. Cointroduction and cooverexpression of each gene with ipdC in the phenylalanine and tyrosine overproducers enhanced the production of 2-phenylethanol and 2-(4-hydroxyphenyl)ethanol from glucose. Introduction of the yahK gene yielded the most efficient production of both aromatic alcohols. During the production of 2-phenylethanol, 2-(4-hydroxyphenyl)ethanol, phenylacetic acid, and 4-hydroxyphenylacetic acid, accumulation of some by-products were observed. Deletion of feaB, pheA, and/or tyrA genes from the chromosomes of the constructed strains resulted in increased desired aromatic compounds with decreased by-products. Finally, each of the six constructed strains was able to successfully produce a different aromatic compound as a major product. We show here that six aromatic compounds are able to be produced from renewable resources without supplementing with expensive precursors.     

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.     

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.     

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.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Effect of Growth Phase Feeding Strategies on Succinate Production by Metabolically Engineered Escherichia coli

Aerobic growth conditions significantly influenced anaerobic succinate production in two-stage fermentation by Escherichia coli AFP111 with knockouts in rpoS, pflAB, ldhA, and ptsG genes. At a low cell growth rate limited by glucose, enzymes involved in the reductive arm of the tricarboxylic acid cycle and the glyoxylate shunt showed elevated activities, providing AFP111 with intracellular redox balance and increased succinic acid yield and productivity.Succinic acid is valued as one of the key basic chemicals used in the preparation of biodegradable polymers or as raw material for chemicals of the C₄ family (8, 19). The fermentative production of succinic acid from renewable resources is environmentally acceptable and sustainable (3). A breakthrough in genetically engineering Escherichia coli (6, 7, 11, 18) for succinate production was the isolation of strain AFP111 (1, 4), a mutant of NZN111 with a spontaneous ptsG mutation (pflAB ldhA double mutant). The process involves a two-stage fermentation, with aerobic cell growth followed by anaerobic conditions for succinate production (16, 21, 22). The aerobically induced enzymes can maintain their activity during the anaerobic phase and significantly affect succinate fermentation (22, 23). Using the best transition time based on the activities of the key enzymes and other physiological states, a two-stage fermentation using the recombinant AFP111 strain harboring pTrc99A-pyc achieved a final succinic acid concentration and productivity of 99.2 g·liter⁻¹ and 1.3 g·liter⁻¹·h⁻¹, respectively (21).Aerobic cell growth is essential for the subsequent anaerobic fermentation. However, few studies have focused on the regulation of aerobic cell growth. As a regulation method, gluconeogenic carbon sources were used instead of glucose for the aerobic growth of Escherichia coli NZN111 and the activities of enzymes that are favorable for the anaerobic synthesis of succinate were enhanced (23, 24). Unfortunately, a gluconeogenic carbon source (e.g., sodium acetate) might increase the osmotic pressure of culture media, which would be detrimental to succinate production (23). As another regulation method, a glucose feeding strategy controlling the glucose concentration at about 0.5 g·liter⁻¹ up to 1 g·liter⁻¹ was reported to prevent excessive formation of acetic acid (16).In this study, we investigated different glucose feeding strategies for the aerobic growth phase of the two-phase process for succinate production by E. coli AFP111. Specifically, we compared several growth rates by using glucose limitation in addition to maximum growth under conditions of excess glucose.E. coli AFP111 [F⁺ λ⁻ rpoS396(Am) rph-1 ΔpflAB::Cam ldhA::Kan ptsG] (4, 16), which was a kind gift from D. P. Clark (Southern Illinois University), was the only strain used in this study. Luria-Bertani (LB) medium (60 ml) was used for inoculum culture in 1,000-ml flasks, and 3 liters of chemically defined medium (13, 14) was used for two-stage culture in a 7-liter fermentor. Two-stage fermentations were divided into three types, based on the glucose feeding strategy used during the aerobic stage. For type I culture, the glucose concentration was maintained at about 20 g·liter⁻¹ during aerobic cell growth. Type II and III cultures comprised a batch process and subsequent glucose-limited fed-batch process (Fig. ​(Fig.1).1). The batch process initially contained 13 g/liter of glucose. The fed-batch process began when the dry cell weight (DCW) reached about 6 g/liter, with type II and type III cultures using a 600 g/liter glucose feed to achieve cell growth rates of 0.15 h⁻¹ and 0.07 h⁻¹, respectively (10). When the DCW reached 12 g·liter⁻¹, the aerobically grown cells were directly transferred to anaerobic conditions (Fig. ​(Fig.1).1). For the anaerobic process, oxygen-free CO₂ was sparged at 0.5 liter·min⁻¹, the pH was controlled between 6.4 and 6.8 with intermittent supplementation of solid magnesium carbonate hydroxide, and the glucose concentration was maintained at about 20 g·liter⁻¹ by supplying glucose in an 800-g·liter⁻¹ solution.Open in a separate windowFIG. 1.Concentrations of glucose (circles), DCW (triangles), and succinic acid (squares) in the three types of two-stage fermentation by AFP111. μ, growth rate.The optical density at 600 nm was used to monitor cell growth, and this value was correlated to DCW. The concentration of glucose was assayed with an enzyme electrode analyzer, and organic acids were quantified by high-performance liquid chromatography (HPLC). The intracellular concentrations of NADH and NAD⁺ were assayed with a cycling method (12). The activities of isocitrate lyase (ICL) (20), pyruvate kinase (PYK) (17), phosphoenolpyruvate (PEP) carboxykinase (PCK) (20, 23), PEP carboxylase (PPC) (23), and malate dehydrogenase (MDH) (23) were measured spectrophotometrically at the end of the aerobic phase and 12 h after the onset of the anaerobic phase.All three types of fermentations were terminated when the succinate concentration increased less than 1 g·liter⁻¹ in 5 h. Type III fermentation was terminated at a final succinic acid concentration of 101.2 g·liter⁻¹ and an anaerobic-phase productivity of 1.89 g·liter⁻¹·h⁻¹ (Fig. ​(Fig.1).1). Trace amounts of by-products (such as acetate, ethanol, and pyruvate) accumulated and did not follow any trend in the anaerobic phase (data not shown).At the end of the aerobic culture phase, the specific enzyme activities of PCK, PYK, and ICL in type III culture were 2.9, 2.5, and 11.4 times higher, respectively, than the activities in type I culture (Table ​(Table1)1) . This phenomenon is consistent with published reports that suggest that the expression of enzymes involved in anaplerotic metabolism and the glyoxylate shunt (5, 15) is elevated in E. coli grown under glucose-limited conditions. These enzymes maintained their activities in the subsequent anaerobic phase (Table ​(Table1)1) and would be central to succinate production (22, 23). The elevated levels of PCK and PPC would provide the reductive branch of the tricarboxylic acid (TCA) cycle with oxaloacetate (OAA) at a higher rate (9), thereby supplying both malate and citrate (Table ​(Table11).

TABLE 1.

Activities of enzymes at the end of the aerobic culture phase and 12 h after the onset of the anaerobic phase

TDP-l-Megosamine Biosynthesis Pathway Elucidation and Megalomicin A Production in Escherichia coli

In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.Most of the deoxy sugars found in natural products belong to the 6-deoxyhexose (6DOH) family (21). Since many of these 6DOHs are essential for the bioactivity of natural compounds, extensive efforts have been made to investigate the relevant genetics, enzymology, and mechanistic features of the biosynthetic pathways leading to these sugars. The amino sugar l-megosamine is found within a family of macrolide compounds produced by the actinomycete Micromonospora megalomicea, named megalomicins A (MegA) (structure 1), B, C1, and C2 (Fig. ​(Fig.11 A) (27). These compounds consist of a 14-membered macrolactone ring carrying three deoxy sugar residues, l-mycarose, d-desosamine, and l-megosamine. The megalomicin congeners differ from each other in the specific acetyl or propionyl groups attached at the 3′′′ or 4′′′ hydroxyls of the mycarose moiety. These macrolides were originally discovered as antibacterial agents which inhibit protein synthesis through selective binding to the bacterial 50S ribosomal subunit in a mode similar to that of erythromycins and other macrolides (25). Due to the similarities with erythromycin in terms of structure, antibacterial activity, and pharmacological properties, megalomicins did not receive much attention until antiviral and antiparasitic activities of these compounds were reported (1, 3). These studies demonstrated that megalomicins interfere with protein trafficking, resulting in an anomalous protein glycosylation (4, 5) that affects the maturation of enveloped viruses, including herpes simplex virus, Semliki Forest virus, vesicular stomatitis virus, and more importantly the human immunodeficiency virus type 1 (HIV-1) (1, 22). In HIV replication, inhibition of gp160 protein processing to gp120 and gp41 resulted in noninfectious virions (22). In addition, megalomicins also showed antiparasitic activity against the epimastigote stage of Trypanosoma cruzi, Leishmania spp., and Plasmodium falciparum, although in this case the mechanism of action still remains unclear (3).Open in a separate windowFIG. 1.(A) Structures of megalomicins and erythromycins. (B) Genetic organization of the meg gene cluster from M. megalomicea. A 12-kb fragment, including putative l-megosamine biosynthesis genes, is indicated.The main structural difference between megalomicins and erythromycins is the presence of the l-megosamine sugar moiety at C-6 (Fig. ​(Fig.1A).1A). Since erythromycin does not exhibit antiparasitic and antiviral activities, the presence of this additional amino sugar in megalomicins could be associated with the differential properties of these compounds (1, 3). Due to the potential pharmacological relevance of megalomicins and the lack of a detailed characterization of the l-megosamine biosynthetic pathway from the megalomicin (meg) gene cluster, an in-depth metabolic route study was deemed warranted.Analysis of the overall organization of the meg gene cluster revealed that l-megosamine biosynthesis genes are grouped together within this gene cluster (Fig. ​(Fig.1B)1B) (25). This was demonstrated by the heterologous expression of a 12-kb DNA fragment that included the putative megosamine biosynthesis genes in the erythromycin producer strain Saccharopolyspora erythraea, which allowed the production of megalomicins in this host (25). Six biosynthetic steps were proposed for the biosynthesis of TDP-l-megosamine (l-Meg) (structure 2) from the intermediate TDP-4-keto-6-deoxy-d-glucose (TKDG) (structure 3). Neither the biosynthesis pathway nor the enzymes involved in each catalytic step have been confirmed.Herein, the investigation focused on the biosynthesis of l-Meg from M. megalomicea by the heterologous expression of meg genes in Escherichia coli. The sequence of enzymatic reactions implicated in this pathway was confirmed by analyzing the TDP-sugar intermediates generated from the expression of operons containing different sets of genes. This methodology allowed the validation of a new pathway for the biosynthesis of l-Meg from the precursor TKDG through the use of five enzymatic steps. Bioconversion experiments furthermore demonstrated that the attachment of l-megosamine to the macrolide intermediate required both a specific glycosyltransferase and a helper protein.     

Evidence for Two Genes Specifically Involved in Unsaturated Fatty Acid Biosynthesis in Escherichia coli

Unsaturated fatty acid auxotrophs of Eschericha coli have been divided into two distinct cistrons by extract complementation and genetic complementation based on abortive transduction. Lesions in one cistron result in the loss of the beta-hydroxydecanoyl thioester dehydrase which produces the first biosynthetic intermediate in unsaturated fatty acid formation. Evidence is presented which indicates that lesions in the second cistron result in the lack of a second enzyme specifically involved in the biosynthesis of unsaturated fatty acids.     

Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.     

Escherichia coli Strains Engineered for Homofermentative Production of d-Lactic Acid from Glycerol

Given its availability and low price, glycerol has become an ideal feedstock for the production of fuels and chemicals. We recently reported the pathways mediating the metabolism of glycerol in Escherichia coli under anaerobic and microaerobic conditions. In this work, we engineer E. coli for the efficient conversion of glycerol to d-lactic acid (d-lactate), a negligible product of glycerol metabolism in wild-type strains. A homofermentative route for d-lactate production was engineered by overexpressing pathways involved in the conversion of glycerol to this product and blocking those leading to the synthesis of competing by-products. The former included the overexpression of the enzymes involved in the conversion of glycerol to glycolytic intermediates (GlpK-GlpD and GldA-DHAK pathways) and the synthesis of d-lactate from pyruvate (d-lactate dehydrogenase). On the other hand, the synthesis of succinate, acetate, and ethanol was minimized through two strategies: (i) inactivation of pyruvate-formate lyase (ΔpflB) and fumarate reductase (ΔfrdA) (strain LA01) and (ii) inactivation of fumarate reductase (ΔfrdA), phosphate acetyltransferase (Δpta), and alcohol/acetaldehyde dehydrogenase (ΔadhE) (strain LA02). A mutation that blocked the aerobic d-lactate dehydrogenase (Δdld) also was introduced in both LA01 and LA02 to prevent the utilization of d-lactate. The most efficient strain (LA02Δdld, with GlpK-GlpD overexpressed) produced 32 g/liter of d-lactate from 40 g/liter of glycerol at a yield of 85% of the theoretical maximum and with a chiral purity higher than 99.9%. This strain exhibited maximum volumetric and specific productivities for d-lactate production of 1.5 g/liter/h and 1.25 g/g cell mass/h, respectively. The engineered homolactic route generates 1 to 2 mol of ATP per mol of d-lactate and is redox balanced, thus representing a viable metabolic pathway.Lactic acid (lactate) and its derivatives have many applications in the food, pharmaceutical, and polymer industries (13, 30). An example is polylactic acid, a renewable, biodegradable, and environmentally friendly polymer produced from d- and l-lactate (19). In this context, biological processes have the advantage of being able to produce chirally pure lactate from inexpensive media containing only the carbon source and mineral salts (43). While lactic acid bacteria traditionally have been used in the production of d-lactate from carbohydrate-rich feedstocks, several laboratories recently have reported alternative biocatalysts (13, 30), many of which are engineered Escherichia coli strains that produce d- or l-lactate (4, 8, 50, 51, 52).Unlike the aforementioned reports, which have dealt with the use of carbohydrates, our work focuses on the use of glycerol as a carbon source for the production of d-lactate. Glycerol has become an inexpensive and abundant substrate due to its generation in large amounts as a by-product of biodiesel and bioethanol production (18, 32, 47). The conversion of glycerol to higher-value products has been proposed as a path to economic viability for the biofuels industry (47). One such product is lactate, whose production could be readily integrated into existing biodiesel and bioethanol facilities, thus establishing true biorefineries.Although many microorganisms are able to metabolize glycerol (25), the use of industrial microbes such as E. coli could greatly accelerate the development of platforms to produce fuels and chemicals from this carbon source. We recently reported on the ability of E. coli to metabolize glycerol under either anaerobic or microaerobic conditions and identified the environmental and metabolic determinants of these processes (9, 11, 28). In one of the studies, the pathways involved in the microaerobic utilization of glycerol were elucidated, and they are shown in Fig. ​Fig.11 (9). A common characteristic of glycerol metabolism under either anaerobic or microaerobic conditions is the generation of ethanol as the primary product and the negligible production of lactate (6, 9, 11, 28). In the work reported here, the knowledge base created by the aforementioned studies was used to engineer E. coli for the efficient conversion of glycerol to d-lactate in minimal medium. The engineered strains hold great promise as potential biocatalysts for the conversion of low-value glycerol streams to a higher-value product like d-lactate.Open in a separate windowFIG. 1.Pathways involved in the microaerobic utilization of glycerol in E. coli (9). Genetic modifications supporting the metabolic engineering strategies employed in this work are illustrated by thicker lines (overexpression of gldA-dhaKLM, glpK-glpD, and ldhA) or cross bars (disruption of pflB, pta, adhE, frdA, and dld). Broken lines illustrate multiple steps. Relevant reactions are represented by the names of the gene(s) coding for the enzymes: aceEF-lpdA, pyruvate dehydrogenase complex; adhE, acetaldehyde/alcohol dehydrogenase; ackA, acetate kinase; dhaKLM, dihydroxyacetone kinase; dld, respiratory d-lactate dehydrogenase; fdhF, formate dehydrogenase, part of the formate hydrogenlyase complex; frdABCD, fumarate reductase; gldA, glycerol dehydrogenase; glpD, aerobic glycerol-3-phosphate dehydrogenase; glpK, glycerol kinase; hycB-I, hydrogenase 3, part of the formate hydrogenlyase complex; ldhA, fermentative d-lactate dehydrogenase; pflB, pyruvate formate-lyase; pta, phosphate acetyltransferase; pykF, pyruvate kinase. Abbreviations: DHA, dihydroxyacetone; DHAP, DHA phosphate; G-3-P, glycerol-3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; P/O, amount of ATP produced in the oxidative phosphorylation per pair of electrons transferred through the electron transport system; QH₂, reduced quinones.     

MocA Is a Specific Cytidylyltransferase Involved in Molybdopterin Cytosine Dinucleotide Biosynthesis in Escherichia coli

We have purified and characterized a specific CTP:molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP:molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase YagTSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl₂ are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 ± 0.01 min⁻¹ was obtained. A 1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 ± 0.02 μm for CTP and 1.17 ± 0.18 μm for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.The biosynthesis of the molybdenum cofactor (Moco)² is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. In Moco the molybdenum atom is coordinated to the dithiolene group of the 6-alkyl side chain of a pterin called molybdopterin (MPT). Moco biosynthesis has been extensively studied in Escherichia coli by using a combination of biochemical, genetic, and structural approaches (1, 2). The biosynthesis of Moco has been divided into four major steps in Escherichia coli: (i) formation of precursor Z (3, 4), (ii) formation of MPT from precursor Z (5, 6), (iii) insertion of molybdenum to form Moco via an adenylylated MPT intermediate (7–9), and (iv) additional modification by covalent addition of GMP to the C4′ phosphate of MPT via a pyrophosphate bond, forming the molybdopterin guanine dinucleotide (MGD) cofactor (10, 11). In E. coli, GMP attachment to Moco is catalyzed by the MobA and MobB proteins (12). Although MobA was shown to be essential for this reaction and acts as a GTP:molybdopterin guanylyltransferase (11), the role of MobB still remains uncertain. From the crystal structure, it was postulated that MobB is an adapter protein acting in concert with MobA to achieve the efficient biosynthesis and utilization of MGD (13). Although MobA was shown to bind MPT, Mo-MPT, and MGD (14), investigations of in vitro studies using purified MobA, MgCl₂, GTP, and either MPT or Mo-MPT showed that MGD was only formed by MobA when the molybdenum atom was already ligated to MPT (15). The formation of bis-MGD is one of the most enigmatic steps in Moco biosynthesis in E. coli. It is still not known whether the two MGD molecules assemble on MobA or instead after the insertion into the respective target proteins like DMSO reductase or nitrate reductase A. In other bacteria like Arthrobacter nicotinovorans, Veillonella atypica, or Oligotropha carboxidovorans, Moco can be further modified by the attachment of CMP to the C4′ phosphate of MPT forming the molybdopterin cytosine dinucleotide (MCD) cofactor (16–18). A specific enzyme catalyzing the CTP:molybdopterin cytidylyltransferase reaction has not been identified so far. For A. nicotinovorans nicotine dehydrogenase and ketone dehydrogenase the involvement of a MobA homologous protein for MCD formation was reported (16); however, it was not shown whether the MobA protein was specifically required for MCD biosynthesis or whether it was also involved in the biosynthesis of MGD in this bacterium. Furthermore, enzymes binding MCD in bacteria usually contain an additional modification at the molybdenum site of Moco, where a terminal oxo-ligand is exchanged by a sulfido ligand, forming sulfurated or mono-oxo Moco (19). Recently, the MCD-containing protein YagTSR was identified and characterized in E. coli as a periplasmic aldehyde oxidoreductase which oxidizes a broad spectrum of aldehydes using ferredoxin as electron acceptor (20). It was shown that for the production of an active form of YagTSR, the YagQ protein was required, which is believed to be a MCD binding chaperone involved in the sulfuration of the Mo site and the insertion of sulfurated MCD into apoYagTSR (20). The majority of the other molybdoenzymes in E. coli were shown to bind the bis-MGD form of Moco, in which molybdenum is coordinated to two MGD moieties. The other exception is the YedY protein, being so far the only E. coli protein binding the Mo-MPT form of Moco (21). However, the physiological role of this protein still remains unclear.Investigations on YagTSR showed that MCD was inserted into YagR independent of the function of MobA, indicating that a so-far unidentified protein is involved in MCD biosynthesis in E. coli (20). Here, we report the identification of the specific CTP:molybdopterin cytidylyltransferase, which we named MocA (formerly named YgfJ by the E. coli nomenclature of genes with unknown function). Purified MocA was shown to catalyze the formation of MCD from Mo-MPT and CTP in vitro. Additionally, we report that a disruption in the mocA gene impaired MCD biosynthesis in E. coli, resulting in an inactive YagTSR protein devoid of Moco.     

CS5 Pilus Biosynthesis Genes from Enterotoxigenic Escherichia coli O115:H40

We have sequenced the entire region of DNA required for the biosynthesis of CS5 pili from enterotoxigenic Escherichia coli O115:H40 downstream of the major subunit gene, designated csfA (for coli surface factor five A). Five more open reading frames (ORFs) (csfB, csfC, csfE, csfF, and csfD) which are transcribed in the same direction as the major subunit and are flanked by a number of insertion sequence regions have been identified. T7 polymerase-mediated overexpression of the cloned csf ORFs confirmed protein sizes based on the DNA sequences that encode them. The expression of only the csf region in E. coli K-12 resulted in the hemagglutination of human erythrocytes and the cell surface expression of CS5 pili, suggesting that the cluster contains all necessary information for CS5 pilus biogenesis and function.     

Identification of a Hotdog Fold Thioesterase Involved in the Biosynthesis of Menaquinone in Escherichia coli

Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.     

Use of Inducible Feedback-Resistant N-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically Engineered Escherichia coli K-12 Strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of “arg-derepressed” E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5α strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroF^FBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.     

Mutations in Escherichia coli aceE and ribB Genes Allow Survival of Strains Defective in the First Step of the Isoprenoid Biosynthesis Pathway

A functional 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is required for isoprenoid biosynthesis and hence survival in Escherichia coli and most other bacteria. In the first two steps of the pathway, MEP is produced from the central metabolic intermediates pyruvate and glyceraldehyde 3-phosphate via 1-deoxy-D-xylulose 5-phosphate (DXP) by the activity of the enzymes DXP synthase (DXS) and DXP reductoisomerase (DXR). Because the MEP pathway is absent from humans, it was proposed as a promising new target to develop new antibiotics. However, the lethal phenotype caused by the deletion of DXS or DXR was found to be suppressed with a relatively high efficiency by unidentified mutations. Here we report that several mutations in the unrelated genes aceE and ribB rescue growth of DXS-defective mutants because the encoded enzymes allowed the production of sufficient DXP in vivo. Together, this work unveils the diversity of mechanisms that can evolve in bacteria to circumvent a blockage of the first step of the MEP pathway.     

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.     

大肠杆菌和志贺氏菌O-抗原糖基转移酶与聚合酶的生物信息学研究

利用生物信息学手段对大肠杆菌和志贺氏菌的 1 1 0个O 抗原糖基转移酶与 39个O 抗原聚合酶的序列进行分析 ,探讨这两种酶的序列和结构特点。统计了其序列一致性 ,密码子使用和 (G C) %含量的特点 ;讨论了O 抗原糖基转移酶和聚合酶对底物的特异性 ;推测了 6组糖基转移酶的功能 ;通过对蛋白拓扑结构的预测 ,发现O 抗原聚合酶中广泛存在一个位于细胞周质中的亲水环 (Loop) ,是可能的功能区域 ;通过对蛋白高级结构的预测 ,发现O 抗原糖基转移酶属于两个不同的蛋白超家族。