Microbial production of 2-deoxyribose 5-phosphate from acetaldehyde and triosephosphate for the synthesis of 2'-deoxyribonucleosides

2-Deoxyribose 5-phosphate was produced from acetaldehyde and dihydroxyacetone phosphate via D-glyceraldehyde 3-phosphate by Klebsiella pneumoniae B-4-4 through deoxyriboaldolase- and triosephosphate isomerase-catalyzing reactions. Under the optimum conditions, 98.7 mM 2-deoxyribose 5-phosphate was produced from 200 mM acetaldehyde and 117 mM dihydroxyacetone phosphate in 2 h with a molar yield of 84%. The 2-deoxyriobse 5-phosphate produced was directly transformed to 2'-deoxyribonucleoside by phosphopentomutase- and nucleoside phosphorylase-catalyzing reactions.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Engineering Escherichia coli for efficient production of 5-aminolevulinic acid from glucose

5-Aminolevulinic acid (ALA) recently received much attention due to its potential applications in many fields. In this study, we developed a metabolic strategy to produce ALA directly from glucose in recombinant Escherichia coli via the C5 pathway. The expression of a mutated hemA gene, encoding a glutamyl-tRNA reductase from Salmonella arizona, significantly improved ALA production from 31.1 to 176 mg/L. Glutamate-1-semialdehyde aminotransferase from E. coli was found to have a synergistic effect with HemA^M from S. arizona on ALA production (2052 mg/L). In addition, we identified a threonine/homoserine exporter in E. coli, encoded by rhtA gene, which exported ALA due to its broad substrate specificity. The constructed E. coli DALA produced 4.13 g/L ALA in modified minimal medium from glucose without adding any other co-substrate or inhibitor. This strategy offered an attractive potential to metabolic production of ALA in E. coli.     

Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.     

Construction of hypervesiculation Escherichia coli strains and application for secretory protein production

Outer membrane vesicles (OMVs) are extracellular vesicles released from the surface of Gram-negative bacteria, including Escherichia coli. Several gene-deficient mutants relating to envelope stress (nlpI and degP) and phospholipid accumulation in the outer leaflet of the outer membrane (mlaA and mlaE) increase OMV production. This study examined the combinatorial deletion of these genes in E. coli and its effect on OMV production. The nlpI and mlaE double-gene-knockout mutant (ΔmlaEΔnlpI) showed the highest OMV production. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based quantitative analysis showed that OMV production by strain ΔmlaEΔnlpI was ~30 times that by the wild-type (WT). In addition, to evaluate the protein secretion capacity of OMVs, a green fluorescent protein (GFP) fused with outer membrane protein W (OmpW) was expressed in OMVs. Western blot analysis showed that GFP secretion through OMVs reached 3.3 mg/L in the culture medium of strain ΔmlaEΔnlpI/gfp, 500 times that for the WT. Our approach using OMVs for extracellular protein secretion in E. coli is an entirely new concept compared with existing secretion systems.     

Crystallization of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Crystals of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli have been grown out of ammonium sulfate by the hanging drop method of vapor diffusion. The crystals belong to the hexagonal space group P6₁22 or P6₅22, with $\text{a = 124}\text{A}\text{?}\text{and}\text{c = 381}\text{A}\text{?}$, and diffract to 3.8 Å resolution.     

Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

Background  

Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies.     

A comprehensive comparison of DNA replication past 2-deoxyribose and its tetrahydrofuran analog in Escherichia coli

Apurinic/apyrimidinic (AP) sites are alkali labile lesions that, when encountered during DNA replication, can block polymerases or potentially result in mutagenic events. Owing to the instability of 2-deoxyribose lesions (AP), a chemically stable tetrahydrofuran analog (F) is often used as a model of abasic sites. A comparison of the two lesions in Saccharomyces cerevisiae revealed that the model lesion and 2-deoxyribose have distinct in vivo effects. Comprehensive comparative analyses of F and AP have not been carried out in Escherichia coli. We conducted a side-by-side investigation of F and AP in E.coli to compare their biological effects and interactions with SOS polymerases. Both lesions were examined in SOS-induced and uninduced cells. Our studies reveal that in uninduced E.coli the effects of individual polymerases in the replication of plasmids containing F or AP are distinct. However, when cells are SOS-induced, the biological effects of F and AP are similar.     

CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli.

Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so.     

Construction of Escherichia coli strains producing L-serine from glucose

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.     

Escherichia coli and its application in a mediated amperometric glucose sensor

Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor. The E. coli modified glucose sensor, which was prepared by immobilizing E. coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme. This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution. The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM. Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current. The E. coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA). When E. coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.     

Purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli.

A procedure for the purification of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli is described. Homogeneous enzyme of specific activity 17.7 units/mg was obtained in 22% yield. The key purification step involves substrate elution of the enzyme from a cellulose phosphate column. The subunit Mr was estimated to be 49 000 by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The native Mr was estimated to be 55 000 by gel filtration, indicating that the enzyme is monomeric.     

Metabolic engineering of Escherichia coli for the production of succinic acid from glucose

Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

CMP-N-acetyl neuraminic-acid synthetase from Escherichia coli: fermentative production and application for the preparative synthesis of CMP-neuraminic acid

In an optimized sorbitol/yeast extract/mineral salt medium up to 12 U/l CMP-N-acetyl-neuraminic-acid (Neu5Ac) synthetase was produced by Escherichia coli K-235 in shake-flask culture. A colony mutant of this strain, E. coli K-235/CS1, was isolated with improved enzyme formation: in shake flasks with a yield of up to 20.8 U/l and 54 mU/mg protein in the cell extract. With this strain 26500 U CMP-Neu5Ac synthetase was produced with a high specific activity (0.128 U/mg) by fed-batch fermentation on 230-1 scale. On a 10-l scale the enzyme yield was 191 U/l culture medium. The enzyme was partially purified by precipitation with polyethyleneglycol resulting in a three- to fourfold enrichment and a recovery rate of more than 80%; most of the CTP hydrolysing enzymes were removed. The native synthetase was deactivated completely by incubation at 45°C for 10 min, but could be stabilized remarkably by glycerol and different salts. The enzyme was used for the preparative synthesis of CMP-Neu5Ac with a conversion yield of 87% based on CTP.     

Overexpression and purification of fructose-1-phosphate kinase from Escherichia coli: application to the assay of fructose 1-phosphate

Fructose 1-phosphate is a metabolite that plays a regulatory role in liver and is best measured using an assay based on its conversion to fructose 1,6-bisphosphate by a bacterial fructose-1-phosphate kinase (Fru1PK). The open reading frame encoding Escherichia coli Fru1PK has been introduced in an expression plasmid (pET3a) based on the T7 promoter-driven system, which was used to overexpress the enzyme. The conditions for the production of soluble Fru1PK were optimized. The purification procedure used involved ammonium sulfate precipitation and chromatography on DEAE-Sepharose and was aimed mostly at stabilizing the enzyme and at freeing Fru1PK from bacterial contaminants that could interfere in the fructose 1-phosphate assay. From a 1-liter culture, more than 50 mg protein is obtained. This preparation can be used in an enzymatic assay that measures specifically fructose 1-phosphate in tissue extracts.