Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.     

Metabolic engineering of Escherichia coli to enhance shikimic acid production from sorbitol

Shikimic acid (SA) is the key synthetic material of Oseltamivir, which is an effective drug for the prevention and treatment of influenza. In this study, to block the downstream metabolic pathway of SA, the shikimate kinase isoenzyme genes aroK and aroL were deleted by Red recombination. Moreover, the key enzyme genes aroG, aroB, tktA and aroE of SA pathway were co-expressed by constructing the recombinant vector pETDuet-GBAE. As a result, SA production of E. coli BW25113 (?aroL/aroK, DE3)/pETDuet-GBAE reached 1,077.6 mg/l when low amounts of sorbitol (5 g/l) were fed in shake flasks. The yield was 3.7 times that when glucose was used (P < 0.05). The results showed that sorbitol was an optimized carbon source for the high efficient accumulation of SA for the first time, which was applicable to use in the industry for high yields and low consumption.     

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Metabolic engineering for the production of shikimic acid in an evolved <Emphasis Type="Italic">Escherichia coli</Emphasis> strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS^-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroG ^fbr, tktA, aroB and aroE, on SA synthesis.     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

A convenient method for multiple insertions of desired genes into target loci on the <Emphasis Type="Italic">Escherichia coli</Emphasis> chromosome

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF ^fbr and tyrA ^fbr or aroF ^fbr and pheA ^fbr ) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.     

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.     

Production of p-Aminobenzoic acid by metabolically engineered Escherichia coli

The production of chemical compounds from renewable resources is an important issue in building a sustainable society. In this study, Escherichia coli was metabolically engineered by introducing T7lac promoter-controlled aroF^fbr, pabA, pabB, and pabC genes into the chromosome to overproduce para-aminobenzoic acid (PABA) from glucose. Elevating the copy number of chromosomal P_T7lac-pabA-pabB distinctly increased the PABA titer, indicating that elevation of 4-amino-4-deoxychorismic acid synthesis is a significant factor in PABA production. The introduction of a counterpart derived from Corynebacterium efficiens, pabAB (ce), encoding a fused PabA and PabB protein, resulted in a considerable increase in the PABA titer. The introduction of more than two copies of P_T7lac-pabAB (ce-mod), a codon-optimized pabAB (ce), into the chromosome of a strain that simultaneously overexpressed aroF^fbr and pabC resulted in 5.1?mM PABA from 55.6?mM glucose (yield 9.2%). The generated strain produced 35?mM (4.8?g?L^?1) PABA from 167?mM glucose (yield 21.0%) in fed-batch culture.     

Microaerobic Conversion of Glycerol to Ethanol in Escherichia coli

Glycerol has become a desirable feedstock for the production of fuels and chemicals due to its availability and low price, but many barriers to commercialization remain. Previous investigators have made significant improvements in the yield of ethanol from glycerol. We have developed a fermentation process for the efficient microaerobic conversion of glycerol to ethanol by Escherichia coli that presents solutions to several other barriers to commercialization: rate, titer, specific productivity, use of inducers, use of antibiotics, and safety. To increase the rate, titer, and specific productivity to commercially relevant levels, we constructed a plasmid that overexpressed glycerol uptake genes dhaKLM, gldA, and glpK, as well as the ethanol pathway gene adhE. To eliminate the cost of inducers and antibiotics from the fermentation, we used the adhE and icd promoters from E. coli in our plasmid, and we implemented glycerol addiction to retain the plasmid. To address the safety issue of off-gas flammability, we optimized the fermentation process with reduced-oxygen sparge gas to ensure that the off-gas remained nonflammable. These advances represent significant progress toward the commercialization of an E. coli-based glycerol-to-ethanol process.     

Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK ⁺) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.     

Structural Analysis of an Extracellular Polysaccharide of Porodisculus pendulus

The aroL gene, encoding shikimate kinase of Brevibacterium lactofermentum, a coryneform glutamic acid-producing bacterium, was cloned. Recombinant plasmids containing the aroL gene caused elevated levels of shikimate kinase synthesis in B. lactofermentum. It was found that in addition to the aroL gene, the aroB and aroE genes, encoding dehydroquinate synthase and shikimate dehydrogenase, respectively, also existed on these recombinant plasmids, in complementation tests with various Escherichia coli and B. lactofermentum aromatic amino acid auxotrophs. The aroL, aroB and aroE genes of B. lactofermentum are located closely on the cloned DNA fragment, in that order. It was shown that at least these three aro genes form a cluster on the chromosome of B. lactofermentum.     

Characterization of Methylglyoxal Synthase from Clostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The K_m and V_max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min⁻¹ μg⁻¹, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.     

Production of glucose-6-phosphate by glucokinase coupled with an ATP regeneration system

A process of glucose-6-phosphate (G-6-P) production coupled with an adenosine triphosphate (ATP) regeneration system was constructed that utilized acetyl phosphate (ACP) via acetate kinase (ACKase). The genes glk and ack from Escherichia coli K12 were amplified and cloned into pET-28a(+), then transformed into E. coli BL21 (DE3) and the recombinant strains were named pGLK and pACK respectively. Glucokinase (glkase) in pGLK and ACKase in pACK were both overexpressed in soluble form. G-6-P was efficiently produced from glucose and ACP using a very small amount of ATP. The conversion yield was greater than 97 % when the reaction solution containing 10 mM glucose, 20 mM ACP-Na₂, 0.5 mM ATP, 5 mM Mg²⁺, 50 mM potassium phosphate buffer (pH 7.0), 4.856 U glkase and 3.632 U ACKase were put into 37 °C water bath for 1 h.     

Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

Improvement of shikimic acid production in <Emphasis Type="Italic">Escherichia coli</Emphasis> with growth phase-dependent regulation in the biosynthetic pathway from glycerol

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33 g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.     

Transposon-generated Tn10 insertion mutations at thearo genes ofEscherichia coli K-12

We have obtained a set ofEscherichia coli K-12 derivatives with transposon-generated Tn10 insertion mutations at thearo genes of their aromatic biosynthetic pathway. Bacteriophage NK561 (Tn10) has been used for transposon mutagenesis ofE. coli, strain BW545. Tetracycline (Tc)-resistant derivatives were screened by their Aro^– phenotype by growth on a minimal medium with adequate requirements. Sixaro mutant types were mapped; two strains werearoA, twoaroD, onearoB oraroE, and onearoC. A selective medium and ad-cycloserine enrichment in the presence of tetracycline were used to select for Aro^–, Tc-sensitive derivatives. The reversion index to aromatic-independent colonies of some derivatives was less than 2 × 10^–11 per bacterium per generation. P1 transduction experiments transferred an aroA::Tn10 insertion fromE. coli BW545 to an enterotoxigenicE. coli strain from porcine origin. Derivatives of this strain beingaro, Tc-sensitive and not reverting toaro ⁺ at a detectable frequency, and many others transduced at will, may prove their usefulness as live vaccines.     

Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria

A production system of UDP-N-acetylglucosamine (UDP-GlcNAc) was established by using recombinant Escherichia coli and Corynebacterium ammoniagenes in combination. E. coli overexpressed the UDP-GlcNAc biosynthetic genes, glmM, glmU, glk, ppa, ack, and pta, whereas C. ammoniagenes contributed to the formation of UTP from orotic acid. Glucose 1,6-diphosphate (Glc-1,6-P₂), which was required for the activity of phosphoglucosamine mutase involved in UDP-GlcNAc biosynthesis, was supplied by phosphoglucomutase and phosphofructokinase. Starting with orotic acid (65 mM) and glucosamine (400 mM), UDP-GlcNAc accumulated at 11.4 mM (7.4 g l^–1) after 8 h.