Metabolic engineering of Escherichia coli for α-farnesene production

Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.     

Promoter engineering of cascade biocatalysis for α-ketoglutaric acid production by coexpressing l-glutamate oxidase and catalase

Applied Microbiology and Biotechnology - Enzymatic transformation is now an attractive alternative for α-ketoglutaric acid (α-KG) production, but the oxidative deamination from l-glutamic...     

Biomimetic Au nanodot synthesis using Escherichia coli secretion and interaction with α-ketoglutaric acid

正Dear Editor,Gold nanostructures have distinctive physicochemical properties, such as quantum size effects, surface plasmon resonance (SPR), high catalytic activity, and self-assembly,so they are one of the most widely studied materials.     

Production of γ-aminobutyric acid in Escherichia coli by engineering MSG pathway

Abstract

The compound γ-aminobutyric acid (GABA) has many important physiological functions. The effect of glutamate decarboxylases and the glutamate/GABA antiporter on GABA production was investigated in Escherichia coli. Three genes, gadA, gadB, and gadC were cloned and ligated alone or in combination into the plasmid pET32a. The constructed plasmids were transformed into Escherichia coli BL21(DE3). Three strains, E. coli BL21(DE3)/pET32a-gadA, E. coli BL21(DE3)/pET32a-gadAB and E. coli BL21(DE3)/pET32a-gadABC were selected and identified. The respective titers of GABA from the three strains grown in shake flasks were 1.25, 2.31, and 3.98?g/L. The optimal titer of the substrate and the optimal pH for GABA production were 40?g/L and 4.2, respectively. The highest titer of GABA was 23.6?g/L at 36?h in batch fermentation and was 31.3?g/L at 57?h in fed-batch fermentation. This study lays a foundation for the development and use of GABA.     

Efficient production of endotoxin depleted bioactive α-hemolysin of uropathogenic Escherichia coli

Abstract

Uropathogenic E. coli (UPEC), especially associated with severe urinary tract infections (UTI) pathologies, harbors an important virulence factor known as α-hemolysin (110?kDa). Hemolytic activity of α-hemolysin (HlyA) requires modification (acylation) of two lysine residues of HlyA by HlyC, part of operon hlyCABD. Most of the previous studies had used whole operon hlyCABD and gene tolC cloning for the production of active α-hemolysin. Studies involving α-hemolysin are limited due to the cumbersome and manual method of purification for this toxin. Here, we report a simple method for production of both active and inactive recombinant α-hemolysin by cloning only hlyA and hlyC genes of operon hlyCABD. Presence of both active and inactive α-hemolysin would be advantageous for functional characterization. After translation, the yield of the purified α-hemolysin was 1?mg/200?ml. Functionality of the recombinant α-hemolysin protein was confirmed using hemolytic assay. This is the first report of the production of active and inactive recombinant α-hemolysin for functional studies.     

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g^–1 dry wt.     

Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

Native to propionibacteria, the Wood–Werkman cycle enables propionate production via succinate decarboxylation. Current limitations in engineering propionibacteria strains have redirected attention toward the heterologous production in model organisms. Here, we report the functional expression of the Wood–Werkman cycle in Escherichia coli to enable propionate and 1-propanol production. The initial proof-of-concept attempt showed that the cycle can be used for production. However, production levels were low (0.17 mM). In silico optimization of the expression system by operon rearrangement and ribosomal-binding site tuning improved performance by fivefold. Adaptive laboratory evolution further improved performance redirecting almost 30% of total carbon through the Wood–Werkman cycle, achieving propionate and propanol titers of 9 and 5 mM, respectively. Rational engineering to reduce the generation of byproducts showed that lactate (∆ldhA) and formate (∆pflB) knockout strains exhibit an improved propionate and 1-propanol production, while the ethanol (∆adhE) knockout strain only showed improved propionate production.     

Metabolic engineering of Escherichia coli for biosynthesis of β-nicotinamide mononucleotide from nicotinamide

The β-nicotinamide mononucleotide (NMN) is a key intermediate of an essential coenzyme for cellular redox reactions, NAD. Administration of NMN is reported to improve various symptoms, such as diabetes and age-related physiological decline. Thus, NMN is attracting much attention as a promising nutraceutical. Here, we engineered an Escherichia coli strain to produce NMN from cheap substrate nicotinamide (NAM) and glucose. The supply of in vivo precursor phosphoribosyl pyrophosphate (PRPP) and ATP was enhanced by strengthening the metabolic flux from glucose. A nicotinamide phosphoribosyltransferase with high activity was newly screened, which is the key enzyme for converting NAM to NMN with PRPP as cofactor. Notably, the E. coli endogenous protein YgcS, which function is primarily in the uptake of sugars, was firstly proven to be beneficial for NMN production in this study. Fine-tuning regulation of ygcS gene expression in the engineered E. coli strain increased NMN production. Combined with process optimization of whole-cell biocatalysts reaction, a final NMN titre of 496.2 mg l^-1 was obtained.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

Metabolic engineering of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement

Fucosyllactoses, including 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2′-FL and 3-FL in engineered Escherichia coli. To boost the production of 2′-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous α1,2-fucosyltransferase and α1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2′-FL and 3-FL. Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2′-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2′-FL and 3-FL biosynthesizing strains. The final concentrations of 2′-FL and 3-FL were 9.12 and 12.43 g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.     

In vitro metabolic engineering for the production of α-ketoglutarate

α-Ketoglutarate (aKG) represents a central intermediate of cell metabolism. It is used for medical treatments and as a chemical building block. Enzymatic cascade reactions have the potential to sustainably synthesize this natural product. Here we report a systems biocatalysis approach for an in vitro reaction set-up to produce aKG from glucuronate using the oxidative pathway of uronic acids. Because of two dehydrations, a decarboxylation, and reaction conditions favoring oxidation, the pathway is driven thermodynamically towards complete product formation. The five enzymes (including one for cofactor recycling) were first investigated individually to define optimal reaction conditions for the cascade reaction. Then, the kinetic parameters were determined under these conditions and the inhibitory effects of substrate, intermediates, and product were evaluated. As cofactor supply is critical for the cascade reaction, various set-ups were tested: increasing concentrations of the recycling enzyme, different initial NAD⁺ concentrations, as well as the use of a bubble reactor for faster oxygen diffusion. Finally, we were able to convert 10 g L⁻¹ glucuronate with 92% yield of aKG within 5 h. The maximum productivity of 2.8 g L⁻¹ h⁻¹ is the second highest reported in the biotechnological synthesis of aKG.     

Overproduction and secretion of α-ketoglutaric acid by microorganisms

This mini-review presents a summary of research results of biotechnological production of alpha-ketoglutaric acid (KGA) by bacteria and yeasts. KGA is of particular industrial interest due to its broad application scope, e.g., as building block chemical for the chemical synthesis of heterocycles, dietary supplement, component of infusion solutions and wound healing compounds, or as main component of new elastomers with a wide range of interesting mechanical and chemical properties. Currently KGA is produced via different chemical pathways, which have a lot of disadvantages. As an alternative several bacteria and yeasts have already been studied for their ability to produce KGA as well as for conditions of overproduction and secretion of this intermediate of the tricarboxylic acid cycle. The aim of this mini-review was to summarize the known data and to discuss the potentials of biotechnological processes of KGA production.     

Engineering central metabolic modules of Escherichia coli for improving β-carotene production

ATP and NADPH are two important cofactors for production of terpenoids compounds. Here we have constructed and optimized β-carotene synthetic pathway in Escherichia coli, followed by engineering central metabolic modules to increase ATP and NADPH supplies for improving β-carotene production. The whole β-carotene synthetic pathway was divided into five modules. Engineering MEP module resulted in 3.5-fold increase of β-carotene yield, while engineering β-carotene synthesis module resulted in another 3.4-fold increase. The best β-carotene yield increased 21%, 17% and 39% after modulating single gene of ATP synthesis, pentose phosphate and TCA modules, respectively. Combined engineering of TCA and PPP modules had a synergistic effect on improving β-carotene yield, leading to 64% increase of β-carotene yield over a high producing parental strain. Fed-batch fermentation of the best strain CAR005 was performed, which produced 2.1 g/L β-carotene with a yield of 60 mg/g DCW.     

Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization

Enhancing the production of α-cyclodextrin glycosyltransferase (α-CGTase) is a key aim in α-CGTase industries. Here, the mature α-cgt gene from Paenibacillus macerans JFB05-01 was redesigned with systematic codon optimization to preferentially match codon frequencies of Escherichia coli without altering the amino acid sequence. Following synthesis, codon-optimized α-cgt (coα-cgt) and wild-type α-cgt (wtα-cgt) genes were cloned into pET-20b(+) and expressed in E.?coli BL21(DE3). The total protein yield of the synthetic gene was greater than wtα-cgt expression (1,710?mg?L^?1) by 2,520?mg?L^?1, with the extracellular enzyme activity being improved to 55.3?U?mL^?1 in flask fermentation. ΔG values at -3 to +50 of the pelB site of both genes were ?19.10?kcal?mol^?1. Functionally, coα-CGTase was equally as effective as wtα-CGTase in forming α-cyclodextrin (α-CD). These findings suggest that preferred codon usage is advantageous for translational efficiency to increase protein expression. Finally, batch fermentation was applied, and the extracellular coα-CGTase enzyme activity was 326?% that of wtα-CGTase. The results suggest that codon optimization is a reasonable strategy to improve the yield of α-CGTase for industrial application.     

Genetic and metabolic engineering for microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural biopolymer of glutamic acid. The repeating units of γ-PGA may be derived exclusively from d-glutamic acid, or l-glutamic acid, or both. The monomer units are linked by amide bonds between the α-amino group and the γ-carboxylic acid group. γ-PGA is biodegradable, edible and water-soluble. It has numerous existing and emerging applications in processing of foods, medicines and cosmetics. This review focuses on microbial production of γ-PGA via genetically and metabolically engineered recombinant bacteria. Strategies for improving production of γ-PGA include modification of its biosynthesis pathway, enhancing the production of its precursor (glutamic acid), and preventing loss of the precursor to competing byproducts. These and other strategies are discussed. Heterologous synthesis of γ-PGA in industrial bacterial hosts that do not naturally produce γ-PGA is discussed. Emerging trends and the challenges affecting the production of γ-PGA are reviewed.     

High-density Escherichia coli cultures for continuous l(−)-carnitine production

The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l⁻¹ h⁻¹). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used. Received: 10 December 1998 / Received revision: 19 February 1999 / Accepted: 20 February 1999