Metabolic Engineering of Escherichia coli for Production of 2-phenylethanol from Renewable Glucose

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like odor and has wide applications. The present work aims to construct a synthetic pathway for 2-PE synthesis from glucose in Escherichia coli. First, the genes adh1 (encoding alcohol dehydrogenase) and kdc (encoding phenylpyruvate decarboxylase) from Saccharomyces cerevisiae S288c and Pichia pastoris GS115 were investigated in E. coli, respectively, and single overexpression of adh1 or kdc significantly increased 2-PE accumulation. When co-overexpressing adh1 and kdc, 2-PE was increased up to 130 from 57 mg/L. Furthermore, by optimizing coordinated expression of the four committed genes aroF, pheA, adh1 and kdc, 2-PE was improved to 285 mg/L which was the highest production of 2-PE by the recombinant E. coli system. In addition, our results also demonstrated that the tyrB gene, which encodes aromatic-amino-acid transaminase, plays an important role on 2-PE synthesis.     

Metabolic Engineering of Escherichia coli Cells for Ethanol Production under Aerobic Conditions

The ethanol production by recombinant Escherichia coli introducing of pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) from Zymomonas mobilis was investigated under aerobic conditions. In aerobic culture (K_La = 1.5 min^-1), the cells expressing pdc and adhB produced 0.4 g l^-1 ethanol when cultured for 18 h. This value was improved in BW25113Δpta/pHfdh/pTadhB-pdc following 4 g l^-1 formate feeding at 0.8 g l^-1 ethanol. In higher oxygenation level (K_La = 6.1 min^-1), the production of ethanol was further enhanced at 1.79 g l^-1 ± 0.37 g l^-1 after 24 h cultivation. Formate was found not detectable at the end of culture, indicating complete degradation this organic acid to regenerate NADH from NAD⁺. The culture strategy was effective to inactivate lactate dehydrogenase, which is major competitor for ethanol production in utilizing NADH.     

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.     

l-Lactate Production from Seaweed Hydrolysate of Laminaria japonica Using Metabolically Engineered Escherichia coli

Renewable and carbon neutral, marine algal biomass could be an attractive alternative substrate for the production of biofuel and various biorefinery products. Thus, the feasibility of brown seaweed (Laminaria japonica) hydrolysate as a carbon source was investigated here for l-lactate production. This work reports the homofermentative route for l-lactate production by introducing Streptococcus bovis/equinus l-lactate dehydrogenase in an engineered Escherichia coli strain where synthesis of the competing by-product was blocked. The engineered strain utilized both glucose and mannitol present in the hydrolysate under microaerobic condition and produced 37.7 g/L of high optical purity l-lactate at 80 % of the maximum theoretical value. The result shown in this study implies that algal biomass would be as competitive with lignocellulosic biomass in terms of lactic acid production and that brown seaweed can be used as a feedstock for the industrial production of other chemicals.     

Machine Learning Analysis of Essential Oils from Cuban Plants: Potential Activity against Protozoa Parasites

Essential oils (EOs) are a mixture of chemical compounds with a long history of use in food, cosmetics, perfumes, agricultural and pharmaceuticals industries. The main object of this study was to find chemical patterns between 45 EOs and antiprotozoal activity (antiplasmodial, antileishmanial and antitrypanosomal), using different machine learning algorithms. In the analyses, 45 samples of EOs were included, using unsupervised Self-Organizing Maps (SOM) and supervised Random Forest (RF) methodologies. In the generated map, the hit rate was higher than 70% and the results demonstrate that it is possible find chemical patterns using a supervised and unsupervised machine learning approach. A total of 20 compounds were identified (19 are terpenes and one sulfur-containing compound), which was compared with literature reports. These models can be used to investigate and screen for bioactivity of EOs that have antiprotozoal activity more effectively and with less time and financial cost.     

Optimization of Culture Conditions for Enhanced Lysine Production Using Engineered Escherichia coli

In this study, culture conditions, including dissolved oxygen (DO) content, presence of osmoprotectants, residual glucose concentration, and ammonium sulfate-feeding strategies, were investigated for decreasing the inhibition effects of acetic acid, ammonium, and osmotic stress on l-lysine fermentation by Escherichia coli. The results revealed that higher DO content and lower residual glucose concentration could decrease acetic acid accumulation, betaine supplementation could enhance osmotic stress tolerance, and variable speed ammonium sulfate-feeding strategy could decrease ammonium inhibition. Thus, with 25 % DO content, 0–5.0 g/L of residual glucose concentration, and 1.5 g/L of betaine supplementation, 134.9 g/L of l-lysine was obtained after 72 h of culture, with l-lysine yield and productivity of 45.4 % and 1.9 g/(L?·?h), respectively.     

Production of Indirubin from Tryptophan by Recombinant Escherichia coli Containing Naphthalene Dioxygenase Genes from Comamonas sp. MQ

Indirubin, a red isomer of indigo, can be used for the treatment of various chronic diseases. However, the microbial production of indirubin did not receive much attention probably due to its low yield compared with indigo. In this study, the recombinant Escherichia coli containing the naphthalene dioxygenase (NDO) genes from Comamonas sp. MQ was used to produce indirubin from tryptophan. To enhance the production of indirubin, the induction conditions for NDO expression were optimized. The optimal induction conditions were carried out with 0.5 mM isopropyl-β-d-thiogalactopyranoside at 30 °C when cells were grown to OD₆₀₀?≈?1.20. Subsequently, the effects of medium composition on indirubin production were investigated by response surface methodology, and 9.37?±?1.01 mg/l indirubin was produced from 3.28 g/l tryptophan. Meanwhile, the indirubin production was further improved by adding 2-oxindole and isatin to the tryptophan medium after induction. About 57.98?±?2.62 mg/l indirubin was obtained by the addition of 500 mg/l 2-oxindole after 1-h induction, which was approximately 6.2-fold to that without additional 2-oxindole. The present study provided a possible way to improve the production of indirubin and should lay the foundation for the application of microbial indirubin production.     

大肠杆菌异源表达pMMO活性中心pmoB及生物催化研究

颗粒甲烷单加氧酶（pMMO）是甲烷氧化菌中催化甲烷氧化生成甲醇的一种酶．Methylococcus capsulatus IMV 3021的pMMO活性位点是pmoB亚基,该亚基是一种可溶性蛋白．我们研究将pmoB亚基进行异源表达及生物催化活性的验证．当培养基中烟酰胺腺嘌呤二核苷酸（NADH）浓度为5 mmol／L时,可以观察到异源表达pmoB亚基具有催化甲烷氧化成甲醇活性,生成甲醇浓度为1．04 mmol／L．研究pMMO活性对于开发能直接将甲烷转化成甲醇的新型、环保催化剂有非常重要意义．     

An Improved Method for the Conjugation of FAB' to B-D-Galactosidase from Escherichia coli

Abstract

An improved method for the conjugation of Fab' to B-D-galactosidase from Escherichia coli was developed. The enzyme with thiol groups was treated with excess of N, N'-o-phenylene-dimaleimide to introduce maleimide groups, and the maleimide-enzyme was reacted with thiol groups in the hinge of Fab' to form Fab'-B-D-galactosidase conjugate. Gel filtration of Fab' was required only once, and the recovery of Fab' in the conjugate was 71–81%, while in the previous method, gel filtration was required twice and the recovery was 31–38 %. Although the enzyme activity was decreased slightly (8–12%) by the dimaleimide treatment, the conjugate was as useful for sandwich enzyme immunoassay as that obtained by the previous method. 1gG also could be conjugated to B-D-galactosidase more efficiently in the present method than in the previous one.     

Fermentation of Biologically Pretreated Wheat Straw for Ethanol Production: Comparison of Fermentative Microorganisms and Process Configurations

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.     

Escherichia coli glucuronylsynthase: an engineered enzyme for the synthesis of beta-glucuronides

The glycosynthase derived from E. coli beta-glucuronidase catalyzes the glucuronylation of a range of primary, secondary, and aryl alcohols with moderate to excellent yields. The procedure provides an efficient, stereoselective, and scalable single-step synthesis of beta-glucuronides under mild conditions.     

Screen-printed electrochemical hybridization biosensor for the detection of DNA sequences from the Escherichia coli pathogen

An electrochemical biosensor for the specific detection of short DNA sequences from the E. coli pathogen is described. This hybridization device relies on the immobilization of a 25-mer oligonucleotide probe, from the E. coli lacZ gene, onto a screen-printed carbon electrode. Chronopotentiometric detection of the Co(bpy)₃⁺³ indicator is used for monitoring the hybridization event. Numerous variables of the assay protocol, including those of the probe immobilization step, the hybridization event, and the indicator association/detection, are characterized and optimized. Hybridization times of 2- and 30-min are sufficient for detecting 300- and 50 ng/mL, respectively, of the E. coli DNA target. Applicability to analysis of untreated environmental water samples is illustrated. Such single-use electrochemical sensors hold great promise for decentralized environmental and food testing for the E. coli pathogen.     

l-Ribose Production from l-Arabinose by Immobilized Recombinant Escherichia coli Co-expressing the l-Arabinose Isomerase and Mannose-6-Phosphate Isomerase Genes from Geobacillus thermodenitrificans

l-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the l-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l l-ribose from 300 g/l l-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co²⁺, with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h^?1 produced an average of 100 g/l l-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for l-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from l-arabinose as the substrate.     

Process Engineering for Production of Chiral Hydroxycarboxylic Acids from Bacterial Polyhydroxyalkanoates

An efficient process for the production of (R)‐hydroxycarboxylic acids (RHAs) from polyhydroxyalkanoates (PHAs) was developed. It involved the synthesis of PHA in bacteria, followed by bringing the culture broth directly to a pH optimal for in vivo PHA degradation, thus avoiding cell collection by centrifugation and pellet resuspension. The optimal pH was maintained to allow maximal release of RHAs. Using this process, cells having a dry weight (w) of 1.8 g · L⁻¹ and 45% (w/w) PHA exhibited a linear PHA degradation rate of about 0.059 g · L⁻¹ · h⁻¹ in the first 9 h. Concomitantly, RHAs were released with a rate of 0.057 g · L⁻¹ · h⁻¹. Further incubation of up to 15 h resulted in almost 90% (w/w) degradation of PHA. Based on this approach in combination with chemostat and a plug flow reactor a continuous process for the production of RHAs could be achieved.

     

GSH Protects the Escherichia coli Cells from High Concentrations of Thymoquinone

The aim of the present study was to evaluate the potential protective effect of glutathione (GSH) on Escherichia coli cells grown in a high concentration of thymoquinone (TQ). This quinone, as the main active compound of Nigella sativa seed oil, exhibits a wide range of biological activities. At low concentrations, it acts as an antioxidant, and at high concentrations, an antimicrobial agent. Therefore, any interactions between thymoquinone and glutathione are crucial for cellular defense against oxidative stress. In this study, we found that GSH can conjugate with thymoquinone and its derivatives in vitro, and only fivefold excess of GSH was sufficient to completely deplete TQ and its derivatives. We also carried out studies on cultures of GSH-deficient Escherichia coli strains grown on a minimal medium in the presence of different concentrations of TQ. The strains harboring mutations in gene ΔgshA and ΔgshB were about two- and fourfold more sensitive (256 and 128 µg/mL, respectively) than the wild type. It was also revealed that TQ concentration has an influence on reactive oxygen species (ROS) production in E. coli strains—at the same thymoquinone concentration, the level of ROS was higher in GSH-deficient E. coli strains than in wild type.     

Potential of Biocellulose Nanofibers Production from Agricultural Renewable Resources: Preliminary Study

In the present preliminary study, we report results for the biocellulose nanofibres production by Gluconacetobacter xylinus. Production was examined by utilizing different feedstocks of single sugars and sugar mixtures with compositions similar to the acid hydrolyzates of different agriculture residues. Profiles for cell proliferation, sugar consumption, and the subsequent pH changes were thoroughly analyzed. Highest biocellulose production of 5.65 g/L was achieved in fructose medium with total sugar consumption of 95.57%. Moreover, the highest production using sugar mixtures was 5.2 g/L, which was achieved in feedstock with composition identical to the acid hydrolyzate of wheat straws. This represented the highest biocellulose yield of 17.72 g/g sugars compared with 14.77 g/g fructose. The lowest production of 1.1 and 1.75 g/L were obtained in xylose and glucose media, respectively, while sucrose and arabinose media achieved relatively higher production of 4.7 and 4.1 g/L, respectively. Deviation in pH of the fermentation broths from the optimum value of 4–5 generally had marked effect on biocellulose production with single sugars in feedstock. However, the final pH values recorded in the different sugar mixtures were ～3.3–3.4, which had lower effect on production hindrance. Analyzing profiles for sugars' concentrations and cell growth showed that large amount of the metabolized sugars were mainly utilized for bacterial cell growth and maintenance, rather than biocellulose production. This was clearly observed with single sugars of low production, while sugar consumption was rather utilized for biocellulose production with sugar mixtures. Results reported in this study demonstrate that agriculture residues might be used as potential feedstocks for the biocellulose nanofibres production. Not only this represents a renewable source of feedstock, but also might lead to major improvements in production if proper supplements and control were utilized in the fermentation process.     

Structural Elucidation of the O-Antigen Polysaccharide from Escherichia coli O181

Shiga-toxin-producing Escherichia coli (STEC) is an important pathogen associated to food-borne infection in humans; strains of E. coli O181, isolated from human cases of diarrhea, have been classified as belonging to this pathotype. Herein, the structure of the O-antigen polysaccharide (PS) from E. coli O181 has been investigated. The sugar analysis showed quinovosamine (QuiN), glucosamine (GlcN), galactosamine (GalN), and glucose (Glc) as major components. Analysis of the high-resolution mass spectrum of the oligosaccharide (OS), obtained by dephosphorylation of the O-deacetylated PS with aqueous 48 % hydrofluoric acid, revealed a pentasaccharide composed of two QuiNAc, one GlcNAc, one GalNAc, and one Glc residue. The ¹H and ¹³C NMR chemical shift assignments of the OS were carried out using 1 D and 2 D NMR experiments, and the OS was sequenced using a combination of tandem mass spectrometry (MS/MS) data and NMR ¹³C NMR glycosylation shifts. The structure of the native PS was determined using NMR spectroscopy, and it consists of branched pentasaccharide repeating units joined by phosphodiester linkages: →4)[α-l-QuipNAc-(1→3)]-α-d-GalpNAc6Ac-(1→6)-α-d-Glcp-(1→P-4)-α-l-QuipNAc-(1→3)-β-d-GlcpNAc-(1→; the O-acetyl groups represent 0.4 equivalents per repeating unit. Both the OS and PSs exhibit rare conformational behavior since two of the five anomeric proton resonances could only be observed at an elevated temperature.