Molar Conversion Ratio Research Articles

Engineering of 4-hydroxyphenylacetate-3-hydroxylase from Escherichia coli for efficient biosynthesis of piceatannol

Piceatannol, a catechol compound, exhibits various health benefits, with great application value in food, medicine, and cosmetics. However, the low natural abundance of piceatannol limits its cost-effective isolation, and chemical synthesis is difficult. Here, we report an efficient biocatalytic system for the production of piceatannol from resveratrol. Structure-based semi-rational engineering was used to generate HpaBCG209F, a novel HpaBC mutant of 4-hydroxyphenylacetate-3-hydroxylase from Escherichia coli. Biocatalytic activity assays revealed that HpaBCG209F exhibits 3.12 times higher activity toward resveratrol than wild-type HpaBC. We also introduced formic acid dehydrogenase (FDH) as an NADH-regeneration system for HpaBCG209F to support piceatannol production. Co-expression of HpaBCG209F and FDH in E. coli BL21(DE3) enabled the generation of 8.23 mM piceatannol from 10.2 mM resveratrol within 9.5 h under optimized conditions, representing an 80.6% molar conversion ratio. This study therefore successfully developed a highly active HpaBC variant for hydroxylating resveratrol to piceatannol, as well as demonstrated a promising biopathway for piceatannol production.

Metabolic Engineering of Escherichia coli for Hyperoside Biosynthesis.

Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L−1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L−1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L−1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L−1·h−1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.

Metabolic Engineering of Escherichia coli for Efficient Biosynthesis of Lacto-N-tetraose Using a Novel β-1,3-Galactosyltransferase from Pseudogulbenkiania ferrooxidans.

Human milk oligosaccharides (HMOs) attract considerable interest in recent years because of their particular role in infant health. Lacto-N-tetraose (LNT), one of the most abundant HMOs, has been commercially added in the infant formula as a functional fortifier. In this study, a novel LNT-producing β-1,3-galactosyltransferase (β-1,3-GalT) from Pseudogulbenkiania ferrooxidans was screened from 14 putative candidates, and a highly LNT-producing metabolically engineered Escherichia coli strain was constructed based on a previously constructed lacto-N-triose II (LNT II)-producing strain, by strengthening UDP-galactose synthesis and introduction of P. ferrooxidans β-1,3-GalT. The engineered strain produced 3.11 and 25.49 g/L LNT in shake-flask and fed-batch cultivation, with the molar conversion ratio of LNT II to LNT of 88.15 and 85.09%, respectively. The productivity and specific yield of LNT in fed-batch cultivation were measured to be 0.61 g/L·h and 0.76 g/g dry cell weight, respectively. To the best of our knowledge, it is the highest LNT yield ever reported.

Enhancing production of 9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD) from phytosterols by metabolic pathway engineering of mycobacteria

9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD), as an important precursor for a large number of steroidal drugs, can be produced by the mycobacterial degradation of phytosterols’ feedstock. Phytosterols are wastes separated from the production of vegetable oil. Three catabolic pathways were involved in the microbial conversion of phytosterols to form 9-OHAD in Mycobacterium neoaurum MS136, namely sterol uptake, sterol side-chain degradation, and steroid nucleus cleavage and degradation. To enhance the production of 9-OHAD, we systematically studied the expression pattern of the genes during the conversion of phytosterols to 9-OHAD. The genes with significant up-regulation (e.g., hsd, cyp125, fadD19, hsd4A and ksh) were overexpressed to examine their effects on the 9-OHAD production. Thus, the recombinant Mycobacterium neoaurum MS136-F was constructed with the overexpression of these four key genes (e.g., hsd, hsd4A, kshA1 and kshB) and the knockout of the gene kstd that led to the degradation of 9-OHAD. Upon adding 13.0 g/L of commercial phytosterols feedstock (including 6.2 g/L β-sitosterol, 3.0 g/L campesterol, 3.3 g/L stigmasterol and 0.5 g/L others), 6.8 g/L 9-OHAD was obtained by this recombinant strain MS136-F after 108 h’ fermentation, which was 1.45-fold of that of the parental M. neoaurum MS136 (4.7 g/L, the molar conversion rate is 77.2%). In the phytosterols feedstock, β-sitosterol and campesterol were converted to 9-OHAD with a much high molar conversion ratio of 98.5%. However, stigmasterol (3.3 g/L) was hardly used by mycobacteria, only 0.3 g/L of which was converted 9-OHAD. This study has a direct implication for the industrial production of 9-OHAD.

Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138

ABSTRACT As a prebiotics, lactosucrose plays an important role in maintaining human gastrointestinal homeostasis. In this study, a thermostable enzyme from Arthrobacter sp. 10138 was screened from six β-fructofuranosidase-producing strains for the lactosucrose production and the coding gene was heterologously expressed in Escherichia coli for efficient expression. Recombinant β-fructofuranosidase was purified and biochemically characterized by MALDI-TOFMS spectrometry. The transfructosylation product by this recombinant enzyme was determined to be lactosucrose rather than other oligosaccharides or polysaccharides by HPLC and LC-MS. Efficient extracellular secretion of β-fructofuranosidase was achieved by the optimization of signal peptide and induction conditions. It was found that with the signal peptide torT, the highest extracellular activity reached 111.01 U/mL, which was 38.4-fold higher than that with the OmpA signal peptide. Under the optimal conditions (pH 6.0, temperature 50°C, enzyme amount 40 μg/ml, sucrose 150 g/L and lactose 150 g/L), 109 g/L lactosucrose was produced with a molar conversion ratio of 49.3%. Here the thermostable β-fructofuranosidase from Arthrobacter sp. 10138 can be used for efficient synthesis of lactosucrose, and this provides a good startpoint for the industrial production of lactosucrose in the future.

Experimental and numerical study of biogas, methane and carbon dioxide produced by pre-treated wheat straw and pre-digested cow dung

ABSTRACTThe biogas constituting majorly CH4 and CO2 has been produced by Ca(OH)2 pre-treated wheat straw with pre-digested cow dung. Some of the key thermodynamic parameters like specific heat capacity, density and heating capacity of the biogas produced have also been calculated per day as well as throughout the hydraulic retention time. The governing equations of biogas with appropriate phase and interfacial conditions describing the physics of the biogas have been derived. The control volume approach has been used to predict the total volume (ml) of biogas, CH4 and CO2 throughout the experiment and on the daily basis. The effects of feedstock, temperature and pressure on the production of biogas, CH4 and CO2 in anaerobic digestion have also been studied. The average number of molar fraction and conversion ratio of CH4 and CO2 are correlated with number of carbon atoms available in feedstock. Numerical calculations by using developed model and Modified Gompertz model have shown proficient agreement with the experimental observations.

Biocatalytic Production of Glucosamine from N-Acetylglucosamine by Diacetylchitobiose Deacetylase.

Glucosamine (GlcN) is widely used in the nutraceutical and pharmaceutical industries. Currently, GlcN is mainly produced by traditional multistep chemical synthesis and acid hydrolysis, which can cause severe environmental pollution, require a long prodution period but a lower yield. The aim of this work was to develop a whole-cell biocatalytic process for the environment-friendly synthesis of glucosamine (GlcN) from N-acetylglucosamine (GlcNAc). We constructed a recombinant Escherichia coli and Bacillus subtilis strains as efficient whole-cell biocatalysts via expression of diacetylchitobiose deacetylase (Dacph) from Pyrococcus furiosus. Although both strains were biocatalytically active, the performance of B. subtilis was better. To enhance GlcN production, optimal reaction conditions were found: B. subtilis whole-cell biocatalyst 18.6 g/l, temperature 40°C, pH 7.5, GlcNAc concentration 50 g/l and reaction time 3 h. Under the above conditions, the maximal titer of GlcN was 35.3 g/l, the molar conversion ratio was 86.8% in 3-L bioreactor. This paper shows an efficient biotransformation process for the biotechnological production of GlcN in B. subtilis that is more environmentally friendly than the traditional multistep chemical synthesis approach. The biocatalytic process described here has the advantage of less environmental pollution and thus has great potential for large-scale production of GlcN in an environment-friendly manner.

Numerical and experimental study of biogas, methane and carbon dioxide produced by pre-treated slurry

ABSTRACTThe biogas constituting majorly CH4 and CO2 has been produced by pre-treated wheat straw with cow dung. Some of the key thermodynamic parameters like specific heat capacity, density and heating capacity of the biogas produced have also been calculated per day as well as throughout the hydraulic retention time. The governing equations of biogas with appropriate phase and interfacial conditions describing the physics of the biogas have been derived. The control volume approach has been used to predict the total volume (ml) of biogas, CH4 and CO2 throughout the experiment and on the daily basis. The effects of feedstock, temperature and pressure on the production of biogas, CH4 and CO2 in anaerobic digestion have also been studied. The average number of molar fraction and conversion ratio of CH4 and CO2 are correlated with number of carbon atoms available in feedstock. Numerical calculations have shown proficient agreement with the experimental observations.HighlightsNumerical and experimental study of biogas, methane and carbon dioxide produced by pre-treated slurry.Thermodynamic parameters like specific heat capacity, density and heating capacity of the biogas have been calculated.The effects of feedstock, temperature and pressure on the production of biogas, methane and carbon dioxide in anaerobic digestion (AD) have been studied.Numerical calculations have shown proficient agreement with the experimental observations of pre-treated wheat straw with fresh cow dung.

Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes—one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)—were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%.

Rational engineering of p-hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway.

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc.

A novel systems dynamics model for simulation of yeast batch, fed-batch and continuous cultures

α-Arbutin is a glycosylated hydroquinone that has an inhibitory function against tyrosinase. The aim of the present study is to develop an efficient and inexpensive method for large-scale production of α-arbutin by using Xanthomonas BT-112 as biocatalyst. To accomplish this goal, various surfactants were tested to enhance the α-arbutin production, and the optimal operational conditions for 30 L jar fermenter were scaled up for a production level of 3000 L with using a constant volumetric oxygen transfer coefficient (KLa) and the volumetric aeration rate per volume unit (Q/V) as scale-up criteria. Under the optimized conditions, the α-arbutin produced in the presence of 0.4% (w/v) Tween-80 was 124.8% higher than that of the control, and the yield of α-arbutin in 3000 L fermenter was 38.2 g/L with a molar conversion ratio of 93.7% based on the amount of hydroquinone supplied. This result is comparable to the results from laboratory-scale fermenter. Hence, 100-fold scale-up was successfully achieved.

Fermentation scale up for α-arbutin production by Xanthomonas BT-112

α-Arbutin is a glycosylated hydroquinone that has an inhibitory function against tyrosinase. The aim of the present study is to develop an efficient and inexpensive method for large-scale production of α-arbutin by using Xanthomonas BT-112 as biocatalyst. To accomplish this goal, various surfactants were tested to enhance the α-arbutin production, and the optimal operational conditions for 30L jar fermenter were scaled up for a production level of 3000L with using a constant volumetric oxygen transfer coefficient (KLa) and the volumetric aeration rate per volume unit (Q/V) as scale-up criteria. Under the optimized conditions, the α-arbutin produced in the presence of 0.4% (w/v) Tween-80 was 124.8% higher than that of the control, and the yield of α-arbutin in 3000L fermenter was 38.2g/L with a molar conversion ratio of 93.7% based on the amount of hydroquinone supplied. This result is comparable to the results from laboratory-scale fermenter. Hence, 100-fold scale-up was successfully achieved.

One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

English

The biodegradation of morpholine has attracted much interest because morpholine causes environmental pollution. Ten species belonging to nine genera were tested for their abilities to degrade morpholine in mineral salts medium containing morpholine (1 g/l). Mycobacterium sp. isolated from polluted water sample collected from Abu Za”baal lakes, effectively utilized morpholine as carbon, nitrogen and energy source. The tested Mycobacterium was able to grow in high concentrations of morpholine but the rapidly increase in pH of the growth medium and accumulation of ammonia inhibited bacterial growth and complete mineralization of morpholine. The molar conversion ratio of morpholine to ammonia was 1:0.89. Growing of the selected bacterium in liquid medium with 1 g/l morpholine at 37°C and pH 6.5, enhanced morpholine degradation. Addition of metyrapone to the growth medium inhibited morpholine degradation. Immobilization of Mycobacterium cells in sodium alginate increased morpholine degradation compared with free cells. At high concentrations of morpholine (4 to 6 g/l), there was a decrease in both cell viability and respiration of Mycobacterium but no genotoxicity was found.   Key words: Morpholine, Mycobacterium, biodegradation, pollution, ammonia, cytochrome P450, metyrapone, immobilization.

Expression of the guanosine-inosine kinase gene from Exiguobacterium acetylicum in Corynebacterium ammoniagenes and phosphorylation of inosine to produce inosine 5′-monophosphate

The guanosine-inosine kinase gene (gsk) isolated from Exiguobacterium acetylicum was expressed in an ATP-regenerating strain, Corynebacterium ammoniagenes. In order to induce its expression, three promoters (those for the Escherichia coli tac, Escherichia coli trp, and Corynebacterium glutamicum odhA gene) with the corresponding ribosome-binding sequences were examined. The E. coli trp promoter was most efficient with regard to inducing the expression of gsk in C. ammoniagenes. Further, the resulting strain, which has both inosine kinase activity and ATP-regenerating activity, was used to induce the phosphorylation of inosine to produce inosine 5′-monophosphate (5′-IMP), which is widely used as a flavor enhancer; approximately 80 g of 5′-IMP/l was produced with a molar conversion ratio of 80%.

Regioselective carboxylation of catechol by 3,4-dihydroxybenzoate decarboxylase of Enterobacter cloacae P

3,4-Dihydroxybenzoate decarboxylase in Enterobacter cloacae P241 was induced by adding 3,4-dihydroxybenzoic acid, 3-hydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or 4-acetamidobenzoic acid to the culture medium. After stabilizing the enzyme activity by adding 5 mM dithiothreitol and 20 mM Na(2)S(2)O(3) to a cell-free extract, catechol at 50 mM was carboxylated in the presence of 3 M KHCO(3) to 3,4-dihydroxybenzoic acid with a molar conversion ratio of 28% after 14 h at 30 degrees C.

Alanine production in an H+-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H(+)-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164-2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H(+)-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H(+)-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.

Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2

We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO(3) in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO(3), respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.

Purification and characterization of 2,6-dihydroxybenzoate decarboxylase reversibly catalyzing nonoxidative decarboxylation

A nonoxidative decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM12048. The enzyme activity was induced specifically by 2,6-dihydroxybenzoate. The purified enzyme was a homotetramer of identical 38 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate without requiring any cofactors. In the presence of KHCO(3), the enzyme also catalyzed the regioselective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate at a molar conversion ratio of 30%.

Isolation of Dimethyl Sulfone-Degrading Microorganisms and Application to Odorless Degradation of Dimethyl Sulfoxide

With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO 2 ) as the sole carbon source, DMSO 2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO 2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/ l ) DMSO was degraded to sulfate ion with 80% molar conversion ratio.