Effective asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate by recombinant E. coli CCZU-A13 in [Bmim]PF6-hydrolyzate media.

It was the first report that the concentrated hydrolyzates from the enzymatic hydrolysis of dilute NaOH (3wt%)-soaking rice straw at 30°C was used to form [Bmim]PF6-hydrolyzate (50:50, v/v) media for bioconverting ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] (>99% e.e.) with recombinant E. coli CCZU-A13. Compared with pure glucose, the hydrolyzates could promote both initial reaction rate and the intracellular NADH content. Furthermore, emulsifier OP-10 (20mM) was employed to improve the reductase activity. Moreover, Hp-ß-cyclodextrin (0.01mol Hp-ß-cyclodextrin/mol COBE) was also added into this bioreaction system for enhancing the biosynthesis of (R)-CHBE from COBE by E. coli CCZU-A13 whole-cells. The yield of (R)-CHBE (>99% e.e.) from 800mM COBE was obtained at 100% in the [Bmim]PF6-hydrolyzate (50:50, v/v) media by supplementation of OP-10 (20mM) and Hp-ß-CD (8mM). In conclusion, an effective strategy for the biosynthesis of (R)-CHBE was successfully demonstrated.

Effective pretreatment of sugarcane bagasse with combination pretreatment and its hydrolyzates as reaction media for the biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by whole cells of E. coli CCZU-K14.

In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Compared with glucose, arabinose and cellobiose couldn't promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of (S)-CHBE (∼500g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of ß-CD (0.4mol ß-CD/mol COBE), l-glutamine (200mM) and glycine (500mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate (S)-CHBE.

Effective pretreatment of dilute NaOH-soaked chestnut shell with glycerol-HClO4-water media: structural characterization, enzymatic saccharification, and ethanol fermentation.

In this study, an effective pretreatment of dilute NaOH-soaked chestnut shell (CNS) with glycerol-HClO4-water (88.8:1.2:10, w/w/w) media at 130 °C for 30 min was successfully demonstrated. Results revealed that the combination pretreatment removed 66.0 % of lignin and 73.7 % of hemicellulose in untreated CNS. The changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS were characterized with Fourier transform infrared spectroscopy, fluorescent microscope, scanning electron microscopy, and X-ray diffraction. Biotransformation of glycerol-HClO4-water pretreated-NaOH-soaked CNS (50 g/L) with a cocktail of enzymes for 72 h, the reducing sugars and glucose were 39.7 and 33.4 g/L, respectively. Moreover, the recovered hydrolyzates containing 20 g/L glucose had no inhibitory effects on the ethanol-fermenting microorganism, and the ethanol production was 0.45 g/g glucose within 48 h. In conclusion, this combination pretreatment shows promise as pretreatment solvent for wheat straw, although the in-depth exploration of this subject is needed.

Enzymatic in situ saccharification of chestnut shell with high ionic liquid-tolerant cellulases from Galactomyces sp. CCZU11-1 in a biocompatible ionic liquid-cellulase media.

In this study, it was the first time to report that the cellulases of Galactomyces sp. CCZU11-1 showed high activity and stability in the culture and reaction media containing IL [Mmim]DMP. Using untreated chestnut shell (CNS) as carbon source in the culture media containing IL [Mmim]DMP (5%, w/v), high activity of FPA (28.6U/mL), xylanase (186.2U/mL), and CMCase (107.3U/mL) were obtained, and 184.9mg/L of total protein was achieved. Furthermore, the changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS utilized with Galactomyces sp. CCZU11-1 were characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. After was enzymatically hydrolyzed with the prepared crude enzymes in IL diluted to 20% (w/v), a high yield of reducing sugars, 62.1%, was obtained. Significantly, Galactomyces sp. CCZU11-1 showed high potential for the efficient transformation of lignocellulosic materials to glucose in a single-step process.

Enhancement of enzymatic saccharification of corn stover with sequential Fenton pretreatment and dilute NaOH extraction.

In this study, an effective method by the sequential Fenton pretreatment and dilute NaOH extraction (FT-AE) was chosen for pretreating corn stover. Before dilute NaOH (0.75 wt%) extraction at 90 °C for 1h, Fenton reagent (0.95 g/L of FeSO4 and 29.8 g/L of H2O2) was employed to pretreat CS at a solid/liquid ratio of 1/20 (w/w) at 35 °C for 30 min. The changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated solid residue were correlated with the enhancement of enzymatic saccharification. After being enzymatically hydrolyzed for 72 h, the reducing sugars and glucose from the hydrolysis of 60 g/L FT-AE-CS pretreated could be obtained at 40.96 and 23.61 g/L, respectively. Finally, the recovered hydrolyzates containing glucose had no inhibitory effects on the ethanol fermenting microorganism. In conclusion, the sequential Fenton pretreatment and dilute NaOH extraction has high potential application in future.

Significantly improving enzymatic saccharification of high crystallinity index's corn stover by combining ionic liquid [Bmim]Cl-HCl-water media with dilute NaOH pretreatment.

In this study, a pretreatment by combining acidified aqueous ionic liquid 1-butyl-3-methylimidazolium chloride (IL [Bmim]Cl) solution with dilute NaOH extraction was employed to pretreat high crystallinity index (CrI) of corn stover before its enzymatic saccharification. After NaOH extraction, [Bmim]Cl-HCl-water (78.8:1.2:20, w/w/w) media was used for further pretreatment at 130 °C for 30 min. After being enzymatically hydrolyzed for 48 h, corn stover pretreated could be biotransformed into reducing sugars in the yield of 95.1%. Furthermore, SEM, XRD and FTIR analyses of untreated and pretreated corn stovers were examined. It was found that the intact structure was disrupted by combination pretreatment and resulted in a porous and amorphous regenerated cellulosic material that greatly improved enzymatic hydrolysis. Finally, the recovered hydrolyzates obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. In conclusion, the combination pretreatment shows high potential application in future.

Effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate by supplementation of l-glutamine, d-xylose and ß-cyclodextrin in n-butyl acetate-water media.

To avoid adding NAD(+) and effectively transform ethyl 4-chloro-3-oxobutanoate, the mixture of l-glutamine (200mM) and d-xylose (250mM) was added into in n-butyl acetate-water (10:90, v/v) biphasic system instead of NAD(+) for increasing the biocatalytic efficiency. To further improve the synthesis of optically pure ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee), ß-cyclodextrin was also added into this reaction media, and ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee) could be effectively synthesized from 800mM ethyl 4-chloro-3-oxobutanoate in the yield of 100% by whole-cells of recombinant E. coli CCZU-A13. Finally, the possible mechanism for improving the reductase activity by supplementation of l-glutamine, d-xylose and ß-CD was proposed. In conclusion, this strategy has high potential for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate (>99% ee).

Biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with an NADH-dependent reductase (ClCR) discovered by genome data mining using a modified colorimetric screening strategy.

An NADH-dependent reductase (ClCR) was discovered by genome data mining. After ClCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-T15 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was screened using a modified high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (S)-CHBE (>99% ee) with the resting cells of E. coli CCZU-T15 was successfully demonstrated in toluene-water (50:50, v/v) biphasic system. Biotransformation of 1000 mM COBE for 24 h in the biphasic system, (S)-CHBE (>99% ee) could be obtained in the high yield of 96.4%. Significantly, E. coli CCZU-T15 shows high potential in the industrial production of (S)-CHBE (>99% ee).

Improved biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by adding L-glutamine plus glycine instead of NAD+ in ß-cyclodextrin-water system.

To reduce dependence on the expensive cofactor and effectively biotransform ethyl 4-chloro-3-oxobutanoate, L-glutamine and glycine were found to enhance the content of intracellular NADH and the reductase activity. Adding the mixture of 200 mM of L-glutamine and 500 mM of glycine to the reaction media, a 1.67-fold of reductase activity was increased over the control without the addition of the two compounds. Moreover, ß-cyclodextrin (0.4 mol ß-cyclodextrin/mol ethyl 4-chloro-3-oxobutanoate) was also added into this reaction media, and the biocatalytic activity of the whole-cell biocatalyst of Escherichia coli CCZU-K14 was increased by 1.34-fold than that without ß-cyclodextrin. In this ß-cyclodextrin-water media containing L-glutamine (200 mM) plus glycine (500 mM), ethyl (S)-4-chloro-3-hydroxybutanoate (>99% ee) could be obtained from 3000 mM ethyl 4-chloro-3-oxobutanoate in the yield of 98.0% after 8h. All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of ethyl (S)-4-chloro-3-hydroxybutanoate.

Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by Rhodococcus sp.

Rhodococcus sp. CGMCC 4911 transformed 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives into corresponding diols. Ethylene sulfate, glycol sulfide, 1,3-PDS, and 1,2-propanediol cyclic sulfate were effectively hydrolyzed with growing cells. (R)-1,2-Propanediol (>99 % e.e.) was obtained at 44 % yield with growing cells. Glycol sulfide, ethylene sulfate, and 1,3-PDS were converted into the corresponding diols at 94.6, 96.3, and 98.3 %, respectively. Optimal reaction conditions with lyophilized resting cells were 30 °C, pH 7.5, and cell dosage 17.9 mg cell dry wt/ml. 1,3-Propanediol was obtained from 50 mM 1,3-PDS at 97.2 % yield by lyophilized cells after 16 h. Lyophilized cells were entrapped in calcium alginate with a half-life of 263 h at 30 °C, and the total operational time of the immobilized biocatalysts could reach over 192 h with a high conversion rate.

Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by toluene-permeabilized cells of Bacillus sp. CCZU11-1.

In this study, it was the first report that Bacillus sp. CCZU11-1 was used for the biotransformation of 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives. The catalytic performance of Bacillus sp. sulfatase in the biotransformation of 1,3-PDS was significantly improved by biocatalyst permeabilization and immobilization. Using cell permeabilization, the hydrolytic activity of the whole-cell biocatalyst was increased by 3.5-fold after 1.5 h of pretreatment with 10 % (v/v) toluene at 30 °C and pH 7.0. Biotransformation of 20 mM 1,3-PDS for 24 h, 1,3-propanediol (1,3-PD) could be obtained in the yield of 97.4 % under the optimized reaction condition. Additionally, the immobilized biocatalysts, permeabilized cells entrapped in calcium alginate, and cross-linked enzyme aggregates were further employed to biotansform 1,3-PDS. Moreover, the total operational time of the immobilized biocatalysts could reach above 240 h with high conversion rate (>90 %).

Discovery of a reductase-producing strain recombinant E. coli CCZU-A13 using colorimetric screening and its whole cell-catalyzed biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate.

An NADH-dependent reductase (SsCR) was discovered by genome data mining. After SsCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-A13 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) was screened using one high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (R)-CHBE (>99% ee) with the resting cells of E. coli CCZU-A13 was successfully demonstrated in n-butyl acetate-water (10:90, v/v) biphasic system. Biotransformation of 600mM COBE for 8h in the biphasic system, (R)-CHBE (>99% ee) could be obtained in the high yield of 100%. Moreover, the broad substrate specificity in the reduction of aliphatic and aromatic carbonyl compounds was also found. Significantly, E. coli CCZU-A13 shows high potential in the industrial production of (R)-CHBE (>99% ee) and its derivatives.

Biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate by NADH-dependent reductase from E. coli CCZU-Y10 discovered by genome data mining using mannitol as cosubstrate.

The reductase (PgCR) from recombinant Escherichia coli CCZU-Y10 displayed high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). To efficiently synthesize (S)-CHBE (>99 % enantiomeric excess (ee)), the highly stereoselective bioreduction of COBE into (S)-CHBE with the whole cells of E. coli CCZU-Y10 was successfully demonstrated in a dibutyl phthalate-water biphasic system. The appropriate ratio of the organic phase to water phase was 1:1 (v/v). The optimum reaction temperature, reaction pH, cosubstrate, NAD(+), and cell dosage of the biotransformation of 100 mM COBE in this biphasic system were 30 °C, 7.0, mannitol (2.5 mmol/mmol COBE), 0.1 µmol/(mmol COBE), and 0.1 g (wet weight)/mL, respectively. Moreover, COBE at a high concentration of (1,000 mM) could be asymmetrically reduced to (S)-CHBE in a high yield (99.0 %) and high enantiometric excess value (>99 % ee). Significantly, E. coli CCZU-Y10 shows high potential in the industrial production of (S)-CHBE (>99 % ee).

Highly efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate and its derivatives by a robust NADH-dependent reductase from E. coli CCZU-K14.

An NADH-dependent reductase (CmCR) from Candida magnoliae was discovered by genome mining for carbonyl reductases. After CmCR was overexpressed in Escherichia coli BL21, a robust reductase-producing strain, recombinant E. coli CCZU-K14, was employed for the efficient synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) from the reduction of ethyl 4-chloro-3-oxobutanoate (COBE). After the optimization, the optimum reaction conditions were obtained. Notably, E. coli CCZU-K14 had broad substrate specificity in reducing both aliphatic and aromatic substrates, and excellent enantioselectivity of CCZU-K14 was observed for most of the tested substrates, resulting in chiral alcohols of over 99.9% ee. Moreover, COBE at a high concentration of (3000mM) could be asymmetrically reduced to (S)-CHBE in the high yield (>99.0%) and high enantiometric excess value (>99.9% ee) after 14h. Significantly, E. coli CCZU-K14 shows high potential in the industrial production of (S)-CHBE and its derivatives (>99.9% ee).