Building Flexible Escherichia coli Modules for Bifunctionalizing n-Octanol: The Byproduct of Oleic Acid Biorefinery.

Artificial biorefinery of oleic acid into 1,10-decanedioic acid represents a revolutionizing route to the sustainable production of chemically difficult-to-make bifunctional chemicals. However, the carbon atom economy is extremely low (56%) due to the formation of unifunctional n-octanol. Here, we report a panel of recombinant Escherichia coli modules for diverse bifunctionalization, where the desired genetic parts are well distributed into different modules that can be flexibly combined in a plug-and-play manner. The designed ω-functionalizing modules could achieve ω-hydroxylation, consecutive ω-oxidation, or ω-amination of n-octanoic acid. By integrating these advanced modules with the reported oleic acid-cleaving modules, high-value C8 and C10 products, including ω-hydroxy acid, ω-amino acid, and α,ω-dicarboxylic acid, were produced with 100% carbon atom economy. These ω-functionalizing modules enabled the complete use of all of the carbon atoms from oleic acid (released from plant oil) for the green synthesis of structurally diverse bifunctional chemicals.

Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium-Chain Fatty Acids.

The P450-mediated terminal hydroxylation of non-activated C-H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7-C8:0 and sequence alignments. Some of the resultant single mutants were advantageous over the wild-type enzyme with higher reaction rates, achieving a complete conversion of n-octanoic acid (C8:0, 1 mM) in a shorter time period. Especially, a single-mutation variant, D258E, showed 3.8-fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium-chain fatty acid C8:0 to the high value-added product 8-hydroxyoctanoic acid.

Biological synthesis of 2,5-bis(hydroxymethyl)furan from biomass-derived 5-hydroxymethylfurfural by E. coli CCZU-K14 whole cells.

Biocatalytic upgrading of bio-based platform chemical 5-hydroxymethylfurfural (5-HMF) to 2,5-bis(hydroxymethyl)furan (BHMF) is currently of great interest due to the product specificity, mild reaction and high efficiency. In this work, 200mM 5-HMF could be effectively biotransformed to BHMF at 90.6% with highly 5-HMF-tolerant recombinant E. coli CCZU-K14 whole cells at pH 6.5 and 30°C under the optimum reaction conditions (cosubstrate glucose 1.0mol glucose/(mol 5-HMF), D-xylose 400mM, l-glutamic acid 250mM, Mg2+ 1.5mM, 0.2mol ß-cyclodextrin/(mol 5-HMF), CTAB (cetyltrimethyl ammonium bromide) 12.5mM, and 0.1g wet cells/mL). It was found that E. coli CCZU-K14 was highly tolerant to 5-HMF (up to 400mM). Effective bioreduction of biomass-derived 5-HMF (≤200) to BHMF was successfully demonstrated in this study. In conclusion, this strategy showed high potential application for the synthesis of BHMF.

Biodegradation of alkali lignin by a newly isolated Rhodococcus pyridinivorans CCZU-B16.

Based on the Prussian blue spectrophotometric method, one high-throughput screening strategy for screening lignin-degrading microorganisms was built on 24-well plate at room temperature. One high activity of alkali lignin-degrading strain Rhodococcus pyridinivorans CCZU-B16 was isolated from soil. After the optimization of biodegradation, 30.2% of alkali lignin (4 g/L) was degraded under the nitrogen-limited condition (30/1 of C/N ratio; g/g) at 30 °C for 72 h. It was found that syringyl (S) units and guaiacyl (G) in lignin decreased after biodegradation. Moreover, the accumulated lipid in cells had a fatty acid profile rich in C16 and C18 with four major constituent fatty acids including palmitic acid (C16:0; 22.4%), palmitoleic acid (C16:1; 21.1%), stearic acid (C18:0; 16.2%), and oleic acid (C18:1; 23.1%). In conclusion, Rhodococcus pyridinivorans CCZU-B16 showed high potential application in future.

Chemical-enzymatic conversion of corncob-derived xylose to furfuralcohol by the tandem catalysis with SO42-/SnO2-kaoline and E. coli CCZU-T15 cells in toluene-water media.

One-pot synthesis of furfuralcohol from corncob-derived xylose was attempted by the tandem catalysis with solid acid SO42-/SnO2-kaoline and recombination Escherichia coli CCZU-T15 whole-cells in the toluene-water media. Using SO42-/SnO2-kaoline (3.5wt%) as catalyst, the furfural yield of 74.3% was obtained from corncob-derived xylose in the toluene-water (1:2, v:v) containing 10mM OP-10 at 170°C for 30min. After furfural liquor was mixed with corncob-hydrolysate from the enzymatic hydrolysis of oxalic acid-pretreated corncob residue, furfural (50.5mM) could be completely biotransformed to furfuralcohol with Escherichia coli CCZU-T15 whole-cells harboring an NADH-dependent reductase (ClCR) in the toluene-water (1:3, v:v) containing 12.5mM OP-10 and 1.6mM glucose/mM furfural at 30°C and pH 6.5. Furfuralcohol was obtained at 13.0% yield based on starting material corncob (100% furfuralcohol yield for bioreduction of furfural step). Clearly, this one-pot synthesis of furfuralcohol strategy shows high potential application for the effective utilization of corncob.

Enzymatic in situ saccharification of sugarcane bagasse pretreated with low loading of alkalic salts Na2SO3/Na3PO4 by autoclaving.

Sugarcane bagasse (SCB) is an abundant, renewable and inexpensive agricultural byproduct for the production of biofuel and other biobased products. To effectively saccharify SCB with cellulases, combination with dilute alkali salts Na2SO3/Na3PO4 (0.4% Na3PO4, 0.03% Na2SO3) at 7.5% sulfidity and hot water (DASHW) in "one-pot" pretreatment media by autoclaving at 110°C for 40min was attempted to pretreat SCB in this study. Furthermore, FT-IR, XRD and SEM were employed to characterize the changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated Na2SO3/Na3PO4-SCB solid residue, which indicated that combination pretreatment could effectively remove lignin and hemicellulose for enhancing enzymatic saccharification. After 72h, the reducing sugars and glucose from the enzymatic in situ hydrolysis of 50g/L Na2SO3/Na3PO4-SCB in dilute Na2SO3/Na3PO4 (0.27% Na3PO4, 0.02% Na2SO3) media were obtained at 33.8 and 21.8g/L, respectively. Finally, the SCB-hydrolysates containing 20g/L glucose were used for ethanol fermentation in the presence of dilute alkali salts. After 48h, the ethanol yield was 0.42g ethanol/g glucose, which represents 82.1% of the theoretical yield. In conclusion, this study provided an effective pretreatment strategy for enhancing SCB's saccharification, which has potential application of other lignocellulosic materials.

Effective enzymatic in situ saccharification of bamboo shoot shell pretreated by dilute alkalic salts sodium hypochlorite/sodium sulfide pretreatment under the autoclave system.

In this study, dilute alkali salts (0.6% NaClO, 0.067% Na2S) pretreatment at 10% sulfidity under the autoclave system at 120°C for 40min was used for pretreating bamboo shoot shell (BSS). Furthermore, FT-IR, XRD and SEM were employed to characterize the changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated BSS solid residue. After 72h, the reducing sugars and glucose from the enzymatic in situ hydrolysis of 50g/L pretreated BSS in dilute NaClO/Na2S media could be obtained at 31.11 and 20.32g/L, respectively. Finally, the obtained BSS-hydrolysates containing alkalic salt NaClO/Na2S resulted in slightly negative effects on the ethanol production. Glucose in BSS-hydrolysates was fermented from 20.0 to 0.17g/L within 48h, and an ethanol yield of 0.41g/g glucose, which represents 80.1% of the theoretical yield, was obtained. This study provided an effective strategy for potential utilization of BSS.

Sequential Aqueous Ammonia Extraction and LiCl/N,N-Dimethyl Formamide Pretreatment for Enhancing Enzymatic Saccharification of Winter Bamboo Shoot Shell.

Effective utilization of winter bamboo shoot shell (BSS) is of great interest, since BSS provides a renewable and inexpensive bioresource for the production of biofuels. In this study, an effective combination pretreatment by the sequential aqueous ammonia (25 wt%) extraction at 50 °C for 24 h and LiCl/N,N-dimethyl formamide (LiCl/DMF) (6 wt% of LiCl) pretreatment at 50 °C for 8 h was used for pretreating BSS. SEM, FTIR, and XRD results indicated that combination pretreatment could effectively remove lignin and change the crystal structure of cellulose for promoting enzymatic saccharification. Additionally, significant linear correlations were found about solid recovery-delignification (R 2 = 0.9235), delignification-reducing sugars (R 2 = 0.9552), and delignification-hemicellulose removal (R 2 = 0.9779) during the combination pretreatment. The reducing sugars and glucose from the hydrolysis of 100 g/L pretreated BSS could be obtained at 72.3 and 40.5 g/L, respectively. Using the recovered BSS-hydrolysates containing 20-50 g/L glucose as carbon source, the ethanol yields at 48 h could be obtained at 84.5-86.1% of the theoretical yield. In conclusion, the sequential ammonia extraction and LiCl/DMF pretreatment has high potential application in future.

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.