Publications by authors named "Jian-He Xu"

153 Publications

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli.

Sci Rep 2021 Mar 5;11(1):5290. Epub 2021 Mar 5.

Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.
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http://dx.doi.org/10.1038/s41598-021-84859-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935960PMC
March 2021

Engineering of an oleate hydratase for efficient C10-Functionalization of oleic acid.

Biochem Biophys Res Commun 2021 01 30;537:64-70. Epub 2020 Dec 30.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, East China University of Science and Technology, Shanghai 200237, China; State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing and Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, Shanghai 200237, China. Electronic address:

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C-functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (PaOH) with significantly improved catalytic efficiency (from 33 s mM to 119 s mM), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered PaOH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L day and 160 g L day, respectively.
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http://dx.doi.org/10.1016/j.bbrc.2020.12.039DOI Listing
January 2021

Engineering Bacillus subtilis Isoleucine Dioxygenase for Efficient Synthesis of (2,3,4)-4-Hydroxyisoleucine.

J Agric Food Chem 2020 Dec 28;68(49):14555-14563. Epub 2020 Nov 28.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.

Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2,3,4)-4-hydroxyisoleucine [(2,3,4)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from , resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. l-Isoleucine (228 mM) was completely converted to (2,3,4)-4-HIL by the best variant with a space-time yield of up to 80.8 g L d, which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2,3,4)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2,3,4)-4-HIL.
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http://dx.doi.org/10.1021/acs.jafc.0c06544DOI Listing
December 2020

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity.

Chembiochem 2021 Apr 14;22(7):1190-1195. Epub 2020 Dec 14.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, P. R. China.

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products ("normal" and "abnormal") when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k /K ) 8.9-fold to 484 s  mM for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L  d . This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.
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http://dx.doi.org/10.1002/cbic.202000478DOI Listing
April 2021

A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.

Chembiochem 2021 Mar 12;22(6):996-1000. Epub 2021 Jan 12.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Collaborative Innovation Centre for Biomanufacturing, Frontiers Science Center for Materiobiology and Dynamic Chemistry, Shanghai, 200237, P. R. China.

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.
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http://dx.doi.org/10.1002/cbic.202000658DOI Listing
March 2021

Catalytic conversion of corncob to furfuryl alcohol in tandem reaction with tin-loaded sulfonated zeolite and NADPH-dependent reductase biocatalyst.

Bioresour Technol 2021 Jan 16;320(Pt A):124267. Epub 2020 Oct 16.

Laboratory of Bioresourse and Bioprocessing, Advanced Catalysis and Green Manufacturing Collaborative Innovation Center, Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology, National-Local Joint Engineering Research Center of Biomass Refining and High-Quality Utilization, Changzhou University, Changzhou, People's Republic of China; Laboratory of Biomass and Bioenergy, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, People's Republic of China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Huaiyin Normal University, Huaian, People's Republic of China. Electronic address:

In this study, tin-loaded sulfonated zeolite (Sn-zeolite) catalyst was synthesized for catalysis of raw corncob (75.0 g/L) to 103.0 mM furfural at 52.3% yield in water (pH 1.0) at 170 °C. This corncob-derived furfural was subsequently biotransformed with recombinant E. coli CG-19 cells coexpressing NADPH-dependent reductase and glucose dehydrogenase at 35 °C by supplementary of glucose (1.5 mol glucose/mol furfural), sodium dodecyl sulfate (0.50 mM) and NADP (1.0 μmol NADP/mmol furfural) in the aqueous catalytic media (pH 7.5). Both sodium dodecyl sulfate (0.50 mM) and Sn (1.0 mM) could promote reductase activity by 1.4-folds. Within 3 h, furfural was wholly catalyzed into furfuryl alcohol. By combining chemical catalysis with Sn-zeolite and biocatalysis with CG-19 cells in one-pot, an effective and sustainable process was established for tandemly catalyzing renewable biomass into furfuryl alcohol under environmentally-friendly way.
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http://dx.doi.org/10.1016/j.biortech.2020.124267DOI Listing
January 2021

Structure-guided engineering of Pseudomonas dacunhael-aspartate β-decarboxylase for l-homophenylalanine synthesis.

Chem Commun (Camb) 2020 Nov 23;56(89):13876-13879. Epub 2020 Oct 23.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Centre for Biomanufacturing, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China.

Structure-guided engineering of Pseudomonas dacunhael-aspartate β-decarboxylase (AspBDC) resulted in a double mutant (R37A/T382G) with remarkable 15 400-fold improvement in specific activity reaching 216 mU mg, towards the target substrate 3(R)-benzyl-l-aspartate. A novel strategy for enzymatic synthesis of l-homophenylalanine was developed by using the variant as a biocatalyst affording 75% product yield within 12 h. Our results underscore the potential of engineered AspBDC for the biocatalytic synthesis of pharmaceutically relevant and value added unnatural l-amino acids.
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http://dx.doi.org/10.1039/d0cc05871hDOI Listing
November 2020

Identification two key residues at the intersection of domains of a thioether monooxygenase for improving its sulfoxidation performance.

Biotechnol Bioeng 2021 02 6;118(2):737-744. Epub 2020 Nov 6.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.

AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMO mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMO mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMO (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the K of the substrate OPS from 34 to 265 μM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.
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http://dx.doi.org/10.1002/bit.27604DOI Listing
February 2021

Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli.

Int J Biol Macromol 2020 Oct 25;160:372-379. Epub 2020 May 25.

Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, 1638 Wolong Road, Nanyang, Henan 473061, People's Republic of China; State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China. Electronic address:

Glutamate decarboxylase (GAD) has the potential of converting L-glutamate to gamma-aminobutyric acid (GABA), which is an important non-proteinogenic amino acid that has a potential use as food additive or dietary supplement for its physiological functions. A novel pyridoxal 5'-phosphate (PLP)-dependent glutamate decarboxylase (LsGAD) was cloned from GRAS (generally recognized as safe) Lactobacillus senmaizukei by genome mining and efficiently expressed in Escherichia coli BL21. The LsGAD displayed excellent temperature property, pH property and kinetic parameters compared with the probe LbGAD and the other GADs. By increasing the copy number of the LsGAD encoding gene, the expression level of LsGAD and the biosynthesis yield of GABA were increased, which was near to 2 times of that was expressed in single copy. These results established a solid foundation for increasing the added value of L-glutamate and the biosynthesis of GABA.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.05.195DOI Listing
October 2020

Rational Engineering of Formate Dehydrogenase Substrate/Cofactor Affinity for Better Performance in NADPH Regeneration.

Appl Biochem Biotechnol 2020 Oct 13;192(2):530-543. Epub 2020 May 13.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.

Formate dehydrogenases are critical tools for nicotinamide cofactor regeneration, but their limited catalytic efficiency (k/K) is a major drawback. A formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) was the first native NADP-dependent formate dehydrogenase reported and has the highest k/K toward NADP (k/K) compared with other FDHs that can utilize NADP as a hydrogen acceptor. However, the substrate and cofactor affinities of BstFDH are inferior to those of other FDHs, making its practical application difficult. Herein, we engineered recombinant BstFDH to enhance its HCOO and NADP affinities. Based on sequence information analysis and homologous modeling results, I124, G146, S262, and A287 were found to affect the binding affinity for HCOO and NADP. By combining these mutations, we identified a BstFDH variant (G146M/A287G) that reduced K to 0.09 mM, with a concomitant decrease in K, and gave 1.6-fold higher k/K than the wild type (WT). Furthermore, BstFDH I124V/G146H/A287G, with the lowest K of 8.51 mM, showed a catalytic efficiency that was 2.3-fold higher than that of the wild type and a decreased K of 0.11 mM. These results are beneficial for improving the performance of NADP-dependent formate dehydrogenase in the NADPH regeneration of various bioreductive reactions and provide a useful guide for engineering of the substrate and cofactor affinity of other enzymes.
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http://dx.doi.org/10.1007/s12010-020-03317-7DOI Listing
October 2020

High level and enantioselective production of L-phenylglycine from racemic mandelic acid by engineered Escherichia coli using response surface methodology.

Enzyme Microb Technol 2020 May 18;136:109513. Epub 2020 Jan 18.

Henan Provincial Engineering Laboratory of Insect Bio-Reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-Line of South-to-North, Nanyang Normal University, 1638 Wolong Road, Nanyang, Henan, 473061, People's Republic of China. Electronic address:

L-Phenylglycine (L-PHG) is a member of unnatural amino acids, and becoming more and more important as intermediate for pharmaceuticals, food additives and agrochemicals. However, the existing synthetic methods for L-PHG mainly rely on toxic cyanide chemistry and multistep processes. To provide green, safe and high enantioselective alternatives, we envisaged cascade biocatalysis for the one-pot synthesis of L-PHG from racemic mandelic acid. A engineered E. coli strain was established to co-express mandelate racemase, D-mandelate dehydrogenase and L-leucine dehydrogenase and catalyze a 3-step reaction in one pot, enantioselectively transforming racemic mandelic acid to give L-PHG (e.e. >99 %). After the conditions for biosynthesis of L-PHG optimized by response surface methodology, the yield and space-time yield of L-PHG can reach 87.89 % and 79.70 g·L·d, which was obviously improved. The high-yielding and enantioselective synthetic methods use cheap and green reagents, and E. coli whole-cell catalysts, thus providing green and useful alternative methods for manufacturing L-PHG.
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http://dx.doi.org/10.1016/j.enzmictec.2020.109513DOI Listing
May 2020

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Chembiochem 2020 09 4;21(18):2680-2688. Epub 2020 Jun 4.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDH ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDH (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDH was also more stable than BmGDH when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDH was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.
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http://dx.doi.org/10.1002/cbic.202000196DOI Listing
September 2020

Stereocomplementary Synthesis of Pharmaceutically Relevant Chiral 2-Aryl-Substituted Pyrrolidines Using Imine Reductases.

Org Lett 2020 05 13;22(9):3367-3372. Epub 2020 Apr 13.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.

Exploring a collection of naturally occurring imine reductases (IREDs) identified two stereocomplementary IREDs with reducing activity toward sterically hindered 2-aryl-substituted pyrrolines. Using ()-selective IR and ()-selective IR, various chiral 2-aryl-substituted pyrrolidines with excellent enantioselectivity (>99% ee) were stereocomplementarily synthesized in good yield (60-80%), demonstrating the feasibility of IREDs for generating pharmaceutically relevant chiral 2-aryl-substituted pyrrolidine intermediates.
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http://dx.doi.org/10.1021/acs.orglett.0c00802DOI Listing
May 2020

High throughput solid-phase screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic β-amino alcohols.

Biotechnol Lett 2020 Aug 26;42(8):1501-1511. Epub 2020 Mar 26.

Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.

Objectives: To screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic β-amino alcohols.

Results: A new strain named Arthrobacter sp. TYUT010-15 with the (R)-selective deamination activity of cyclic β-amino alcohol has been isolated from nature via a high throughput solid-phase screening method. The reaction conditions of TYUT010-15 were optimized. Using the resting cell of TYUT010-15 as the catalyst, kinetic resolution of trans-2-aminocyclopentanol, trans-2-aminocyclohexanol and cis-1-amino-2-indanol was carried out to afford (1S, 2S)-trans-2-aminocyclopentanol, (1S, 2S)-trans-2-aminocyclohexanol and (1R, 2S)-cis-1-amino-2-indanol in > 99% ee and 49.6-50% conversion. Four aromatic β-amino alcohols and two amines were also resolved, (S)-β-amino alcohols and (R)-amines were obtained in > 99% ee. Preparation experiment was conducted with 200 mM (23.2 g L) racemic trans-2-aminocyclohexanol, yielding the desired (1S, 2S)-trans-2-aminocyclohexanol in 40% isolated yield, > 99% ee and 5.8 g L d space time yields.

Conclusions: This study provides a high throughput solid-phase method for screening of bacteria with cyclic amino alcohol deamination activity and a first example for practical preparation of chiral cyclic β-amino alcohol by Arthrobacter sp. TYUT010-15.
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http://dx.doi.org/10.1007/s10529-020-02869-2DOI Listing
August 2020

Attenuated substrate inhibition of a haloketone reductase via structure-guided loop engineering.

J Biotechnol 2020 Jan 19;308:141-147. Epub 2019 Dec 19.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, China. Electronic address:

Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2',4'-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a K of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter k/K of L211H was 3.1 × 10 s mM, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.
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http://dx.doi.org/10.1016/j.jbiotec.2019.12.011DOI Listing
January 2020

Coevolution of the Activity and Thermostability of an ϵ-Keto Ester Reductase for Better Synthesis of an (R)-α-Lipoic Acid Precursor.

Chembiochem 2020 05 5;21(9):1341-1346. Epub 2020 Mar 5.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for BiomanufacturingTechnology, East China University of Science and Technology, Shanghai, 200237, P. R. China.

In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2 towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg -from 120 U mg in the case of the wild-type enzyme (CpAR2 )-and the half-deactivating temperature (T , for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2 . Consequently, only 2 g L of lyophilized E. coli cells harboring CpAR2 and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L  d ). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.
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http://dx.doi.org/10.1002/cbic.201900693DOI Listing
May 2020

An Ammonium-Formate-Driven Trienzymatic Cascade for ω-Transaminase-Catalyzed ()-Selective Amination.

J Org Chem 2019 11 6;84(22):14987-14993. Epub 2019 Nov 6.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing , East China University of Science and Technology , 130 Meilong Road , Shanghai 200237 , China.

()-Amination mediated by ()-specific ω-transaminases generally requires costly d-alanine in excess to obtain the desired chiral amines in high yield. Herein, a one-pot, trienzymatic cascade comprising an ()-specific ω-transaminase, an amine dehydrogenase, and a formate dehydrogenase was developed for the economical and eco-friendly synthesis of ()-chiral amines. Using inexpensive ammonium formate as the sole sacrificial agent, the established cascade system enabled efficient ω-transaminase-mediated ()-amination of various ketones, with high conversions and excellent (>99%); water and CO were the only waste products.
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http://dx.doi.org/10.1021/acs.joc.9b02445DOI Listing
November 2019

Protein engineering for bioreduction of carboxylic acids.

J Biotechnol 2019 Sep 17;303:53-64. Epub 2019 Jul 17.

Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD, United Kingdom. Electronic address:

Carboxylic acids (CAs) are widespread in Nature. A prominent example is fatty acids, a major constituent of lipids. CAs are potentially economical precursors for bio-based products such as bio-aldehydes and bio-alcohols. However, carboxylate reduction is a challenging chemical transformation due to the thermodynamic stability of carboxylate. Carboxylic acid reductases (CARs), found in bacteria and fungi, offer a good solution to this challenge. These enzymes catalyse the NADPH- and ATP-dependent reduction of aliphatic and aromatic CAs. This review summarised all the protein engineering work that has been done on these versatile biocatalysts to date. The intricate catalytic mechanism and structure of CARs prompted us to first examine their domain architecture to facilitate the subsequent discussion of various protein engineering strategies. This then led to a survey of assays to detect aldehyde formation and to monitor aldenylation activity. Strategies for NADPH and ATP regeneration were also incorporated, as they are deemed vital to developing preparative-scale biocatalytic process and high-throughput screening systems. The objectives of the review are to consolidate CAR engineering research, stimulate interest, discussion or debate, and advance the field of bioreduction.
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http://dx.doi.org/10.1016/j.jbiotec.2019.07.001DOI Listing
September 2019

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY).

Bioresour Bioprocess 2019 31;6(1):20. Epub 2019 May 31.

1Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD UK.

Background: Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from strain PC15.

Result: After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in / ) and higher HO tolerance (sevenfold improvement based on IC).

Conclusion: To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.
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http://dx.doi.org/10.1186/s40643-019-0255-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6544594PMC
May 2019

Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase.

Appl Environ Microbiol 2019 06 16;85(11). Epub 2019 May 16.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China

Baeyer-Villiger monooxygenases (BVMOs) are an emerging class of promising biocatalysts for the oxidation of ketones to prepare corresponding esters or lactones. Although many BVMOs have been reported, the development of highly efficient enzymes for use in industrial applications is desirable. In this work, we identified a BVMO from (BVMO) with a high affinity toward aliphatic methyl ketones ( < 3.0 μM). The enzyme was highly soluble and relatively stable, with a half-life of 23 h at 30°C and pH 7.5. The most effective substrate discovered so far is 2-hexanone ( = 2.1 s; = 1.5 μM). Furthermore, BVMO exhibited excellent regioselectivity toward most aliphatic ketones, preferentially forming typical (i.e., normal) products. Using the newly identified BVMO as the catalyst, a high concentration (26.0 g/liter; 200 mM) of methyl levulinate was completely converted to methyl 3-acetoxypropionate after 4 h, with a space-time yield of 5.4 g liter h Thus, BVMO is a promising biocatalyst for the synthesis of 3-hydroxypropionate from readily available biobased levulinate to replace the conventional fermentation. BVMOs are emerging as a green alternative to traditional oxidants in the BV oxidation of ketones. Although many BVMOs are discovered and used in organic synthesis, few are really applied in industry, especially in the case of aliphatic ketones. Herein, a highly soluble and relatively stable monooxygenase from (BVMO) was identified with high activity and excellent regioselectivity toward most aliphatic ketones. BVMO possesses unusually high substrate loading during the catalysis of the oxidation of biobased methyl levulinate to 3-hydroxypropionic acid derivatives. This study indicates that the synthesis of 3-hydroxypropionate from readily available biobased levulinate by BVMO-catalyzed oxidation holds great promise to replace traditional fermentation.
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http://dx.doi.org/10.1128/AEM.00239-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532031PMC
June 2019

One-Pot Synthesis of Phenylglyoxylic Acid from Racemic Mandelic Acids via Cascade Biocatalysis.

J Agric Food Chem 2019 Mar 5;67(10):2946-2953. Epub 2019 Mar 5.

Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North , Nanyang Normal University , 1638 Wolong Road , Nanyang , Henan 473061 , People's Republic of China.

Phenylglyoxylic acid (PGA) are key building blocks and widely used to synthesize pharmaceutical intermediates or food additives. However, the existing synthetic methods for PGA generally involve toxic cyanide and complex processes. To explore an alternative method for PGA biosynthesis, we envisaged cascade biocatalysis for the one-pot synthesis of PGA from racemic mandelic acid. A novel mandelate racemase named ArMR showing higher expression level (216.9 U·mL fermentation liquor) was cloned from Agrobacterium radiobacter and identified, and six recombinant Escherichia coli strains were engineered to coexpress three enzymes of mandelate racemase, d-mandelate dehydrogenase and l-lactate dehydrogenase, and transform racemic mandelic acid to PGA. Among them, the recombinant E. coli TCD 04, engineered to coexpress three enzymes of ArMR, LhDMDH, and LhLDH, can transform racemic mandelic acid (100 mM) to PGA with 98% conversion. Taken together, we provide a green approach for one-pot biosynthesis of PGA from racemic mandelic acid.
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http://dx.doi.org/10.1021/acs.jafc.8b07295DOI Listing
March 2019

Enzymatic synthesis of 10-oxostearic acid in high space-time yield via cascade reaction of a new oleate hydratase and an alcohol dehydrogenase.

J Biotechnol 2019 18;306S:100008. Epub 2019 May 18.

State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, PR China. Electronic address:

Hydroxy fatty acids and carbonyl fatty acids are important multi-functional materials for the manufacture of fragrances and other fine chemicals. In this study, a novel oleate hydratase (PaOH) was cloned from Paracoccus aminophilus DSM 8538 and solubly expressed in Escherichia coli. The recombinant PaOH efficiently catalyzed the hydration of oleic acid, with a specific activity of 5.21 U mg protein. Enzymatic hydration of oleic acid was optimized for the production of 10-hydroxystearic acid. Under the optimal conditions, a pilot reaction was performed on a 1-L scale, where 90 g L of oleic acid was converted into 10-hydroxystearic acid by 10 g L of lyophilized enzyme within 4 h, achieving a conversion of 96.1% and a space-time yield of up to 552 g L d. The resultant 10-hydroxystearic acid was further converted into 10-oxostearic acid, via enzymatic cascade with a secondary alcohol dehydrogenase (MlADH) from Micrococcus luteus WIUJH20, using a lactate dehydrogenase (LdLDH) from Lactobacillus delbrueckii to drive the co-enzyme regeneration. The cascade reaction was carried out stepwise in one pot. Total conversion reached 95.0% after 10 h reaction at a scale of 1 L, with a space time yield of 217 g L d. The final yield of 10-oxostearic acid isolated was 52.2% (mol mol), with a purity of >99.0%.
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http://dx.doi.org/10.1016/j.btecx.2019.100008DOI Listing
May 2019

Enantioselective synthesis of enantiopure β-amino alcohols via kinetic resolution and asymmetric reductive amination by a robust transaminase from Mycobacterium vanbaalenii.

J Biotechnol 2019 Jan 13;290:24-32. Epub 2018 Dec 13.

Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.

Chiral β-amino alcohols are very important chiral building block for preparing bioactive compounds for use in pharmaceutical and fine chemical industries. Synthesis of chiral β-amino alcohols by transaminase is big challenging due to the strict substrate specificities and very low activity of the enzyme. In this work, a (R)-selective ω-transaminase (MVTA) from Mycobacterium vanbaalenii was employed as a biocatalyst for the first time for the synthesis of chiral β-amino alcohol via kinetic resolution and asymmetric reductive amination. The enzyme was purified and characterized. Kinetic resolution of a set of racemic β-amino alcohols including two cyclic β-amino alcohols by MVTA was demonstrated, affording (R)-β-amino alcohols, (1S, 2S)-trans-2-aminocyclopentanol and (1R, 2S)-cis-1-amino-2-indanols in >99% ee and 50-62% conversion. Asymmetric reductive amination of three α-hydroxy ketones (10-300 mM) by MVTA was conducted, (S)-β-amino alcohols were obtained with >99% ee and 80-99% conversion. Preparation experiment for the reductive amination of 200 mM 2-hydroxyacetophenone by the resting cells of recombinant E. coli (MVTA) was proceeded smoothly and product (S)-2-amino-2-phenylethanol was obtained with 71% isolated yield, >99% ee and 68.6 g/L/d volumetric productivity. The current research proved that the MVTA is a robust enzyme for the preparation of chiral β-amino alcohol with high volumetric productivity.
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http://dx.doi.org/10.1016/j.jbiotec.2018.12.003DOI Listing
January 2019

Structural investigation of the enantioselectivity and thermostability mechanisms of esterase RhEst1.

J Mol Graph Model 2018 10 8;85:182-189. Epub 2018 Sep 8.

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China. Electronic address:

The esterase RhEst1 can catalyze the asymmetric hydrolysis of ethyl (±)-2,2-dimethylcyclopropane carboxylate (DmCpCe), yielding a pharmaceutically relevant (S)-carboxylic acid. A triple mutant RhEst1 showed a 5-fold increase in the catalytic activity but a significant decrease in the enantioselectivity. Further optimization studies led to a new enzyme with an additional A143T mutation, which showed both increased catalytic activity and recovered enantioselectivity as well as improved thermostability. To reveal the detailed structural mechanisms for these improved properties, we performed all-atom molecular dynamics simulations on the wild type and two mutants A147I/V148F/G254A and A143T/A147I/V148F/G254A RhEst1, in complex with R-DmCpCe and S-DmCpCe substrates, respectively. The structural stability of the enzyme variants was investigated with the residue interaction network analysis. In RhEst1, S-DmCpCe was observed to adopt a more "activated" conformation than R-DmCpCe, with the active site residues better prearranged for the reaction, leading to the improved enantioselectivity towards S-DmCpCe. The mutations in the two mutants, especially A143T, could lead to different motion patterns in the cap domain, thus affecting the structure of the substrate entrance tunnel. The residue interaction networks analysis showed an increased number of interactions in RhEst1 and RhEst1 as compared to the wild type enzyme, especially the π-π stacking interactions between Phe148 and the nearby residues, providing an explanation for the increased thermostability of the two mutant enzymes observed experimentally. Our work provides essential molecular insights into the substrate binding, enantioselectivity and structural stability of esterase RhEst1, which will facilitate the development of more efficient RhEst1 variants for pharmaceutical applications.
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http://dx.doi.org/10.1016/j.jmgm.2018.08.010DOI Listing
October 2018

Direct Access to Medium-Chain α,ω-Dicarboxylic Acids by Using a Baeyer-Villiger Monooxygenase of Abnormal Regioselectivity.

Chembiochem 2018 10 20;19(19):2049-2054. Epub 2018 Aug 20.

State Key Laboratory of Bioreactor Engineering and, Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, P.R. China.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium-chain α,ω-dicarboxylic acids through a Baeyer-Villiger oxidation-hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long-chain aliphatic keto acids (C -C ) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω-dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω-dicarboxylic acids from renewable oils and fats.
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http://dx.doi.org/10.1002/cbic.201800318DOI Listing
October 2018

Discovery of Two Native Baeyer-Villiger Monooxygenases for Asymmetric Synthesis of Bulky Chiral Sulfoxides.

Appl Environ Microbiol 2018 07 2;84(14). Epub 2018 Jul 2.

State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, China

Two Baeyer-Villiger monooxygenases (BVMOs), designated BVMO and BVMO, were discovered from and , respectively. Both monooxygenases displayed novel features for catalyzing the asymmetric sulfoxidation of bulky and pharmaceutically relevant thioethers. Evolutionary relationship and sequence analysis revealed that the two BVMOs belong to the family of typical type I BVMOs and the subtype ethionamide monooxygenase. Both BVMOs are active toward medium- and long-chain aliphatic ketones as well as various thioether substrates but are ineffective toward cyclohexanone, aromatic ketones, and other typical BVMO substrates. BVMO and BVMO showed the highest activities (0.117 and 0.025 U/mg protein, respectively) toward thioanisole among the tested substrates. Furthermore, these BVMOs exhibited distinct activity and excellent stereoselectivity toward bulky and prochiral prazole thioethers, which is a unique feature of this family of BVMOs. No native enzyme has been reported for the asymmetric sulfoxidation of bulky prazole thioethers into chiral sulfoxides. The identification of BVMO and BVMO provides an important scaffold for discovering enzymes capable of asymmetrically oxidizing bulky thioether substrates by genome mining. Baeyer-Villiger monooxygenases (BVMOs) are valuable enzyme catalysts that are an alternative to the chemical Baeyer-Villiger oxidation reaction. Although BVMOs display broad substrate ranges, no native enzymes were reported to have activity toward the asymmetric oxidation of bulky prazole-like thioether substrates. Herein, we report the discovery of two type I BVMOs from (BVMO) and (BVMO) which are able to catalyze the asymmetric sulfoxidation of bulky prazole thioethers (proton pump inhibitors [PPIs], a group of drugs whose main action is a pronounced and long-lasting reduction of gastric acid production). Efficient catalysis of omeprazole oxidation by BVMO was developed, indicating that this enzyme is a promising biocatalyst for the synthesis of bulky and pharmaceutically relevant chiral sulfoxide drugs. These results demonstrate that the newly identified enzymes are suitable templates for the discovery of more and better thioether-converting BVMOs.
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http://dx.doi.org/10.1128/AEM.00638-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6029080PMC
July 2018

Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids.

Appl Microbiol Biotechnol 2018 Jul 10;102(14):6047-6056. Epub 2018 May 10.

Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.

A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30-40 °C, and highly active over a wide range of pH values (pH 6.0-10.0). In addition, the HpN could tolerate against several hydrophilic organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a K of 4.2 mM and a k of 170 s, the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor. Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73-99%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.
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http://dx.doi.org/10.1007/s00253-018-9057-7DOI Listing
July 2018

Bioamination of alkane with ammonium by an artificially designed multienzyme cascade.

Metab Eng 2018 05 22;47:184-189. Epub 2018 Feb 22.

Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China. Electronic address:

Biocatalytic C-H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an "Artificially Bioamination Pathway" for C-H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450 mutant, an alcohol dehydrogenase ScCR from Streptomyces coelicolor and an amine dehydrogenase EsLeuDH from Exiguobacterium sibiricum, constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes.
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http://dx.doi.org/10.1016/j.ymben.2018.02.009DOI Listing
May 2018

Biosynthesis of Phenylglyoxylic Acid by LhDMDH, a Novel d-Mandelate Dehydrogenase with High Catalytic Activity.

J Agric Food Chem 2018 Mar 27;66(11):2805-2811. Epub 2018 Feb 27.

Henan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North , Nanyang Normal University , 1638 Wolong Road , Nanyang , Henan 473061 , People's Republic of China.

d-Mandelate dehydrogenase (DMDH) has the potential to convert d-mandelic acid to phenylglyoxylic acid (PGA), which is a key building block in the field of chemical synthesis and is widely used to synthesize pharmaceutical intermediates or food additives. A novel NAD-dependent d-mandelate dehydrogenase was cloned from Lactobacillus harbinensi (LhDMDH) by genome mining and expressed in Escherichia coli BL21. After being purified to homogeneity, the oxidation activity of LhDMDH toward d-mandelic acid was approximately 1200 U·mg, which was close to four times the activity of the probe. Meanwhile, the k/ K value of LhDMDH was 28.80 S·mM, which was distinctly higher than the probe. By coculturing two E. coli strains expressing LhDMDH and LcLDH, we developed a system for the efficient synthesis of PGA, achieving a 60% theoretical yield and 99% purity without adding coenzyme or cosubstrate. Our data supports the implementation of a promising strategy for the chiral resolution of racemic mandelic acid and the biosynthesis of PGA.
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http://dx.doi.org/10.1021/acs.jafc.7b05835DOI Listing
March 2018

A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase.

Chembiochem 2018 03 19;19(5):521-526. Epub 2018 Jan 19.

Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, 200237, China.

Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.
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http://dx.doi.org/10.1002/cbic.201700609DOI Listing
March 2018
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