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Bioproduction of optical pure (R)-citronellal from (E/Z)-citral at high substrate loading remains challenging. Low catalytic efficiency of (R)-stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure-based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards (E/Z)-citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site-saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the (E/Z)-citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency (kcat /Km ) for (Z)-citral and (E)-citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in (E/Z)-citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled (E)-citral and (Z)-citral to bind with a smaller distance to the key hydrogen donors at a modified (R)-selective binding mode. The variant Y84V was then co-expressed with glucose dehydrogenase from Bacillus megaterium in E. coli D4, in which competing prim-alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l-1 (E/Z)-citral was completely converted into (R)-citronellal with 95.4% ee value and a high space-time yield of 121.6 g l-1 day-1 . The work highlights the synthetic potential of Y84V, which enabled the highest productivity of (R)-citronellal from (E/Z)-citral in high enantiopurity so far.

Background d-Xylonic acid is a versatile platform chemical with broad potential applications as a water reducer and disperser for cement and as a precursor for 1,4-butanediol and 1,2,4-tributantriol. Microbial production of d-xylonic acid with bacteria such as Gluconobacter oxydans from inexpensive lignocellulosic feedstock is generally regarded as one of the most promising and cost-effective methods for industrial production. However, high substrate concentrations and hydrolysate inhibitors reduce xylonic acid productivity. Results The d-xylonic acid productivity of G. oxydans DSM2003 was improved by overexpressing the mGDH gene, which encodes membrane-bound glucose dehydrogenase. Using the mutated plasmids based on pBBR1MCS-5 in our previous work, the recombinant strain G. oxydans/pBBR-R3510-mGDH was obtained with a significant improvement in d-xylonic acid production and a strengthened tolerance to hydrolysate inhibitors. The fed-batch biotransformation of d-xylose by this recombinant strain reached a high titer (588.7 g/L), yield (99.4%), and volumetric productivity (8.66 g/L/h). Moreover, up to 246.4 g/L d-xylonic acid was produced directly from corn stover hydrolysate without detoxification at a yield of 98.9% and volumetric productivity of 11.2 g/L/h. In addition, G. oxydans/pBBR-R3510-mGDH exhibited a strong tolerance to typical inhibitors, i.e., formic acid, furfural, and 5-hydroxymethylfurfural. Conclusion Through overexpressing mgdh in G. oxydans, we obtained the recombinant strain G. oxydans/pBBR-R3510-mGDH, and it was capable of efficiently producing xylonic acid from corn stover hydrolysate under high inhibitor concentrations. The high d-xylonic acid productivity of G. oxydans/pBBR-R3510-mGDH made it an attractive choice for biotechnological production.

Objectives: 3,4-Dihydroxybutyric acid (3,4-DHBA) is a multi-functional C4 platform compound with wide applications in the synthesis of materials and pharmaceuticals. Currently, although the biosynthetic pathway for the production of 3,4-DHBA has been developed, low productivity still hampers its use on large scales. Here, a non-natural four-steps biosynthetic pathway was established in recombinant E. coli with a combinatorial strategy.Results: Firstly, several aldehyde dehydrogenases (ALDHs) were screened and characterized for catalyzing the dehydrogenation of 3,4-dihydroxybutanal (3,4-DHB) to 3,4-DHBA through in vitro enzyme assays. Secondly, a recombinant E. coli was successfully constructed to generate 3,4-DHBA from D-xylose by introducing the pathway containing BsGDH, YagF, PpMdlC and ALDH into E. coli with 3.04 g/L 3,4-DHBA obtained. Then, disruption of competing pathways by deleting xylA, ghrA, ghrB and adhP genes contributed to increase the accumulation of 3,4-DHBA by 87%. Final, fusion expression of PpMdlC and YagF resulted in an enhancement of 3,4-DHBA titer (7.71 g/L), as the highest titer reported so far.Conclusions: These results showed that deleting competing pathways and constructing fusion protein could significantly improve the 3,4-DHBA titer in engineered E. coli.

A cytosolic medium-chain alcohol dehydrogenase Gox0313 from Gluconobacter oxydans DSM2003 was heterologously expressed in Escherichia coli BL21 (DE3) and the resulting proteins were purified and characterized. The recombinant enzyme was confirmed to have a good ability to selectively oxidize the terminal hydroxyl group of varied aliphatic and aromatic diols to the corresponding hydroxyl aldehydes, with no oxidative activities toward hydroxyl aldehydes in the presence of NAD+. This enzyme could not oxidize sec-alcohols. Among the primary diols, ethylene glycol was efficiently converted to glycolaldehyde by Gox0313 coupled with NADH oxidase-2 (NOX-2) from Lactobacillus brevis ATCC367 for NAD+ regeneration. Under the optimal conditions using this enzyme-coupled system (1 U/mL Gox0313 and 4 U/mL NOX-2), 42.32 mM glycolaldehyde was produced from 50 mM ethylene glycol in 8 h with a yield of 83.2%. When the enzyme concentration of Gox0313 was increased to 120 U/mL in the reaction, 500 mM of ethylene glycol was converted to 484.2 mM of glycolaldehyde in 14 h, resulting in a yield of 96.8%. With respect to the superior catalytic activity to primary diols, Gox0313 might be a potentially promising biocatalyst for bioproduction of glycolaldehyde and other hydroxyl aldehydes.

To characterize a novel ene-reductase from Meyerozyma guilliermondii and achieve the ene-reductase-mediated reduction of activated C=C bonds. The gene encoding an ene-reductase was cloned from M. guilliermondii. Sequence homology analysis showed that MgER shared the maximal amino acid sequence identity of 57 % with OYE2.6 from Scheffersomyces stipitis. MgER showed the highest specific activity at 30 °C and pH 7 (100 mM sodium phosphate buffer), and excellent stereoselectivities were achieved for the reduction of (R)-carvone and ketoisophorone. Under the reaction conditions (30 °C and pH 7.0), 150 mM (R)-carvone could be completely converted to (2R,5R)-dihydrocarvone within 22 h employing purified MgER as catalyst, resulting in a yield of 98.9 % and an optical purity of >99 % d.e. MgER was characterized as a novel ene-reductase from yeast and showed great potential for the asymmetric reduction of activated C=C bonds of α,β-unsaturated compounds.

Background (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol [(R)-3,5-BTPE] is a valuable chiral intermediate for Aprepitant (Emend) and Fosaprepitant (Ivemend). Biocatalyzed asymmetric reduction is a preferred approach to synthesize highly optically active (R)-3,5-BTPE. However, the product concentration and productivity of reported (R)-3,5-BTPE synthetic processes remain unsatisfied. Results A NADPH-dependent carbonyl reductase from Lactobacillus kefir (LkCR) was discovered by genome mining for reduction of 3,5-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent enantioselectivity. In order to synthesize (R)-3,5-BTPE efficiently, LkCR was coexpressed with glucose dehydrogenase from Bacillus subtilis (BsGDH) for NADPH regeneration in Escherichia coli BL21 (DE3) cells, and the optimal recombinant strain produced 250.3 g/L (R)-3,5-BTPE with 99.9% ee but an unsatisfied productivity of 5.21 g/(L h). Then, four different linker peptides were used for the fusion expression of LkCR and BsGDH in E. coli to regulate catalytic efficiency of the enzymes and improved NADPH-recycling efficiency. Using the best strain (E. coli/pET-BsGDH-ER/K(10 nm)-LkCR), up to 297.3 g/L (R)-3,5-BTPE with enantiopurity >99.9% ee was produced via reduction of as much as 1.2 M of substrate with a 96.7% yield and productivity of 29.7 g/(L h). Conclusions Recombinant E. coli/pET-BsGDH-ER/K(10 nm)-LkCR was developed for the bioreduction of 3,5-BTAP to (R)-3,5-BTPE, offered the best results in terms of high product concentration and productivity, demonstrating its great potential in industrial manufacturing of (R)-3,5-BTPE. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0585-5) contains supplementary material, which is available to authorized users.