Protein engineering is a powerful strategy for enhancing the properties of enzymes for industrial applications. However, thermostabilizing an enzyme via this strategy while simultaneously improving its activity is challenging due to the well-known stability–activity trade-off. Herein, using native ketoreductase LbCR, thermostability and activity were evolved separately by directed evolution, generating mutations V198I and M154I/A155D with increased thermostability and mutations A201D/A202L with increased enzymatic activity. On the basis of additivity and cooperative mutational effects, variants LbCRM6 (M154I/A155D/A201D/A202L) and LbCRM8 (M154I/A155D/V198I/A201D/A202L) with simultaneously improved thermostability and activity were subsequently constructed by combining mutations. Analysis of variant structures demonstrated that increased thermostability was largely attributed to rigidification of flexible loops around the active site through the formation of additional hydrogen bonds and hydrophobic interactions. The best variant LbCRM8 displayed a 1944-fold increase in half-life at 40 °C and a 3.2-fold improvement in catalytic efficiency compared with the wide-type enzyme. Using only 1 g L–1 of lyophilized E. coli cells coexpressing this LbCRM8 and glucose dehydrogenase BmGDH as a catalyst, t-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate up to 300 g L–1 loading was completely reduced within 6 h at 40 °C, yielding the corresponding t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate (ATS-7) with >99.5% de and a space-time yield of up to 1.05 kg L–1 day–1. These results demonstrated that LbCRM8 is an attractive biocatalyst for the synthesis of ATS-7, an advanced chiral intermediate for the production of the cholesterol-lowering drug atorvastatin.

Pamiparib, an investigational Poly (ADP-ribose) polymerase (PARP) inhibitor in clinical development, demonstrates excellent selectivity for both PARP1 and PARP2, and superb anti-proliferation activities in tumor cell lines with BRCA1/2 mutations or HR pathway deficiency (HRD). Pamiparib has good bioavailability and is 16-fold more potent than olaparib in an efficacy study using BRCA1 mutated MDA-MB-436 breast cancer xenograft model. Pamiparib also shows strong anti-tumor synergy with temozolomide (TMZ), a DNA alkylating agent used to treat brain tumors. Compared to other PARP inhibitors, pamiparib demonstrated improved penetration across the blood brain barrier (BBB) in mice. Oral administration of pamiparib at a dose as low as 3 mg/kg is sufficient to abrogate PARylation in brain tumor tissues. In SCLC-derived, TMZ-resistant H209 intracranial xenograft model, combination of pamiparib with TMZ overcomes its resistance and shows significant tumor inhibitory effects and prolonged life span. Our data suggests that combination of pamiparib with TMZ has unique potential for treatment of brain tumors. Currently, the combination therapy of pamiparib with TMZ is evaluated in clinical trial [NCT03150862].

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Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases.

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Fermented vegetable-fruit beverages are a popular fermented food, with many potential health benefits. In this study, two commercial Lactobacillus plantarum strains were selected to ferment a beverage containing apples, pears, and carrots. The metabolites and antioxidant activities were examined during the fermentation process. Results showed that lactic acid and acetic acid accumulated gradually, whereas malic acid decreased. Glucose and fructose increased from 0.48 and 14.8 g/L to 7.7 and 20.8 g/L, respectively, while sucrose decreased slightly. Ascorbic acid also increased continuously during the fermentation to 90.74 mg/100 mL. DPPH and ABTS radical scavenging activity and FRAP reached their maximum value after 4-8 days. The accumulation of TPC, TFC, and SOD reached their maximum value on the 8th day of fermentation. Our study revealed that the L. plantarum-fermented vegetable-fruit beverage showed significant antioxidant activity, which is helpful in evaluating the fermentation end-point and developing a high-quality fermented beverage.

β-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) family 17 β-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (β/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear β-1,3-glucan and the remaining glucan is transferred to the end of another β-1,3-glucan acceptor. The first crystal structure of a GH family 17 β-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.