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.

t-Butyl-6-cyano-(3R,5R)-dihydroxyhexanoate is an advanced chiral precursor for the synthesis of the side chain pharmacophore of cholesterol-lowering drug atorvastatin. Herein, a robust carbonyl reductase (LbCR) was newly identified from Lactobacillus brevis, which displays high activity and excellent diastereoselectivity toward bulky t-butyl 6-cyano-(5R)-hydroxy-3oxo-hexanoate (7). The engineered Escherichia coli cells harboring LbCR and glucose dehydrogenase (for cofactor regeneration) were employed as biocatalysts for the asymmetric reduction of substrate 7. As a result, as much as 300 g L −1 of water-insoluble substrate was completely converted to the corresponding chiral diol with >99.5% de in a space−time yield of 351 g L −1 d −1 , indicating a great potential of LbCR for practical synthesis of the very bulky and bi-chiral 3,5-dihydroxy carboxylate side chain of best-selling statin drugs.

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NK cells express activating receptors such as CD16a (FcγRIIIA), NKG2D, SLAM family members and the natural cytotoxicity receptors (NCRs). Full activation of NK cells require co-engagement of different activating receptors. NKp46, a member of NCRs, is a 46 kDa glycoprotein and belongs to immunoglobulin (Ig) superfamily, NKp46 is frequently expressed on tumor infiltrating lymphocytes. Activation of NKp46 with antibody triggers not only NK cell cytotoxicity but also cytokine release. Taken together, NKp46 is a promising target for anti-tumor therapy. Here we described the discovery of fully human NKp46 antibody by phage display approach with common light chain and naïve human B cell library. Bispecific antibody has been researched for many years and very limited number of bispecific antibodies are currently in the market. Heavy and light chain miss pairing is the bottleneck of bispecific antibody discovery and development. To overcome the hurdle, we generated a fully human common light chain phage display library with two commonly used light chain sequences combined with heavy chain fragment from human naïve scFv library. Two light chain sequences were selected based on the approved antibodies sequences and the expression levels. The phage library was then used for panning against recombinant human NKp46 extracellular domain protein and CHOK1 cells overexpressing human NKp46. After conversion to full IgG format, 4 candidates bind specifically to NKp46 and cross react with both human and cyno NKp46. Affinities of the candidates were comparable to bench mark antibodies as determined by FACS. To summarize, we have generated fully human common light chain phage display library that can be used for bispecific antibody discovery. The anti-NKp46 antibodies discovered can be further developed for NK cell engager and NK cell redirecting bispecific antibody. Citation Format: Teddy Yang, Seng Zhu, Jing Gao, Jingyun Yao, Xumin Gong. Discovery of NKp46 antibody for NK cell engager using common light chain phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 524.