No abstract

Ketoreductase (KRED)-catalyzed asymmetric reduction of prochiral ketones is an attractive method to synthesize chiral alcohols. Herein, two KREDs LfSDR1-V186A/E141I and CgKR1-F92I with complementary stereopreference were identified towards reduction of apremilast...

Switching cofactor preference of oxidoreductases from NADPH to NADH by rational engineering, replacing the expensive cofactor NADP + with the cheap cofactor NAD + , is a focus of attention in the industrial application of oxidoreductases. This study focuses on the reversal of cofactor preference for shortchain dehydrogenases/reductases (SDRs). Combined with bioinformatics analyses and in silico analyses, a small and smart mutant library (Mu1-Mu3) of LfSDR1 was rationally designed and constructed. Thus, the excellent NADH-dependent recombinant LfSDR1-V186A/G92V/E141L/G38D/T15A variant (Mu2) was obtained. Meanwhile, novel enzymatic processes for synthesis of the key intermediates [(R)-2 and (S)-4] of telotristat ethyl and crizotinib were successfully created, which mainly relied on Mu2 coupled with an FDH-catalyzed cofactor regeneration system. A co-expressed E. coli whole-cell biocatalyst containing the genes of Mu2 and PpFDH was developed to reduce ketones 1 and 3. Finally, ketone 1 was almost completely converted into the product (R)-2 with a space-time yield of 115.7 g•L À 1 •d À 1 and a 98.8 % ee value.

Background Anoxia is characterized by changes in the morphology, metabolism, and function of tissues and organs due to insufficient oxygen supply or oxygen dysfunction. Gentiana straminea Maxim (G.s Maxim) is a traditional Tibetan medicine. Our previous work found that G.s Maxim mediates resistance to hypoxia, and we found that the ethyl acetate extract had the best effect. Nevertheless, the primary anti-hypoxia components and mechanisms of action remain unclear. Methods Compounds from the ethyl acetate extraction of G.s Maxim were identified using UPLC-Triple TOF MS/MS. Then Traditional Chinese Medicine Systematic Pharmacology Database was used to filtrate them. Network pharmacology was used to forecast the mechanisms of these compounds. Male specific pathogen-free Sprague Dawley rats were randomly divided into six groups: (1) Control; (2) Model; (3) 228 mg/kg body weight Rhodiola capsules; (4) 6.66 g/kg body weight the G.s Maxim’s ethyl acetate extraction; (5) 3.33 g/kg body weight the G.s Maxim’s ethyl acetate extraction; (6) 1.67 g/kg body weight the G.s Maxim’s ethyl acetate extraction. After administering intragastric ally for 15 consecutive days, an anoxia model was established using a hypobaric oxygen chamber (7000 m, 24 h). Then Histology, enzyme-linked immunosorbent assays, and western blots were performed to determine these compounds’ anti-hypoxic effects and mechanisms. Finally, we performed a molecular docking test to test these compounds using Auto Dock. Results Eight drug-like compounds in G.s Maxim were confirmed using UPLC-Triple TOF MS/MS and Lipinski’s rule. The tumor necrosis factor (TNF) signaling pathway, the hypoxia-inducible factor 1 (HIF-1) signaling pathway, and the nuclear factor kappa-B (NF-κB) signaling pathway was signaling pathways that G.s Maxim mediated anti-anoxia effects. The critical targets were TNF, Jun proto-oncogene (JUN), tumor protein p53 (TP53), and threonine kinase 1 (AKT1). Animal experiments showed that the ethyl acetate extraction of G.s Maxim ameliorated the hypoxia-induced damage of hippocampal nerve cells in the CA1 region and reversed elevated serum expression of TNF-α, IL-6, and NF-κ B in hypoxic rats. The compound also reduced the expression of HIF-1α and p65 and increased the Bcl-2/Bax ratio in brain tissue. These findings suggest that G.s Maxim significantly protects against brain tissue damage in hypoxic rats by suppressing hypoxia-induced apoptosis and inflammation. Ccorosolic acid, oleanolic acid, and ursolic acid had a strong affinity with core targets. Conclusions The ethyl acetate extraction of G.s Maxim mediates anti-hypoxic effects, possibly related to inhibiting apoptosis and inflammatory responses through the HIF-1/NF-κB pathway. The primary active components might be corosolic, oleanolic, and ursolic acids.