Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid for producing L-phenylglycine, was directly identified from 4,695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4,253.8 U·mg-1 . Through reaction process optimization, a high-concentration substrate (0.7 M) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 M benzoylformic acid could be aminated to L-phenylglycine with conversion of > 99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of L-phenylglycine. This article is protected by copyright. All rights reserved.

Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformations and high GC content. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA), which includes the measurement of their binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer, termed ARapt1 with the estimated Kd value of 5.5 ± 3 nm, was chosen as the most prominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L-1 from the 12 g L-1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.