Discovery of L-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids.

Beta-hydroxy non-standard amino acids (ß-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for ß-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of ß-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineered Escherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells.

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C-C bond formation. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions - the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard ß-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

Low-Turnover Drug Molecules: A Current Challenge for Drug Metabolism Scientists.

In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for low-turnover (slowly metabolized) drug molecules. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a maximum of 1 hour with liver microsomes to 4 hours with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compounds that are slowly metabolized. Thus, the challenge of accurately estimating low human clearance with confidence has emerged to be among the top challenges that drug metabolism scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metabolism of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodology using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addition, more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HµREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metabolism scientists with the most up-to-date experimental options for dealing with the complex issue of low-turnover drug candidates.

Conference report: a hitchhiker's guide to outsourcing ADME studies: the inside of outsourcing.

This report gives a summary of the key points raised during a roundtable discussion convened at the American Association of Pharmaceutical Scientists 2012 Annual Meeting and Exposition held in Chicago on 17 October 2012. The science of ADME continues to grow, as does the impact of these studies on drug development. Understanding ADME requires efforts from several scientific specialties. With reductions in pharmaceutical company R&D staff there has been a corresponding growth in CROs with the capabilities and expertise to perform ADME work. This roundtable explored the challenges inherent in understanding ADME and the issues that arise when ADME studies shift from in-house study directors to external scientists working within the business model of a CRO. Pharmaceutical industry scientists and procurement specialists can satisfy their expectations by awareness of the growing expertise within CROs and the need for open communication among all partners involved in outsourced work.

Stereochemistry of 1,2-elimination reactions at the E2-E1cB interface--tert-butyl 3-tosyloxybutanoate and its thioester.

Experimental data on the stereoselectivity of base-catalyzed 1,2-elimination reactions that produce conjugated carbonyl compounds are scarce in spite of the importance of these reactions in organic and biochemistry. As part of a comprehensive study in this area, we have synthesized stereospecifically-deuterated beta-tosyloxybutanoate esters and thioesters and studied the stereoselectivity of their elimination reactions under non-ion pairing conditions. With the availability of both the (2R*,3R*) and (2R*,3S*) diastereomers the innate stereoselectivity could be determined unambiguously. (1)H and (2)H NMR data show that these substrates produce 5-6% syn elimination, the usual amount for acyclic substrates undergoing E2 reactions. Contrary to earlier suggestions, activation by a carbonyl group has virtually no influence upon the stereoselectivity. Elimination of the (2R*,3R*) diastereomer of the beta-tosyloxyester and thioester produces 21-25% of the (Z)-alkene, much more than observed with a poorer beta-nucleofuge. A relatively large amount of (Z)-alkene product seems to be a good marker for an E2 pathway, in which the transition state is E1cB-like, rather than an E1cB(irrev) mechanism. Syn KIE values were higher than those for anti elimination for the esters as well as the thioesters. Experimental challenges to the synthesis of stereospecifically-deuterated beta-tosyloxyesters are discussed.