Catalytic anti-Markovnikov oxidation of alkene feedstocks could simplify synthetic routes to many important molecules and solve a long-standing challenge in chemistry. Here we report the engineering of a cytochrome P450 enzyme by directed evolution to catalyze metal-oxo-mediated anti-Markovnikov oxidation of styrenes with high efficiency. The enzyme uses dioxygen as the terminal oxidant and achieves selectivity for anti-Markovnikov oxidation over the kinetically favored alkene epoxidation by trapping high-energy intermediates and catalyzing an oxo transfer, including an enantioselective 1,2-hydride migration. The anti-Markovnikov oxygenase can be combined with other catalysts in synthetic metabolic pathways to access a variety of challenging anti-Markovnikov functionalization reactions.

By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and systematic comparisons of these critical parameters, we developed a toolkit for characterizing any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS performance by comparing how the fluorescence of strains carrying plasmids encoding a fused RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an OTS, and (4) characterize evolved variants of amberless E. coli with improved growth rates.

We report a simple, one‐step enzymatic synthesis of the blue fluorescent noncanonical amino acid β‐(1‐azulenyl)‐l‐alanine (AzAla). By using an engineered tryptophan synthase β‐subunit (TrpB), stereochemically pure AzAla can be synthesized at scale starting from commercially available azulene and l‐serine. Mutation of a universally conserved catalytic glutamate in the active site to glycine has only a modest effect on native activity with indole but abolishes activity on azulene, suggesting that this glutamate activates azulene for nucleophilic attack by stabilization of the aromatic ion.

The metal}othionein-A gene in the metallothionein gene family of the sea urchin Strongylocentrotus purpuratus (SpMTA gene) was sequenced and found to contain three coding exons plus a 3' entirely noncoding exon. Putative a and 0 MT domains were encoded, by its exons 2 and 3, respectively, in reverse of the order in vertebrate metallothionein genes. The SpMTA promoter was characterized through the expression of recombinant constructs containing various portions of the proximal 678-base-pair (bp) 5'-flanking region of the SpMTA gene. Zygotes injected with constructs were cultured to the blastula stage in the presence of a heavy-metal chelator and then incubated in the presence or absence of cadmium. The longest constructs were expressed only when heavy-metal ion was present. Two putative metal-responsive elements (MREs a and b) within 240 bp of the transcription start site resembled mammalian MREs in their critical 8-bp cores (TGCRCNCS)

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a ‘partner’ function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take ~3–5 d, whereas a whole directed evolution can be performed in 3–10 rounds, depending on selection efficiency.