Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention.1 Since evolutionary success is dependent on the total number of rounds performed,2 a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness.3 While researchers have accelerated individual steps in the evolutionary cycle,4–9 the only previous example of continuous directed evolution was the landmark study of Joyce,10 who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in E. coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerases that recognize a distinct promoter, initiate transcripts with A instead of G, and initiate transcripts with C. In one example, PACE executed 200 rounds of protein evolution over the course of eight days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than one week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

Phage-assisted continuous evolution (PACE) uses a modified filamentous bacteriophage life cycle to dramatically accelerate laboratory evolution experiments. In this work we expand the scope and capabilities of the PACE method with two key advances that enable the evolution of biomolecules with radically altered or highly specific new activities. First, we implemented small molecule-controlled modulation of selection stringency that enables otherwise inaccessible activities to be evolved directly from inactive starting libraries through a period of evolutionary drift. Second, we developed a general negative selection that enables continuous counter-selection against undesired activities. We integrated these developments to continuously evolve mutant T7 RNA polymerase enzymes with ∼10,000-fold altered, rather than merely broadened, substrate specificities during a single three-day PACE experiment. The evolved enzymes exhibit specificity for their target substrate that exceeds that of wild-type RNA polymerases for their cognate substrates, while maintaining wild-type-like levels of activity.

Elucidation of natural product biosynthetic pathways provides important insights about the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here we show full reconstitution in vitro of an unusual multi-step oxidative cascade for post-assembly line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and an FAD-dependent oxidase (TamL), which act co-dependently through repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a heretofore unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C-10 oxidation of TirE.

The novel dienoyl tetramic acids tirandamycin C (1) and tirandamycin D (2) with activity against vancomycin-resistant Enterococcus faecalis (VRE) were isolated from the marine environmental isolate Streptomyces sp. 307-9, which also produces the previously identified compounds tirandamycin A (3) and B (4). Spectroscopic analysis of 1 and 2 indicated structural similarity to 3 and 4, with differences only in the pattern of pendant oxygenation on the bicyclic ketal system. The isolation of these putative biosynthetic intermediates was enabled by their sequestration on an adsorbent resin during early stationary-phase fermentation.Tetramic acids comprise a growing set of natural products containing a 2,4-pyrrolidinedione ring system formed from the condensation of an amino acid to a polyketide-derived acyl chain ( Figure 1). 1 These compounds continue to generate significant interest due to their broad structural diversity and breadth of biological activities, with representative examples including the HIV-1 integrase inhibitor equisetin, 2 the mycotoxin cyclopiazonic acid, 3 and the first discovered tetramic acid antibiotic streptolydigin. 4,5 Structurally similar to streptolydigin, the antibiotic tirandamycin A (3) was first isolated from the culture broth of the terrestrial bacterium Streptomyces tirandis in 1971, 6 and again in 1976 from Streptomyces flaveolus along with tirandamycin B (4). 7 These compounds exhibited antimicrobial activity against Grampositive bacteria, and in vitro activity against bacterial RNA polymerase. 8,9 The related compounds tirandalydigin, 10 BU-2313, 11 and nocamycin II 12 have since been characterized, all of which share a bicyclic ketal system and dienoyl tetramic acid moiety characteristic of streptolydigin and tirandamycin. Interest in the biosynthesis of these compounds prompted feeding studies in the streptolydigin system, the results of which support a hybrid polyketidenonribosomal peptide origin. 13 The biogenesis of these compounds would be further informed by the identification of pathway intermediates, but to date these efforts have been quite limited. We report here the use of established resin capture methods to isolate and identify the two new natural products tirandamycin C (1) and tirandamycin D (2) presumed to be key biosynthetic intermediates in the pathway to 3 and 4.During a screen to discover new natural products from marine-derived actinomycetes with activity against vancomycin-resistant Enterococcus faecalis (VRE), we isolated from marine sediments a bacterium of Streptomyces-like morphology designated as strain Streptomyces sp. 307-9. Analysis of extracts from shake-flask fermentation identified the major anti-VRE * To whom correspondence should be addressed: . davidhs@umich.edu † These authors contributed equally to this work. Supporting Information Available: 1 H, COSY, HSQC, HMBC spectra for 1-3, NOESY of 1, 1 H and 13 C of 4; maps of 2D NMR correlations; anti-VRE bioassay data, representative HPLC chromatograms of tirandamycin metabolite profiles; ...