Summary paragraph Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to functions that are new to biology1–3. Yet only a subset of these engineered enzymes can function in living systems4–7. Finding enzymatic pathways that forge chemical bonds not found in biology is particularly difficult in the cellular environment, as this hinges on the discovery not only of new enzyme activities but also reagents that are simultaneously sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution, and generalisation of a fully genetically-encoded platform for producing chiral organoboranes in bacteria. Escherichia coli harbouring wild-type cytochrome c from Rhodothermus marinus8 (Rma cyt c) were found to form carbon–boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into B–H bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram scale biosynthesis, offering up to 15300 turnovers, 6100 h–1 turnover frequency, 99:1 enantiomeric ratio (e.r.), and 100% chemoselectivity. The enantio-preference of the biocatalyst could also be switched to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace which rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than that of known chiral catalysts for the same class of transformation9–11. This tunable method for manipulating boron in cells opens a whole new world of boron chemistry in living systems.

Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10,000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.

Directed evolution is a method to tune the properties of enzymes for use in organic chemistry and biotechnology, to study enzyme mechanisms, and to shed light on darwinian evolution in nature. In order to enhance its efficacy, iterative saturation mutagenesis (ISM) was implemented. This involves: 1) randomized mutation of appropriate sites of one or more residues; 2) screening of the initial mutant libraries for properties such as enzymatic rate, stereoselectivity, or thermal robustness; 3) use of the best hit in a given library as a template for saturation mutagenesis at the other sites; and 4) continuation of the process until the desired degree of enzyme improvement has been reached. Despite the success of a number of ISM-based studies, the question of the optimal choice of the many different possible pathways remains unanswered. Here we considered a complete 4-site ISM scheme. All 24 pathways were systematically explored, with the epoxide hydrolase from Aspergillus niger as the catalyst in the stereoselective hydrolytic kinetic resolution of a chiral epoxide. All 24 pathways were found to provide improved mutants with notably enhanced stereoselectivity. When a library failed to contain any hits, non-improved or even inferior mutants were used as templates in the continuation of the evolutionary pathway, thereby escaping from the local minimum. These observations have ramifications for directed evolution in general and for evolutionary biological studies in which protein engineering techniques are applied.

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

A central goal in molecular evolution is to understand the ways in which genes and proteins evolve in response to changing environments. In the absence of intact DNA from fossils, ancestral sequence reconstruction (ASR) can be used to infer the evolutionary precursors of extant proteins. To date, ancestral proteins belonging to eubacteria, archaea, yeast and vertebrates have been inferred that have been hypothesized to date from between several million to over 3 billion years ago. ASR has yielded insights into the early history of life on Earth and the evolution of proteins and macromolecular complexes. Recently, however, ASR has developed from a tool for testing hypotheses about protein evolution to a useful means for designing novel proteins. The strength of this approach lies in the ability to infer ancestral sequences encoding proteins that have desirable properties compared with contemporary forms, particularly thermostability and broad substrate range, making them good starting points for laboratory evolution. Developments in technologies for DNA sequencing and synthesis and computational phylogenetic analysis have led to an escalation in the number of ancient proteins resurrected in the last decade and greatly facilitated the use of ASR in the burgeoning field of synthetic biology. However, the primary challenge of ASR remains in accurately inferring ancestral states, despite the uncertainty arising from evolutionary models, incomplete sequences and limited phylogenetic trees. This review will focus, firstly, on the use of ASR to uncover links between sequence and phenotype and, secondly, on the practical application of ASR in protein engineering.

Mutants of the lipase from Bacillus subtilis, previously engineered for enhanced thermostability using directed evolution based on the B-FIT method, show significantly increased tolerance to hostile organic solvents.

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The commercially available laccase from Myceliophthora thermophila was evaluated as catalyst for the polymerization of acrylamides. Using the so-called laccase mediator system (LMS), comprising laccase and -diketones, polymerization reactions can be performed using molecular oxygen as terminal electron acceptor. Thus, the LMS can substitute for diazo-or peroxocompounds as radical starters. Factors influencing the efficiency of the LMS, polymer properties, and the stability of the biocatalyst were investigated. Optimal reaction conditions were slightly acidic reaction media at elevated temperatures (around 50°C). The enzyme is active and stable in the presence of high concentrations of water-soluble and water-insoluble cosolvents but is inactivated by acrylates. The average polymer weight can efficiently be controlled via the ratio of monomer to enzyme. The mediator ( -diketone) concentration had no significant influence on the polymer properties. Laccase-catalyzed oxidation appears to be rate-limiting step of the overall reaction. The ambivalent role of molecular oxygen for reaction initiation as well as inhibitor of the polymerization reaction was investigated. Current limitations of the LMS are analyzed and an improved setup comprising physical separation of the enzymatic initiation reaction from the polymerization via immobilized enzymes is proposed. Thus, not only the biocatalyst is highly stabilized but also the product properties can be controlled.