Revealing Additional Stereocomplementary Pairs of Old Yellow Enzymes by Rational Transfer of Engineered Residues.

Every year numerous protein engineering and directed evolution studies are published, increasing the knowledge that could be used by protein engineers. Here we test a protein engineering strategy that allows quick access to improved biocatalysts with very little screening effort. Conceptually it is assumed that engineered residues previously identified by rational and random methods induce similar improvements when transferred to family members. In an application to ene-reductases from the Old Yellow Enzyme (OYE) family, the newly created variants were tested with three compounds, revealing more stereocomplementary OYE pairs with potent turnover frequencies (up to 660 h-1 ) and excellent stereoselectivities (up to >99 %). Although systematic prediction of absolute enantioselectivity of OYE variants remains a challenge, "scaffold sampling" was confirmed as a promising addition to protein engineers' collection of strategies.

Bioorthogonal Enzymatic Activation of Caged Compounds.

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics.

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and ß-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s.

Laboratory-Scale Hydroxylation of Steroids by P450BM3 Variants.

This chapter describes the asymmetric hydroxylation of steroids on laboratory preparative scale, using engineered variants of P450BM3 (CYP102A1) as enzyme catalyst. The following protocol covers the creation of an Escherichia coli BL21-Gold (DE3) expression strain, including necessary control experiments like plasmid preparation, test expression, and creation of storage cultures, to verify successful experimental access to recombinant expressed P450BM3 variants. The recombinant expressed P450BM3 variants are obtained as cleared cell lysate and used in a biotransformation setup to hydroxylate 2.8 mg and up to 15 mg testosterone in the presented protocol. Since P450BM3 depends on NADPH as an electron source for the reaction, a glucose and glucose dehydrogenate based recycling system is added to the reaction. The protocol further includes liquid-liquid extraction of hydroxytestosterone and directs the experimenter to compound purification via column chromatography.

Speeding up directed evolution: Combining the advantages of solid-phase combinatorial gene synthesis with statistically guided reduction of screening effort.

Efficient and economic methods in directed evolution at the protein, metabolic, and genome level are needed for biocatalyst development and the success of synthetic biology. In contrast to random strategies, semirational approaches such as saturation mutagenesis explore the sequence space in a focused manner. Although several combinatorial libraries based on saturation mutagenesis have been reported using solid-phase gene synthesis, direct comparison with traditional PCR-based methods is currently lacking. In this work, we compare combinatorial protein libraries created in-house via PCR versus those generated by commercial solid-phase gene synthesis. Using descriptive statistics and probabilistic distributions on amino acid occurrence frequencies, the quality of the libraries was assessed and compared, revealing that the outsourced libraries are characterized by less bias and outliers than the PCR-based ones. Afterward, we screened all libraries following a traditional algorithm for almost complete library coverage and compared this approach with an emergent statistical concept suggesting screening a lower portion of the protein sequence space. Upon analyzing the biocatalytic landscapes and best hits of all combinatorial libraries, we show that the screening effort could have been reduced in all cases by more than 50%, while still finding at least one of the best mutants.

Iterative saturation mutagenesis: a powerful approach to engineer proteins by systematically simulating Darwinian evolution.

Iterative saturation mutagenesis (ISM) is a widely applicable and powerful strategy for the efficient directed evolution of enzymes. First, one or more amino acid positions from the chosen enzyme are assigned to multi-residue sites (i.e., groups of amino acids or "multisites"). Then, the residues in each multisite are mutated with a user-defined randomization scheme to all canonical amino acids or a reduced amino acid alphabet. Subsequently, the genes of chosen variants (usually the best but not necessarily) are used as templates for saturation mutagenesis at other multisites, and the process is repeated until the desired degree of biocatalyst improvement has been achieved. Addressing multisites iteratively results in a so-called ISM scheme or tree with various upward branches or pathways. The systematic character of ISM simulates in vitro the natural process of Darwinian evolution: variation (library creation), selection (library screening), and amplification (template chosen for the next round of randomization). However, the main feature of ISM that distinguishes it from other directed evolution methods is the systematic probing of a defined segment of the protein sequence space, as it has been shown that ISM is much more efficient in terms of biocatalyst optimization than random methods such as error-prone PCR. In addition, ISM trees have also shed light on the emergence of epistasis, thereby rationally improving the strategies for evolving better enzymes. ISM was developed to improve catalytic properties such as rate, substrate scope, stereo- and regioselectivity using the Combinatorial Active-site Saturation Test (CAST), as well as chemical and thermal stability employing the B-Factor Iterative Test (B-FIT). However, ISM can also be invoked to manipulate such protein properties as binding affinity among other possibilities, including protein-protein interactions. Herein, we provide general guidelines for ISM, using CAST as the case study in the quest to enhance the activity and regioselectivity of the monooxygenase P450BM3 toward testosterone.