Engineering Biocatalysts for the C-H Activation of Fatty Acids by Ancestral Sequence Reconstruction.

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids, but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the subterminal positions of fatty acids. Herein we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multidomain, self-sufficient P450 monooxygenases. We resurrected 11 catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from subterminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multidomain enzymes to develop active, self-sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP).

Ancestral sequence reconstruction is a technique that is gaining widespread use in molecular evolution studies and protein engineering. Accurate reconstruction requires the ability to handle appropriately large numbers of sequences, as well as insertion and deletion (indel) events, but available approaches exhibit limitations. To address these limitations, we developed Graphical Representation of Ancestral Sequence Predictions (GRASP), which efficiently implements maximum likelihood methods to enable the inference of ancestors of families with more than 10,000 members. GRASP implements partial order graphs (POGs) to represent and infer insertion and deletion events across ancestors, enabling the identification of building blocks for protein engineering. To validate the capacity to engineer novel proteins from realistic data, we predicted ancestor sequences across three distinct enzyme families: glucose-methanol-choline (GMC) oxidoreductases, cytochromes P450, and dihydroxy/sugar acid dehydratases (DHAD). All tested ancestors demonstrated enzymatic activity. Our study demonstrates the ability of GRASP (1) to support large data sets over 10,000 sequences and (2) to employ insertions and deletions to identify building blocks for engineering biologically active ancestors, by exploring variation over evolutionary time.

Genetically programmed chiral organoborane synthesis.

Recent advances in enzyme engineering and design have expanded nature's catalytic repertoire to functions that are new to biology. However, only a subset of these engineered enzymes can function in living systems. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus (Rma cyt c) were found to form carbon-boron bonds in the presence of borane-Lewis base complexes, through carbene insertion into boron-hydrogen 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, providing up to 15,300 turnovers, a turnover frequency of 6,100 h-1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned 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 that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.

The strigolactone (SL) class of phytohormones shows broad chemical diversity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the diversification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several diversifying oxidation reactions on the structure. Evidence was obtained for functional divergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone diversification in early land plants, which may have extended to the diversification of strigol-type SLs.

Determinants of thermostability in the cytochrome P450 fold.

Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~40°C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Effect of Binding on Enantioselectivity of Epoxide Hydrolase.

Molecular dynamics simulations and free energy calculations have been used to investigate the effect of ligand binding on the enantioselectivity of an epoxide hydrolase (EH) from Aspergillus niger. Despite sharing a common mechanism, a wide range of alternative mechanisms have been proposed to explain the origin of enantiomeric selectivity in EHs. By comparing the interactions of ( R)- and ( S)-glycidyl phenyl ether (GPE) with both the wild type (WT, E = 3) and a mutant showing enhanced enantioselectivity to GPE (LW202, E = 193), we have examined whether enantioselectivity is due to differences in the binding pose, the affinity for the ( R)- or ( S)- enantiomers, or a kinetic effect. The two enantiomers were easily accommodated within the binding pockets of the WT enzyme and LW202. Free energy calculations suggested that neither enzyme had a preference for a given enantiomer. The two substrates sampled a wide variety of conformations in the simulations with the sterically hindered and unhindered carbon atoms of the GPE epoxide ring both coming in close proximity to the nucleophilic aspartic acid residue. This suggests that alternative pathways could lead to the formation of a ( S)- and ( R)-diol product. Together, the calculations suggest that the enantioselectivity is due to kinetic rather than thermodynamic effects and that the assumption that one substrate results in one product when interpreting the available experimental data and deriving E-values may be inappropriate in the case of EHs.

Exploring the past and the future of protein evolution with ancestral sequence reconstruction: the 'retro' approach to protein engineering.

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.

Learning epistatic interactions from sequence-activity data to predict enantioselectivity.

Enzymes with a high selectivity are desirable for improving economics of chemical synthesis of enantiopure compounds. To improve enzyme selectivity mutations are often introduced near the catalytic active site. In this compact environment epistatic interactions between residues, where contributions to selectivity are non-additive, play a significant role in determining the degree of selectivity. Using support vector machine regression models we map mutations to the experimentally characterised enantioselectivities for a set of 136 variants of the epoxide hydrolase from the fungus Aspergillus niger (AnEH). We investigate whether the influence a mutation has on enzyme selectivity can be accurately predicted through linear models, and whether prediction accuracy can be improved using higher-order counterparts. Comparing linear and polynomial degree = 2 models, mean Pearson coefficients (r) from [Formula: see text]-fold cross-validation increase from 0.84 to 0.91 respectively. Equivalent models tested on interaction-minimised sequences achieve values of [Formula: see text] and [Formula: see text]. As expected, testing on a simulated control data set with no interactions results in no significant improvements from higher-order models. Additional experimentally derived AnEH mutants are tested with linear and polynomial degree = 2 models, with values increasing from [Formula: see text] to [Formula: see text] respectively. The study demonstrates that linear models perform well, however the representation of epistatic interactions in predictive models improves identification of selectivity-enhancing mutations. The improvement is attributed to higher-order kernel functions that represent epistatic interactions between residues.

Simulating compatible solute biosynthesis using a metabolic flux model of the biomining acidophile, Acidithiobacillus ferrooxidans ATCC 23270.

Halotolerant, acidophilic, bioleaching microorganisms are crucial to biomining operations that utilize saline water. Compatible solutes play an important role in the adaptation of these microorganisms to saline environments. Acidithiobacillus ferrooxidans ATCC 23270, an iron- and sulfur-oxidizing acidophilic bacterium, synthesizes trehalose as its native compatible solute but is still sensitive to salinity. Recently, halotolerant bioleaching bacteria were found to use ectoine as their key compatible solute. Previously, bioleaching bacteria were recalcitrant to genetic manipulation; however, recent advancements in genetic tools and techniques allow successful genetic modification of A. ferrooxidans ATCC 23270. Therefore, this study aimed to test, in silico, the effect of native and synthetic compatible solute biosynthesis by A. ferrooxidans ATCC 23270 on its growth and metabolism. Metabolic network flux modelling was used to provide a computational framework for the prediction of metabolic fluxes during production of native and synthetic compatible solutes by A. ferrooxidans ATCC 23270, in silico. Complete pathways for trehalose biosynthesis by the bacterium are proposed and captured in the updated metabolic model including a newly discovered UDP-dependent trehalose synthesis pathway. Finally, the effect of nitrogen sources on compatible solute production was simulated and showed that using nitrogen gas as the sole nitrogen source enables the ectoine-producing 'engineered' microbe to oxidize up to 20% more ferrous iron in comparison to the native microbe that only produces trehalose. Therefore, the predictive outcomes of the model have the potential to guide the design and optimization of a halotolerant strain of A. ferrooxidans ATCC 23270 for saline bioleaching operations.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

Many pathways in laboratory evolution can lead to improved enzymes: how to escape from local minima.

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.

Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary pathways.

In a previous directed evolution study, the B-FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis. It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial; in the B-FIT method the criterion is residues characterized by highest B factors available from X-ray crystallography data. In the present study, B-FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger. Several rounds of ISM resulted in the best variant showing a 21 °C increase in the T(60)(50) value, an 80-fold improvement in half-life at 60 °C, and a 44 kcal mol(-1) improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements; these were characterized by 10-14 °C increases in T(60)(50), 20-30-fold improvement in half-lives at 60 °C and 15-20 kcal mol(-1) elevations in activation energy. Unexpectedly, in the ISM process the best variants were obtained from essentially neutral or even inferior mutant parents, that is, when a given library contains no improved mutants. This constitutes a practical way to escape from what appear to be local minima ("dead ends") in the fitness landscape-a finding of notable significance in directed evolution.

Potential of Acidithiobacillus ferrooxidans to Grow on and Bioleach Metals from Mars and Lunar Regolith Simulants under Simulated Microgravity Conditions.

The biomining microbes which extract metals from ores that have been applied in mining processes worldwide hold potential for harnessing space resources. Their cell growth and ability to extract metals from extraterrestrial minerals under microgravity environments, however, remains largely unknown. The present study used the model biomining bacterium Acidithiobacillus ferrooxidans to extract metals from lunar and Martian regolith simulants cultivated in a rotating clinostat with matched controls grown under the influence of terrestrial gravity. Analyses included assessments of final cell count, size, morphology, and soluble metal concentrations. Under Earth gravity, with the addition of Fe3+ and H2/CO2, A. ferrooxidans grew in the presence of regolith simulants to a final cell density comparable to controls without regoliths. The simulated microgravity appeared to enable cells to grow to a higher cell density in the presence of lunar regolith simulants. Clinostat cultures of A. ferrooxidans solubilised higher amounts of Si, Mn and Mg from lunar and Martian regolith simulants than abiotic controls. Electron microscopy observations revealed that microgravity stimulated the biosynthesis of intracellular nanoparticles (most likely magnetite) in anaerobically grown A. ferrooxidans cells. These results suggested that A. ferrooxidans has the potential for metal bioleaching and the production of useful nanoparticles in space.

Iterative saturation mutagenesis accelerates laboratory evolution of enzyme stereoselectivity: rigorous comparison with traditional methods.

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.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates.

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.

In a quest for engineering acidophiles for biomining applications: challenges and opportunities.

Biomining with acidophilic microorganisms has been used at commercial scale for the extraction of metals from various sulfide ores. With metal demand and energy prices on the rise and the concurrent decline in quality and availability of mineral resources, there is an increasing interest in applying biomining technology, in particular for leaching metals from low grade minerals and wastes. However, bioprocessing is often hampered by the presence of inhibitory compounds that originate from complex ores. Synthetic biology could provide tools to improve the tolerance of biomining microbes to various stress factors that are present in biomining environments, which would ultimately increase bioleaching efficiency. This paper reviews the state-of-the-art tools to genetically modify acidophilic biomining microorganisms and the limitations of these tools. The first part of this review discusses resilience pathways that can be engineered in acidophiles to enhance their robustness and tolerance in harsh environments that prevail in bioleaching. The second part of the paper reviews the efforts that have been carried out towards engineering robust microorganisms and developing metabolic modelling tools. Novel synthetic biology tools have the potential to transform the biomining industry and facilitate the extraction of value from ores and wastes that cannot be processed with existing biomining microorganisms.

Computational tools for directed evolution: a comparison of prospective and retrospective strategies.

Directed evolution methods have proved to be highly effective in the design of novel proteins and in the generation of large libraries of diverse sequences. However, searching through the vast number of mutants produced during such experiments in order to find the best represents a daunting and difficult task. In recent years, a number of computational tools have been developed to provide guidance during this exploratory process. It can, however, be unclear as to which tool or tools best complement the chosen library design strategy. In this review, we describe and critically evaluate some of the more notable tools in this area, discussing the rationale behind each, the requirements for their implementation, and potential issues faced when using them. Some examples of their application in an experimental setting are also provided. The tools have been classified based on contrasting strategies as to how they function: prospective tools SCHEMA and OPTCOMB use extant sequence and structural data to predict optimal locations for crossover sites, whereas retrospective tools ProSAR and ASRA use property data from the mutant library to predict beneficial mutations and features. From our evaluation, we suggest that each tool can play a role in the design process; however this is largely dictated by the data available and the desired experimental strategy for the project.

Increasing the stability of an enzyme toward hostile organic solvents by directed evolution based on iterative saturation mutagenesis using the B-FIT method.

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.