Engineering biosynthetic enzymes for industrial natural product synthesis.

Covering: 2000 to 2020 Natural products and their derivatives are commercially important medicines, agrochemicals, flavors, fragrances, and food ingredients. Industrial strategies to produce these structurally complex molecules encompass varied combinations of chemical synthesis, biocatalysis, and extraction from natural sources. Interest in engineering natural product biosynthesis began with the advent of genetic tools for pathway discovery. Genes and strains can now readily be synthesized, mutated, recombined, and sequenced. Enzyme engineering has succeeded commercially due to the development of genetic methods, analytical technologies, and machine learning algorithms. Today, engineered biosynthetic enzymes from organisms spanning the tree of life are used industrially to produce diverse molecules. These biocatalytic processes include single enzymatic steps, multienzyme cascades, and engineered native and heterologous microbial strains. This review will describe how biosynthetic enzymes have been engineered to enable commercial and near-commercial syntheses of natural products and their analogs.

Effect of Lymphedema Treatment for Management of Acute Pilon Fractures.

Pilon fractures are high-energy injuries that often result in considerable edema and compromise of the soft tissue envelope of the ankle. These injuries are typically staged with an external fixator until the soft tissue is amenable for definitive fixation. This study was conducted to determine the effects of lymphedema treatment for the management of pilon fractures. Patients who underwent open reduction and internal fixation of pilon fractures between 2007 and 2014 at the authors' level II trauma center were identified by Current Procedural Terminology codes indicative of placement of an external fixator (20690) and open reduction and internal fixation of a pilon fracture (27826, 27827, or 27828). The primary efficacy endpoint to determine negative outcomes was 90 days after definitive fixation. Eighty-two patients with 84 pilon fractures met inclusion criteria. Forty-eight ankles (57%) received lymphedema treatment. There were no significant differences in population demographics between the control and treatment groups. Median times to internal fixation in the control and treatment groups were 20 days (inter-quartile range, 15.5-30 days) and 11 days (interquartile range, 6-18 days), respectively. This difference was statistically significant (P=.001). Additionally, there was no significant difference in the overall incidence of wound complications between the control and treatment groups (P=.246). Compression wrapping for posttraumatic edema was effective in reducing the time needed for soft tissues to be appropriate for definitive surgical fixation of pilon fractures without increasing the risk of wound complications. These promising results warrant future study. [Orthopedics. 2017; 40(4):e668-e674.].

Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas.

Carbonic anhydrase (CA) is one of nature's fastest enzymes and can dramatically improve the economics of carbon capture under demanding environments such as coal-fired power plants. The use of CA to accelerate carbon capture is limited by the enzyme's sensitivity to the harsh process conditions. Using directed evolution, the properties of a ß-class CA from Desulfovibrio vulgaris were dramatically enhanced. Iterative rounds of library design, library generation, and high-throughput screening identified highly stable CA variants that tolerate temperatures of up to 107 °C in the presence of 4.2 M alkaline amine solvent at pH >10.0. This increase in thermostability and alkali tolerance translates to a 4,000,000-fold improvement over the natural enzyme. At pilot scale, the evolved catalyst enhanced the rate of CO2 absorption 25-fold compared with the noncatalyzed reaction.

Efficient, chemoenzymatic process for manufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2". During the discovery and development stages, the P2 fragment was produced by a classical resolution approach. As the drug candidate advanced through clinical trials and approached regulatory approval and commercialization, Codexis and Schering-Plough (now Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.

Biotechnology for the acceleration of carbon dioxide capture and sequestration.

The potential for enzymatic acceleration of carbon dioxide capture from combustion products of fossil fuels has been demonstrated. Carbonic anhydrase (CA) accelerates post combustion CO(2) capture, but available CAs are woefully inadequate for the harsh conditions employed in most of these processes. In this review, we summarize recent approaches to improve CA, and processes employing this enzyme, to maximize the benefit from this extremely fast biocatalyst. Approaches to overcoming limitations include sourcing CAs from thermophilic organisms, using protein engineering to evolve thermo-tolerant enzymes, immobilizing the enzyme for stabilization and confinement to cooler regions and process modifications that minimize the (thermo-, solvent) stress on the enzyme.

Engineered enzymes for chemical production.

In order to enable competitive manufacturing routes, most biocatalysts must be tailor-made for their processes. Enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high activity and selectivity on non-natural substrates and toleration of high concentrations of organic media over the wide range of conditions (decreasing substrate, increasing product concentrations, solvents, etc.,) that will be present over the course of a manufacturing process. With the advances in protein engineering technologies, a variety of enzyme properties can be altered simultaneously, if the appropriate screening parameters are employed. Here we discuss the process of directed evolution for the generation of commercially viable biocatalysts for the production of fine chemicals, and how novel approaches have helped to overcome some of the challenges.

Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the "peroxide shunt" pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites.

New tension band material for fixation of transverse olecranon fractures: a biomechanical study.

This study tested the use of braided polyethylene cable as an option for repairing transverse olecranon fractures. Six cadaveric elbows underwent a transverse olecranon osteotomy followed by fixation with tension band constructs using 18-gauge wire and Secure-Strand (U.S. Surgical, North Haven, Conn). Distraction forces up to 450 N were applied to the triceps tendon while measuring fracture displacement with an extensometer. The average maximal fracture gap with the standard AO tension band technique using stainless steel wire was 0.66 +/- 0.43 mm, as opposed to 0.68 +/- 0.45 mm with braided polyethylene cable. A paired t test indicated no significant difference between the two materials. These results support the feasibility of braided polyethylene cable as an alternative to the standard steel-wire tension band.