Synthetic Biochemistry Industry Challenges
Problem 1: Biofilms as ‘Colony Factories’
The idea of cell factories for the construction of small molecules is a well established concept and it starting to gain traction as a commercial proposition (egArtemisinin). Cell factories represent a highly sustainable approach to the future manufacture of many chemicals (including biofuels and especially pharmaceutical small molecules).
Much of the current global interest in biofilms has focused on ways to control them – understandable as globally they are believed to cause tens of billions annually in damages and are highly persistent and difficult to eliminate. However, we are interested not in their control but in extending the concept of cell factories to one of colony factories where a biochemical pathway could be established in bacteria that form biofilms or even be effectively divided across two or more different microbes that form parts of a single biofilm.
Exemplification of the proposal
Ideally a project around biofilms would have two parts:
- Demonstration that a desired biochemical (synthetic biology designed) pathway can be engineered into a biofilm (eg a short artificial pathway that demonstrates a functional group interconversion that would normally require several discrete chemical steps to perform would be highly desirable).
- Using the innate ability of a biofilm to effectively fix cells to surfaces and the robustness generally exhibited by the biofilm should allow it to grow on beads, pipes or mixers within pipes to enable chemical transformations to occur within impacting function of the biofilm.
Expected output of the research
A short cascade of biochemical reactions would be designed to enable a functional group interversion of an easily-analysed model compound using a biofilm ‘colony factory’ approach and the biofilm would be demonstrated to remain viable during the biotransformation process.
Problem 2: Enzymatic Halogenation
Halogenated aromatic compounds are fundamental building blocks in chemical synthesis and are also found in numerous pharmaceuticals and agrochemicals. In a pharmaceutical context, halogen substitution can drastically impact the pharmacology of organic molecules while in a synthetic context, halogenated compounds are frequently employed as key intermediates for cross-coupling reactions.
Chemical halogenation often requires the employment of activated starting materials and long, multistep reactions schemes. Precedence exists for enzymatic halogenation, but the limited substrate scope as well as problematic cofactor requirements have hindered their adoption in biocatalysis. We are interested in the exploration and expansion of the substrate space of this class of enzymes as well as in the demonstration of successful enzymatic halogations for pharmaceutically relevant compounds.
Exemplification of the proposal
Ideally, a project examining enzymatic halogenation would:
- Seek to identify and characterize new halogenases. Characterization should include an explicit description and characterization of substrate scope, cofactor regeneration systems, and activity profiles.
- Demonstrate improved or expanded halogenation activity (perhaps accomplished through directed evolution) on a range of pharmaceutically-relevant compounds.
Expected output of the research
The identification and directed evolution of halogenases for the regioselectivehalogenation of chemically useful small molecules or larger, biologically active molecules.
- D. R. M. Smith, S. Gruschow, R. J. M. Goss. Curr. Opin. Chem. Biol.2013, 17, 276-283.
- K. H. Van Pee. Methods Enzymol. 2012, 516, 237-257.
- J. T. Payne, C.B. Poor, J. C. Lewis Angew. Chem. Int. Ed. 2015, 54, 4226-4230.
Problem 3: Enzymatic Phosphorylation
The generation of phosphate esters from the corresponding alcohols is a reaction of interest due to the presence of these functional groups in numerous biologically active molecules. Phosphorylation by chemical methods is well precedented, but tends to involve laborious protecting group strategies as well as toxic reagents that can potentially lead to unwanted byproducts that are difficult to remove. On the biological side, examples of enzymatic phosphorylation by ATP-dependent kinases are numerous and generally well-characterized mechanistically. However, the dependence of members of this enzyme class on ATP as a stoichiometric phosphate donor, their inhibition by phosphorylated compounds, and their generally limited sustrate scope, make them a less tractable option for the generation of phosphorylated molecules.
There have been some documented examples of the enzymatic phosphorylation of sugars and other alcohol-containing small molecules that use pyrophosphate as a phosphate source. These examples are typically phosphatase (phosphate-hydrolyzing) enzymes that are able to catalyze phosphate transfer while suppressing this hydrolysis activity. Substrate scope, stereoselectivity, and regioselectivity are just some of the challenges that remain to be solved in this emerging class of enzymes. We are interested in the development of new biocatalytic methods for the introduction of phosphate esters to offer new chemical routes for the generation of phosphate-containing compounds.
Exemplification of the proposal
Ideally, a project examining enzymatic phosphorylation will:
- Identify enzymes capable of phosphorylating a wide-range of alcohol substrates. Characterization of this substrate scope as well as the mechanistic basis for any regio- and stereoselectivity should be included.
- Involve a non-ATP source of phosphate or address the obvious scaleability issues that arise from this phosphate source.
Expected output of the research
The identification of phosphotransferases capable of generating phosphate esters from alcohols with varying regio-, chemo- and stereoselectivity.
- T. van Herk, A. F. Hartog, A. M. van der Burg, R. Wever. Adv. Synth. Catal.2005, 347, 1155-1162.
- Y. Mihara, T. Utagawa, H. Yamada, Y. Asano. Appl. Environ. Microbiol.2000,66, 2811-2816
Problem 4: Efficient Enzymatic Alcohol Oxidations
The oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones is an underrepresented reaction class in the manufacture of pharmaceutical APIs. Chemical oxidations typically involve toxic byproducts and require elevated temperatures and pressures, making scale-up challenging from a number of vantage points. The ability to carry out alcohol oxidations in a safe, scalable, and more environmentally benign fashion would offer a number of useful synthetic opportunities in drug manufacture.
The development of biocatalytic systems for the oxidation of alcohols has previously been demonstrated. Due to their high regio- and stereoselectivity as well as their ability to function under relatively mild conditions, these enzymatic systems in theory can help overcome a number of the challenges associated with chemical oxidations. Nonetheless, there are a number of challenges associated with biocatalytic oxidations that include a limited substrate scope and their requirement for robust and process-ready cofactor recycling systems to make them economically viable. We are interested in the development of biocatalytic alcohol oxidation systems that will be able to function on a wide variety of small molecule substrates and that employ cofactor regeneration systems that make these processes economically viable at scale.
Exemplification of the proposal
Ideally, a project examining enzymatic alcohol oxidation systems would seek to:
- Identify enzymes (dehydrogenases, oxidases, ketoreductases) that are able to catalyze the oxidation of a wide variety of primary and secondary alcohols to aldehydes and ketones, respectively.
- Couple these oxidative enzymes with an appropriately developed cofactor recycling systems that function robustly under multiple process-like conditions.
- Demonstrate scaleability of the enzymatic process.
Expected output of the research
A series of enzymatic alcohol oxidation systems was generated and characterized that showed robust activity on a panel of diverse alcohol scaffolds.
- J. Brummund, T. Sonke, M. Muller, Org. Process Res. Dev.2014, in press.
Problem 5: Towards the Discovery of New Nitroreductases
Nitroaromatic compounds represent a valuable source of amines, which are used as intermediates in the synthesis of a wide range of chiral chemicals and pharmaceuticals. Yet, chemical procedures for preparing amines from nitro derivatives are not environmentally or energetically friendly. High temperature and pressure are usually required, with hydrogen chloride and tin being used as catalysts in ethanol.[1] Recently, less-harsh approaches have been employed such as platinum oxide and aqueous ferric chloride,[2] as well as Pd on charcoal,[3] sodium hydrosulfite[4] or Samarium.[5] However, most of these methods still rely on metals that can be toxic and/or expensive. Biotransformations leading to aromatic amines represent a milder and more environmentally friendly approach, compared to traditional chemical synthesis. Moreover, biocatalysis becomes even more valuable when substrates bear other sensitive functionalities.
Exemplification of the proposal
In many bacteria (and to a lesser extent in eukaryotes), nitroreductases act as detoxification enzymes on a broad range of nitroaromatic compounds to deliver the corresponding amines, thus finding applications as biosensors in bioremediation and biocatalysis, but also in health since these enzymes are used to activate prodrugs in chemotherapeutic tumor treatments.[6] So far, the use of nitroreductases in organic synthesis has been limited to the use of Salmonella typhimurium for the synthesis of unsaturated carbonyl compounds, nitroalkenes, and some nitroaromatic substrates.[7] However, the efficiency is suboptimal owing to the nonenzymatic side reactions in which nitrozo and hydroxylamine intermediates are involved. In addition, significant amounts of azoxy aromatic intermediates are usually formed, a fact that explains the low yield of amine obtained.7 More recently reports have emerged of the engineering of ene-reductases towards nitroreductase functionality.[8] Reaction engineering is also feasible to alter the product ratio but reaction stalling at the hydroxylamine stage remains an issue (A. Bommarius, unpublished results)
Two general types of nitroreductases can be distinguished, according to their oxygen sensitivity. TypeI nitroreductases are oxygen-insensitive, while type II are oxygen sensitive, acting via different electron transfer mechanisms. In E. coli there are mainly two nitroreductases, which are responsible for the reduction of nitroaromatic compounds: NfsA[9] (major) and NfsB[10] (minor), so-called after their respective molecular weights of 27 and 24 kDa, respectively.[11]
Expected output of the research
Enzymatic nitroreduction would provide a green improvement to current chemical reduction methods. In order to be a practical approach, complete conversion to the aniline/amine needs to be achieved and substrate scope needs to be expanded. Proposals to address either full reduction or enhancing substrate scope or both are sought.
[1]J. D.Riedel,Patent: DE408665.
[2]ICI Americas Inc.Patent: US5095038A1, 1992.
[3]Takeda Chemical Industries, Ltd.Patent: US6235789B1, 2001.
[4] C. T. Redemann, C. E. RedemannOrg. Synth.1955, 3, 69.
[5]M. K. Basu, F. F. Becker, B. K. Banik, Tetrahedron Lett. 2000, 41, 5603-5606.
[6] See: a) K. Durchschein, M. Hall, K. Faber, Green Chem.2013, 15, 1764-1772; b) M. Kulkarni, A. Chaudhari, J. Environ. Manage.2007, 85, 496-512; c) Nitroreductases: enzymes with environmental, biotechnological and clinical importance in Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology, Ed A. Mendez-Vilas, Vol 2, 2010, 1008-1019.
[7]Y. Yanto, M. Hall, A. S. Bommarius, Org. Biomol. Chem. 2010, 8, 1826-1832.
[8]J.T. Park, L. M. Gomez Ramos, A. S. Bommarius, ChemBioChem2015, 16, 811-818.
[9]S. Zenno, H. Koike, A. N. Kumar, R. Jayaraman, M. Tanokura, K. Saigo, J. Bacteriol. 1996,178, 4508-4514.
[10]S. Zenno, H. Koike, M. Tanokura, K. Saigo, J. Biochem. 1996, 120, 736-744.
[11]J. Rau, A. Stolz, Appl. Environ. Microbiol.2003, 69, 3448-3455.