Synthetic Biology and Directed Evolution of Enzymes

Lead Research Organisation: Imperial College London
Department Name: Life Sciences

Abstract

A considerable amount of work has been applied in finding new enzymes or modifying them to improve some of their useful properties and overcoming the limitations of natural biocatalysis and biotransformation processes. So far, one of the most successful methods to accomplish this is Directed Evolution. This approach includes genetic modification and rational protein engineering to modify enzymes at will, making them more effective in carrying out a process of interest. This is a powerful tool that is usually combined with screening techniques to select the variants of interest out of a large library of mutants. However, to date, screenings are usually laborious, time-consuming, and require intensive work. This PhD proposal will present three different methods for Directed Evolution, which will speed up the screening and selection steps.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/N503873/1 05/10/2015 04/10/2019
1658622 Studentship BB/N503873/1 03/10/2015 30/09/2019
 
Description During my PhD I developed a novel selection system for directed evolution approaches that can be easily applied to a wide range of biomolecules and properties. Such a system bypasses the limitations of most current selection methods, which can only be applied to a relatively narrow range of biomolecules or selected properties, and therefore helps to overcome the main bottleneck of directed evolution: the identification of the variants of interest with the desired enhanced or novel properties.

The selection system is based on an engineered mutant E. coli strain with metabolic defects that cause impaired growth under anaerobic conditions. The metabolic defect can be complemented by adding a given substrate to the culture medium and transforming mutant cells with a biomolecule or a combination of biomolecules whose activity leads to a biotransformation of the substrate added to the culture medium. The system therefore allows to select, for example, novel enzyme variants with improved properties or able to act on novel substrates at will by simply creating a library of a parental enzyme, transforming mutant cells with the library, and growing them anaerobically in the presence of the substrate towards which activity is desired.

The large potential of the selection system was demonstrated by using it to select variants of industrial or pharmaceutical interest of a variety of different enzymes with different novel properties, including relaxed or reversed cofactor specificity, improved kinetic parameters (such as higher kcat, improved substrate affinity or reduced substrate inhibition) and altered substrate specificity.

Firstly, I obtained mutant variants of NADP-dependent alcohol dehydrogenases with reversed cofactor specificity and improved kinetic parameters. The variants contained different types of mutations in the Rossmann fold, which I characterized structurally by means of X-ray crystallography to better understand the molecular basis for the alteration of cofactor preference caused by modifications of the Rossmann fold.

I then obtained variants of another type of enzymes, imine reductases, of high industrial interest thanks to their ability to synthesize chiral amines either by hydrogenation of the corresponding pro-chiral imine or by performing a reductive amination of the corresponding ketone. The selected variants had the ability to use NAD instead of NADP as the cofactor (unlike all known natural imine reductases) and displayed a relief in the substrate inhibition compared with other previously engineered variants.

Finally, I engineered variants of Enterobacter cloacae nitroreductase, one of the potential candidates to be employed for cancer chemotherapy in combination with nitroaromatic prodrugs. I developed two types of variants: a variant with improved kinetic parameters towards substrates already accepted by the native enzyme, and a variant able to act on substrates not accepted by the native enzyme.

I also demonstrated the suitability of the selection system to be applied to other types of biomolecules and more complex systems such as entire metabolic pathways by using it to select optimized variants of an isopropanol-production pathway leading to a maximal isopropanol titer amongst a combinatorial library of variants differing in the regulatory elements controlling the expression of each gene. We have also devised schemes that would allow for selection of many other types of biomolecules (not necessarily proteins), including transporters, binding partners with specific properties, enzymes acting on DNA or RNA and nucleic acids with specific catalytic or binding properties.
Exploitation Route The selection system is ready to be applied to a wide variety of cases. The procedure is described in the patent application already published, and will also be presented in a paper currently in preparation.

Commercial uses should first be agreed and licensed. Our already published patent application provides the grounds for doing so. As previously stated, we are indeed currently undergoing negotiations to license industrial partners to apply the selection method and to use the enzyme variants we have already selected with it.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Transport
 
Description Biochemical Society General Travel Grant
Amount £400 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2018 
End 06/2018
 
Description Metabolic Engineering 12 Junior Researcher Grant
Amount $400 (USD)
Organisation American Institute of Chemical Engineers 
Sector Charity/Non Profit
Country United States
Start 06/2018 
End 06/2018
 
Title A broadly applicable artificial selection system for biomolecule evolution 
Description The developed selection system for directed evolution approaches can be easily applied to a wide range of biomolecules and properties. Such a system bypasses the limitations of most current selection methods, which can only be applied to a relatively narrow range of biomolecules or selected properties, and therefore helps to overcome the main bottleneck of directed evolution: the identification of the variants of interest with the desired enhanced or novel properties. I will aim to give a brief overview of my doctoral research. The selection system is based on an engineered mutant E. coli strain with metabolic defects that cause impaired growth under anaerobic conditions. The metabolic defect can be complemented by adding a given substrate to the culture medium and transforming mutant cells with a biomolecule or a combination of biomolecules whose activity leads to a biotransformation of the substrate added to the culture medium. The system therefore allows to select, for example, novel enzyme variants with improved properties or able to act on novel substrates at will by simply creating a library of a parental enzyme, transforming mutant cells with the library, and growing them anaerobically in the presence of the substrate towards which activity is desired. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact The large potential of the selection system was demonstrated by using it to select variants of industrial or pharmaceutical interest of a variety of different enzymes with different novel properties, including relaxed or reversed cofactor specificity, improved kinetic parameters (such as higher kcat, improved substrate affinity or reduced substrate inhibition) and altered substrate specificity. Firstly, I obtained mutant variants of NADP-dependent alcohol dehydrogenases with reversed cofactor specificity and improved kinetic parameters. I then obtained variants of another type of enzymes, imine reductases, of high industrial interest thanks to their ability to synthesize chiral amines either by hydrogenation of the corresponding pro-chiral imine or by performing a reductive amination of the corresponding ketone. The selected variants had the ability to use NAD instead of NADP as the cofactor (unlike all known natural imine reductases) and displayed a relief in the substrate inhibition compared with other previously engineered variants. Finally, I engineered variants of Enterobacter cloacae nitroreductase, one of the potential candidates to be employed for cancer chemotherapy in combination with nitroaromatic prodrugs. I developed two types of variants: a variant with improved kinetic parameters towards substrates already accepted by the native enzyme, and a variant able to act on substrates not accepted by the native enzyme. I also demonstrated the suitability of the selection system to be applied to other types of biomolecules and more complex systems such as entire metabolic pathways by using it to select optimized variants of an isopropanol-production pathway leading to a maximal isopropanol titer amongst a combinatorial library of variants differing in the regulatory elements controlling the expression of each gene. We have also devised schemes that would allow for selection of many other types of biomolecules (not necessarily proteins), including transporters, binding partners with specific properties, enzymes acting on DNA or RNA and nucleic acids with specific catalytic or binding properties. 
URL https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019243821&tab=PCTBIBLIO&_cid=P11-K7NGK5-8...
 
Title METHOD OF SELECTING A POLYPEPTIDE OF INTEREST 
Description The invention relates to methods for identifying polypeptides and polynucleotides of interest, be they novel or variant polypeptides and polynucleotides, by expressing a plurality of polypeptides in an obligate or facultative anaerobe that is incapable of, or displays a reduction in, the oxidation of NADH and/or NADPH under anaerobic fermentation conditions and selecting an obligate or facultative anaerobe that grows or displays a growth advantage under said conditions. The invention is also concerned with novel enzymes per se, and their use in enzymatic production processes. 
IP Reference WO2019243821 
Protection Patent application published
Year Protection Granted 2019
Licensed No
Impact The intellectual property corresponding to the newly developed selection method is now protected. Additionally, another patent application concerning specific enzyme variants developed with the selection method has been filed. We are currently undergoing talks to license the usage of the selection method to discover new enzymes, as well as the usage of some of the already selected enzyme variants, with industrial partners (commercial in confidence at the moment).