Novel monooxygenase biocatalysts from the environment and the laboratory
Lead Research Organisation:
University of Warwick
Department Name: Biological Sciences
Abstract
Methane oxidising bacteria are very important in the environment since they are a key link in the global biogeochemical methane cycle and oxidise methane in many environments such as wetlands, paddy field soils and landfills before this methane is released into the environment. They thereby mitigating the effects of this potent greenhouse gas and reduce global warming. Methane monooxygenase (MMO) is a bacterial enzyme that catalyses the first step in methane oxidation by bacteria. It is of great interest to chemists because it oxidises methane to methanol at ambient temperatures and pressures, a reaction normally requiring high temperatures and pressures and expensive catalysts. MMO is very unusual in that it will also oxidise very many other alkanes, alkenes and aromatic compounds and their substituted derivatives and therefore it has great potential for use as a biocatalyst in biotransformations and bioremediation in 'green chemistry' reactions that are less polluting than traditional chemical routes. The structure of MMO has been the subject of considerable interest for biologists because of this broad substrate specificity and one aim has been to try to understand how the structure of the enzyme allows the catalysis of such a wide range of compounds and how changing its structure by mutagenesis, forced evolution or construction of mutant and hybrid enzymes will alter its catalytic utility. This is an ambitious grant proposal from world experts in the molecular biology and biochemistry of MMO. We propose to construct key mutants in the active site of MMO and to examine the effects on catalysis of key substrates and to manipulate this enzyme in order to be able to define the pathway of entry of substrates into the active site and to generate novel recombinant enzymes which are able to oxidise new substrates. We also aim to define how the different components of the enzyme interact with each other and how the mechanisms of substrate entry and electron transfer pathways to the site of oxidation in MMO are carried out. In a novel approach, we also wish to carry out 'gene mining' from the environment to capture DNA sequences that encode MMO or related di-iron centre monooxygenases in order to be able to construct new and exciting biocatalysts. This will involve the use of a technique called DNA-Stable Isotope Probing (DNA-SIP) which we originally developed in order to be able to define the population structure of active methane oxidising bacteria in the environment. This involves feeding 13C-substrates such as methane to bacteria contained within environmental samples such as soils. Only the active methanotrophs with MMO will be labelled with this heavy stable isotope. We can then isolate the heavy DNA (containing the whole genomes of methanotrophs and related bacteria) encoding MMO and its relatives from all of the DNA from the thousands of non-methanotrophic bacteria present in soil by density gradient centrifugation. By use of the polymerase chain reaction (PCR), we can then isolate novel MMO sequences from previously uncultivated bacteria which can subsequently be stitched into plasmids that we have developed which allow us to recreate novel MMOs with unusual biocatalytic properties. Analysis of these recombinant enzymes will shed light on the mechanism of action of MMO and also generate new and novel biocatalysts with potential for use in industry in non-polluting biotransformation reactions.
Technical Summary
This is an ambitious grant proposal to study the structure and function of the soluble, methane monooxygenase enzyme (sMMO) from methanotrophs. sMMO is an extremely versatile biocatalyst. Its biological function is to catalyse the first step in methane oxidation, methane to methanol. However, this enzyme is remarkable in that it also co-oxidises over 150 alkanes, alkenes, aromatic compounds and their substituted derivatives, making it an extremely versatile biocatalyst with huge industrial potential for green chemistry reactions in biotransformation and bioremediation. This catalytic versatility is of great interest to us and we seek to define the structure of the active site and to elucidate how substrates enter the active site, define the interactions of subunits that enable this wide substrate range and investigate how hydrogen tunnelling occurs and how intermediates in the oxidation of methane are formed. This will be achieved by the construction and analysis of a wide range of sMMO mutants using a homologous expression system that we developed which enables us to generate large quantities of highly active sMMO proteins. We will couple this with a unique approach involving DNA-Stable Isotope probing, pioneered in our lab for the analysis of active methanotroph populations in the environment. This gives immediate access to MMO enzymes and related di-iron centre monooxygenases present in the uncultivated majority of methanotrophs and related organisms in the environment. This form of gene mining gives us access to unique MMO gene sequences and their homologs which can then be amplified by PCR and cloned into our sMMO expression vectors, thereby generating new and novel biocatalysts based on sMMO which may have huge potential in biotransformation and bioremediation reactions. Analysis of these recombinant sMMOs, which may have considerable industrial potential, will also reveal important features which define the catalytic utility of this fascinating enzyme.
Organisations
People |
ORCID iD |
John Murrell (Principal Investigator) |
Publications
Flickinger, Michael C.
(2010)
Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology
Grob C
(2015)
Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph.
in Environmental microbiology
Khmelenina V
(2018)
Aerobic Utilization of Hydrocarbons, Oils and Lipids
Lock M
(2017)
Mutagenesis and expression of methane monooxygenase to alter regioselectivity with aromatic substrates.
in FEMS microbiology letters
Moussard H
(2010)
Stable Isotope Probing and Related Technologies
Murrell J
(2010)
Handbook of Hydrocarbon and Lipid Microbiology
Murrell JC
(2009)
Microbial biotechnology meets environmental microbiology.
in Microbial biotechnology
Murrell, W. G.; Whiteley, Andrew S.
(2010)
Stable Isotopes in Microbial Molecular Ecology
Nichol T
(2015)
Controlling the Activities of the Diiron Centre in Bacterial Monooxygenases: Lessons from Mutagenesis and Biodiversity
in European Journal of Inorganic Chemistry
Sigdel S
(2015)
Molecular dynamics simulation to rationalize regioselective hydroxylation of aromatic substrates by soluble methane monooxygenase.
in Bioorganic & medicinal chemistry letters
Description | Chinese Academy of Science Award Lecture to general public |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Fighting climate change with microbiology. Chinese Academy of Sciences Distinguished Professorship Award Lecture, Nanjing, China, September, 2015 |
Year(s) Of Engagement Activity | 2015 |
Description | EMBO Conference presentation |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Biogeochemical cycling of atmospheric trace gases. EMBO, Heidelberg, Germany, October 2015 |
Year(s) Of Engagement Activity | 2015 |
Description | Industrial engagement National Biotechnology Institute Montreal Canada |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Research presentation Methanotrophs and methane monooxygenases. NRC-BRI Montreal, April 2015 |
Year(s) Of Engagement Activity | 2015 |
Description | Research presentations at national and international conferences |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | At least 10 research presentations at National and International scientific meetings, workshop and end-user engagement activities, including China, USA, Australia, Germany. Audiences included researchers, postgraduate students, industrialists, policy makers, government organizations |
Year(s) Of Engagement Activity | 2015,2016 |
Description | School Visit Stalham Norfolk |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Fighting climate change with microbiology Talk and discussion. approx. 100 pupils attended. Highly engaged in lecture and debate |
Year(s) Of Engagement Activity | 2016 |
Description | School visit |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Stimulated interest in environmental microbiology Increased applications to UEA |
Year(s) Of Engagement Activity | 2014 |
Description | School visit Ipswich |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | Over 100 sixth form students attended my lecture on Fighting climate change with microbiology |
Year(s) Of Engagement Activity | 2016 |
Description | University Open Days and Summer Schools |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Presentations on research at Open Days, School Visits, Summer Schools (approximately three per year) |
Year(s) Of Engagement Activity | 2015,2016 |