14TSB_SynBio A toolchest for rapid bootstrapping of novel chassis organisms
Lead Research Organisation:
University College London
Department Name: Biochemical Engineering
Abstract
New organisms for the expression of recombinant genes are needed. Organisms that have a greater resistance to the
conditions in biocatalysis for the synthesis of pharmaceutical compounds are needed. We want to move from a
petrochemical based chemical and pharmaceutical synthesis to a more bio-based economy and to do this we need much
better and more rapid tools for building biochemical and metabolic pathways in a new organism (chassis).
The proposed project will develop rapid ways of getting high level expression in any new chassis organism. The various
steps of enzyme expression will be dissected using our new tools and knowledge of how genes are expressed. This will
lead to modular parts for the expression of biocatalysis genes and pathways in any new chassis. We will test this
development pathway in some selected chassis that are examples of novel organisms more fitted to the bioprocess
conditions for the bio-synthesis of pharmaceutical compounds.
conditions in biocatalysis for the synthesis of pharmaceutical compounds are needed. We want to move from a
petrochemical based chemical and pharmaceutical synthesis to a more bio-based economy and to do this we need much
better and more rapid tools for building biochemical and metabolic pathways in a new organism (chassis).
The proposed project will develop rapid ways of getting high level expression in any new chassis organism. The various
steps of enzyme expression will be dissected using our new tools and knowledge of how genes are expressed. This will
lead to modular parts for the expression of biocatalysis genes and pathways in any new chassis. We will test this
development pathway in some selected chassis that are examples of novel organisms more fitted to the bioprocess
conditions for the bio-synthesis of pharmaceutical compounds.
Technical Summary
We will develop rapid tools, techniques and materials to enable the construction of novel expression systems in a new
chassis. The chassis organisms will be chosen from several Gram positive and Gram negative bacteria whose characteristics make them more suited to industrial bioprocess conditions than E. coli.
Promoters from highly expressed genes will be searched for using bioinformatics tools developed by Synthase. These promoters will be synthesised, tested in the new chassis with a reporter gene and modularised for later use in building expression constructs.
Ribosome binding sites will be similarly designed synthesised, tested and modularised.
Genes to be expressed will be chosen with regard to the chassis. For example enzymes for biocatalysis and for building pathways for chiral chemical synthesis will be designed for a chassis that can tolerate organic solvents or high concentrations of the substrate. Genes will be codon optimised for the new host and the new expression systems will be subjected to multifactorial experimental design to iterate between testing, design and implementation to deliver robust expression systems that can be rapidly built for a new chassis.
chassis. The chassis organisms will be chosen from several Gram positive and Gram negative bacteria whose characteristics make them more suited to industrial bioprocess conditions than E. coli.
Promoters from highly expressed genes will be searched for using bioinformatics tools developed by Synthase. These promoters will be synthesised, tested in the new chassis with a reporter gene and modularised for later use in building expression constructs.
Ribosome binding sites will be similarly designed synthesised, tested and modularised.
Genes to be expressed will be chosen with regard to the chassis. For example enzymes for biocatalysis and for building pathways for chiral chemical synthesis will be designed for a chassis that can tolerate organic solvents or high concentrations of the substrate. Genes will be codon optimised for the new host and the new expression systems will be subjected to multifactorial experimental design to iterate between testing, design and implementation to deliver robust expression systems that can be rapidly built for a new chassis.
Planned Impact
The impact of this work will be in the growing bio-economy whjere we want to decrease our reliance on petrochemical based feedstocks for the production of all chemicals, pharmaceuticals and polymers. If we want to increase the use of biologically derived materials both as inputs and for the generation of materials by biocatalysis and through new synthetic metabolic pathways, then these new tools will aid these goals.
The UK fine chemical and pharmaceutical industry would benefit by having an increased number of tools and methods for it to use for the bio-synthesis of compunds.
The UK economy would benefit by keeping the manufacture and research in these areas, within the UK and also to keep in a leading position in the new technologies in synthetic biology.
The UK fine chemical and pharmaceutical industry would benefit by having an increased number of tools and methods for it to use for the bio-synthesis of compunds.
The UK economy would benefit by keeping the manufacture and research in these areas, within the UK and also to keep in a leading position in the new technologies in synthetic biology.
People |
ORCID iD |
| John Ward (Principal Investigator) |
Publications
Grant C
(2014)
Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.
in Scientific reports
| Description | New promoters and translation initiation signals have been developed for the expression of genes in Pseudomonas putida and Pichia. |
| Exploitation Route | Companies and researchers could use the new expression sinals in a modular way to express genes for synthetic biology and industrial biotechnology in new chassis. |
| Sectors | Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
| URL | http://www.ucl.ac.uk/biochemeng/people/academic/ward-j |
| Description | Innovate UK Health and Life Sciences Round 1 |
| Amount | £585,195 (GBP) |
| Funding ID | 84474-536277 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 05/2017 |
| End | 04/2019 |
| Title | New promoters for expression in Pseudomonas putida and other pseudomonas species. |
| Description | The primary goals were to assess what was required to engineer non model organisms, including mechanisms for generating critical genetic parts such as promoters. The project contributed significantly to gaining knowledge about these issues, and has generated large libraries of novel promoters for Pseudomonas putida and Pichia pastoris. Most of Synthace's work prior to this project was in the model system Escherichia coli, where the company has considerable capabilities. However, it had become apparent through our engagement with multiple industry sectors that this capability would be insufficient for addressing a large number of opportunities in pharmaceuticals, agrochemicals and industrial biotechnology sectors. The project generated novel tools and protocols for the engineering of Pseudomonas putida and Pichia pastoris. UCL and Synthace collaborated to produce optimised Pseudomonas protocols, and a set of novel Pseudomonas promoters were identified and comprehensively characterised for their function under a wide range of conditions. Synpromics contributed to part of the project with the goal of applying it's synthetic promoter technology to develop a panel of auto-inducible gene promoters for Pichia pastoris that could be used for production of high value compounds and proteins. Several novel promoters with activities that were stimulated at different points in the growth cycle of P. putida were discovered. The project, therefore, met all the deliverables and milestones that were set out in the second level plan. Additionally a strategy to facilitate commercial exploitation of the project output has been facilitated. We have tested a fundamental method for identifying promoters in non model prokaryotes. This will have great potential application as the field moves into increasing numbers of organisms. |
| Type Of Material | Biological samples |
| Year Produced | 2015 |
| Provided To Others? | Yes |
| Impact | This enabled Synthace to generate more investor funding. The project gave the more general benefits of giving both companies experience with a more broad array of organisms. In Synthace's case, this increased credibility directly contributed to signing a major deal with Dow Agrosciences to develop automated strain engineering methods. In addition, this project shows the applicability of the Synpromics's synthetic promoter technology to the yeast Pichia pastoris. Previous projects showed the development of synthetic promoters in plants and mammalian cells. Expression systems using Pichia pastoris are of great commercial interest thus having the capability to construct and identify gene promoters for this organism is of high value for the technology portfolio of the company. The consortium has already started actively exploiting the results from this project through new technology offerings, which have already contributed to new deals and negotiations with target companies. In addition a publication is in early stages, which we intend to use to publicise some of the advances in the project. There is some technical work that The number of synthetic promoters identified exceeded expectation with an discovery rate far greater than observed in previous projects. Thus the follow on work could include the validation of gene promoter expression strength and inducibility of a great number of synthetic promoters for Pichia pastoris bio-production. We have incorporated the designs and concepts discovered in this research to our work in other areas e.g. for the expression of genes in Schizosaccharomyces pombe and for developing synthetic operons for expression of cytochrome P450 genes with Hypha Discovery and for work with a consortium of universities for expression in continuous culture of E. coli. In summary, we anticipate that this could be the beginning of substantial new areas of business for both company participants, so will be subject to substantial further development in other species to make the most of this opportunity |
| Description | An EngD project between UCL and Synthace. |
| Organisation | Synthace |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | I hosted an EngD student in my lab. Dr Aisha Asra carried out an EngD on Novel Chassis for engineered pathways. As metabolic engineering becomes more complex and more diverse enzymes are utilised in biocatalytic and biosynthetic processes; host organisms for these reactions must be developed that are robust under the conditions required for the reactions or process of interest. My project will elucidate new expression systems in alternative host organisms to E. coli. The project was to develop molecular biology tools for the expression of engineered metabolic pathways in Pseudomonas putida and investigate the usefulness of P. putida as an alternative host to E. coli for engineered biosynthetic pathways for the production of novel molecules that may be of industrial interest. The outcomes: • Finding strong promoters and genetic elements for the best expression of multi enzyme pathways in P. putida. • Cloning and transforming DNA into P. putida. • Producing engineered pathways that produce novel and interesting molecules that may have relevance to industry. 2-hydroxymuconic semialdehyde (2-HMSA) is produced from a truncate of the TOL metacleavage pathway which I have produced in E. coli. I have screened several of the transaminases from the UCL library for uptake of 2-HMSA as a substrate; results have been successful. The Kpn_00799 transaminase in particular appears to have excellent activity with 2-HMSA. A truncated segment of the TOL pathway was cloned into a broad host vector for the expression of this pathway in Pseudomonas putida using the ideas developed in the TSB project. |
| Collaborator Contribution | Synthace hosted Aisha for several months to develop cloning for Pseudomonas and Streptomyces for the expression of proteins and enzymes at the Synthace facilities. |
| Impact | Outputs and outcomes are that Aisha has presented her work orally and by posters at 3 conferences. The data generated is being used in other PhD and research programmes. |
| Start Year | 2013 |