NPIF: A synthetic biology approach to engineering novel commercial variants of cyanobacterial pigment proteins

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

Cyanobacteria are an established bioplatform for the production of commercially important pigment proteins, such as phycoerythrin and C-phycocyanin (C-PC), which are used widely in the food, nutritional, analytical and pharmaceutical industries(1). Scottish Bioenergy (http://www.scotbio.com/) view C-PC as an outstanding candidate for exploitation and aims to produce high-quality C-PC at industrial scale. Scottish Bioenergy has already invested in significant R&D to improve upstream and downstream processing and to produce new cyanobacterial strains with increased C-PC yields. Structurally, C-PC consists of a covalently bonded complex of two apoproteins and phycocyanobilin chromophores, which together form the most abundant pigment protein of the light-harvesting phycobilisome complex(2). However, C-PC is both heat- and light-sensitive, which places considerable constraints on storage and usage. This project proposes to use a synthetic biology approach to engineer commercially relevant cyanobacterial strains to produce new variants of C-PC with increased thermotolerance, light resistance and improved fluorophore properties(3). Synthetic C-PC variants will reduce downstream costs and are predicted to have significant market value.

Training


This project is an outstanding training opportunity to engage with a wide variety of molecular and physiological approaches, and to be involved in commercial translation activities. You will develop expertise in cyanobacterial-based molecular skills, including DNA, RNA and protein analyses (e.g. qRT-PCR, Western blot, ion exchange chromatography), and photosynthetic physiology and fluorescence techniques. The project will employ directed evolution approaches to evolve proteins involved in C-PC biosynthesis for C-PCs with improved characteristics. Promising protein variants will be integrated into commercially relevant strains to test their performance and physiological impact. Thus, you will engineer, test and model the performance of new cyanobacteria strains expressing industrially favourable C-PC forms. There will be abundant opportunity for you to present your research at regular meetings, including national and international conferences.

Integration with Scottish Bioenergy and Alignment to the UK Industrial Strategy

The outlined project links directly with the commercial goals of Scottish Bioenergy. Novel pigment producing strains will be tested first under lab conditions, and then at pre-commercial scale with Scottish Bioenergy. The candidate will maintain close ties through regular planned monthly meetings, to discuss progress. The candidate will perform a dedicated 3-month placement at Scottish Bioenergy in year 4 (first or second quarter) of the project to test the performance of the most promising strains under pre-commercial quantities. Growth and product yields will be compared under light and temperature conditions optimised in lab-based bioreactors. The project is directly relevant to several of the pillars of the UK industrial strategy: research that will directly contribute to commercialisation of a high value product, skills development, supporting businesses to grow, and cultivating world-leading sectors (Scotland has a growing IB sector for industrial scale production of microalgae and a strong allied academic sector).

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/R505511/1 01/10/2017 30/09/2021
1948588 Studentship BB/R505511/1 01/10/2017 30/09/2021 Anton Puzorjov
 
Description 1. A novel set of synthetic biology tools for cyanobacteria (CyanoGate) has been developed and made available to the public
2. Novel commercially relevant cyanobacterial strains have been developed with the capability to produce variants of CPC with increased thermostability
3. On the 4th of March 2020 we submitted a review paper to the Journal of Experimental Botany (JXB). The review provides in-depth coverage of the light-harvesting complexes, including CPC, found in extreme environments.
Exploitation Route Novel cyanobacterial strains might be used for commercial production of novel CPC with improved thermostability.
Once published, the review paper could be used by the research community to further explore the genetic diversity provided by the organisms from extreme environments and their applicability to the industry.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Research outputs have contributed to a Proof of Concept Algae-UK grant application with my industrial partner ScotBio to develop mesophilic CPC strain for commercial use in the food, beverage and textile industries
First Year Of Impact 2019
Sector Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Title CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax 
Description By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile set of advanced DNA assembly tools called CyanoGate. We described the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact The publication has been cited 15 times within the first year following the publication. The system is publicly available through a nonprofit plasmid repository (http://www.addgene.org/browse/article/28196941/) and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community. 
URL http://www.plantphysiol.org/content/180/1/39.abstract
 
Title Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit 
Description We presented a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? Yes  
Impact The online article and the video guide have been viewed over 1,500 times within the first 4 months following the publication. 
URL https://www.jove.com/video/60451/genetic-modification-cyanobacteria-conjugation-using-cyanogate
 
Description Collaboration with the Institute for Bioengineering (Dr Katherine Dunn) 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution We initiated this collaboration and conducted all experimental work.
Collaborator Contribution The collaborator provided training and access to the state-of-the-art equipment to measure the stability of fluorescent proteins.
Impact We conducted a comprehensive analysis of thermostability of novel fluorescent pigment proteins which will contribute to a publication in a high impact journal.
Start Year 2019
 
Description Collaboration with the School of Chemistry (Prof Anita C Jones) 
Organisation University of Edinburgh
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We initiated this collaboration and conducted all experimental work.
Collaborator Contribution The collaborator provided training and access to the state-of-the-art equipment to measure the efficiency of the energy transfer in novel cyanobacterial strains.
Impact We conducted a comprehensive analysis of the energy transfer in novel cyanobacterial strains which will contribute to a publication in a high impact journal.
Start Year 2019
 
Description Industrial partnership with ScotBio 
Organisation ScotBio
Country United Kingdom 
Sector Private 
PI Contribution The knowledge, expertise and equipment available in our lab contribute to the development and testing of new products for the industrial partner.
Collaborator Contribution The partner provided us with the growth equipment and expertise to set up an upscale production in our lab.
Impact I have created a superior version of a blue pigment-protein (phycocyanin) with a significantly improved thermostability. This has led to a Proof of Concept Algae-UK grant application as well as to an MSc student project. It has also helped to increase engagement with the new industries, such as the textile industry, which has not been considered previously.
Start Year 2017
 
Description Bioinformatics Workshop at Eastwood High School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We conducted a workshop on bioinformatics for 15 final year pupils at Eastwood High School. The pupils learnt how to query biological sequence databases by performing BLAST searches. They used the BLAST computer program on the National Center for Biotechnology Information's Web server. They also learnt how to do a BLAST search by using the LINUX command line. They then learnt how to interpret the biological implications of the results, with particular reference to mutations and function. Key Concepts Bioinformatics - Sequence databases - Fasta format - BLAST - DNA sequences - Protein sequences - Substitution, insertion and deletion mutations - Pseudogenes - GUI - Command line - Scientific investigative approaches. The students reported improved understanding of the topic and increased interest towards studying STEM subjects.
Year(s) Of Engagement Activity 2018
 
Description PCR workshops at the National Museum of Scotland 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Hands-on biotechnology workshops delivered for 40 senior school pupils. 'A Question of Taste', enables pupils to carry out PCR (polymerase chain reaction). PCR, an important technique in biotechnology, is part of the curriculum but pupils rarely have the opportunity to carry it out themselves. Pupils are studying Higher or Advanced Higher Biology (aged 15 - 17). Pupils discovered and compared their version of a bitter taste receptor gene (their genotype) to their ability to taste the bitter compound (their phenotype). They carried out the laboratory techniques of DNA extraction, PCR, restriction enzyme digestion and gel electrophoresis. At the end of the workshop, pupils reported improved understanding of the topic (PCR) and increased interest in studying STEM subjects.
Year(s) Of Engagement Activity 2019