Round 1


ALGEBRA: ALGal Environmental and Biotechnological Risk Assessment

Mike Allen (Plymouth Marine Laboratory)


The naturally high metabolic diversity of microalgae, coupled with high growth rates and ability to grow in seawater (and in industrial waste water streams or grey waters), removes a significant reliance on the world’s increasingly valuable freshwater supplies. The industrial biotechnology sector has been quick to respond to such opportunities and recent years have seen a rise in interest in commercial production of microalgal biomass for its associated metabolic products and services. Whilst many key species have become successfully established as suitable for mass culture (predominantly aquaculture related), the advent of the genomic era has heralded a new dawn in microalgae potential by allowing the combination and selection of key physiological characteristics with modified metabolic activities. Put simply, genetically modified (GM) and synthetic microalgae have the potential to revolutionise food, feed, fuel and pharmaceutical production.

However, commercialisation of such microalgae will require the culturing of GM microbes on an unprecedented scale. Much can be learnt from existing large-scale closed culture practises exploiting GM bacterial and yeast strains, however currently the majority of economically viable industrial culture of microalgae occurs in an open setting through the use of raceway ponds which house millions of litres of culture. Even at smaller scales, the utilisation of photobioreactors to expose cells to light effectively, agitate high volumes to enhance nutrient mixing and for the removal of the toxic oxygen produced by these phototrophic organisms, creates a significant barrier to commercialisation when these organisms are genetically modified. There is a fundamental lack of information and assessment tools available to researchers, industrial developers or regulators on the risks associated with their large scale propagation. This project seeks to identify, characterise, assess and scrutinise the factors that will need to be considered in order to progress GM microalgae scale up in both closed and open systems.

 


Unlocking the potential of Dunaliella through development of transformation-based strategies for gene manipulation

Tom Bibby (University of Southampton)


There is growing interest in the potential of photosynthetic microbes to be used and developed as feedstocks for the production of fuel and/or fine chemicals for industrial applications. Photosynthetic microbes rely only on light, water, carbon dioxide and nutrients to generate their cellular biomass, which comprises a complex mix of potentially valuable products/fuels. As such, they can be grown efficiently with a low carbon footprint and even coupled to outputs of industry to reduce the carbon output of existing processes.

The development of such biotechnologies from photosynthetic microbes will reduce our reliance on products and energy derived from fossil fuels. Development of photosynthetic microbes is, however, limited owing to the lack of genetic tools available for the generation of genetically modified and regulated cell lines. To address this problem, this study brings together scientists from the academic and commercial sectors with expertise in the growth and physiological and molecular analysis of photosynthetic microbes. The proposal aims specifically to develop a robust and efficient genetic transformation protocol for Dunaliella tertiolecta, a species identified as a promising candidate for biotechnological development. In addition, we aim to use this technology to knock down the expression of genes we have identified previously as potentially limiting growth of Dunaliella in dense populations required for commercial growth. Combined success of this project will represent a transformative step in the biotechnological development of Dunaliella and major progress in the potential commercial value of this species. Furthermore, the knowledge gained from this proposal will be of value in the biotechnological development of other microalgal species.

 


Exploiting the genetic potential of Galdieria sulphuraria for industrial biotechnology

Seth Davis (University of York)


The unicellular red alga Galdieria sulphuraria (Galdieria) exhibits wide metabolic versatility and displays enormous capacity to thrive at temperatures above 50 °C and highly acidic conditions. It has a broad metabolic repertoire allowing vigorous growth on virtually any sugar, sugar-alcohol or organic acid source. It is a metabolic workhorse. We seek to harness these abilities and develop Galdieria for industrial biotechnological applications. We have assembled an interdisciplinary team including geneticists, biochemists and a chemist to screen a targeted collection of Galdieria strains that display differing growth behaviours and metabolite preferences for their potential in disparate Industrial Biotechnology applications. Our specific goals are to identify strains best capable of using waste from the food industry and biofuel community, and assess the nature of metabolic conversions to determine the utility and benefit of resultant useful products. This includes an examination of the diverse Galdieria genomes to explore the “industrial space” of isolates of this species with an intention of creating recombinant proteins of unique benefits, as they are expected to be heat and acid tolerant. Genome editing will be developed in Galdieria to further the exploitation of this red alga using systems- and synthetic-biology approaches. We have established supplier connections with various UK SMEs who will donate starting materials and we will engage with the micro-SME Starbons to examine the ability of Galdieria to convert these low-value waste materials into higher value polysaccharides and other high-value chemicals, such as protein-based antioxidants. For example, preliminary work revealed the capacity of some strains of Galdieria support growth on barley-malt permeate waste, and we believe it has the capacity to convert these to useful industrial products. We will define the identity of these potentially economically beneficial constituents, whether they are protein- or metabolic-product based. A specific effort will target the enormously stable antioxidants Galdieria cells generate as compounds of clear economic utility, with relevance to the cosmetic and food industries. Proof of concept funding will underpin a subsequent Industrial Biotechnology Catalyst proposal aimed at producing industrial strains of Galdieria and the work proposed here isolates those specific avenues of highest economic potential.

 


Exploring chlorophyll-f and associated metabolism for improved intensive cultivation of cyanobacteria

Carole Llewellyn (Swansea University)


The ability of photosynthetic microorganisms to use sunlight and CO2 to produce molecules that have use as sustainable chemicals to replace existing petroleum based chemicals is proving attractive to industry. The relative ease with which cyanobacteria can be metabolically engineered compared to microalgae, make cyanobacteria a particularly attractive proposition. In addition, some cyanobacteria may have significant advantages in their ability to grow in low light conditions.

Cultivation of photosynthetic microorganisms can be a challenge because as soon as cell densities increase, shading and light penetration is an issue. This is a particular challenge in the large and intensive scale required for industry. Recent discoveries have shown that some cyanobacteria are adapted to live in low light levels using an unusual type of chlorophyll (chlorophyll-f) to capture light from the far end of the red spectrum. This extends the photosynthetic range for maximum use of solar energy available in enclosed photobioreactors. We have found chlorophyll-f in a strain of cyanobacteria that we have shown to be suitable for industrial scale-up, and indeed chlorophyll-f may be contributing to the suitability of this strain for cultivation at high densities in the UK.

Currently, we do not know by how much of an advantage it is to have chlorophyll-f and by how much it could help in the intensive cultivation that is required by industry. In this project we will explore chlorophyll-f and associated metabolism in this industrially relevant strain of cyanobacterial we will do this by undertaking experiments comparing growth and metabolism under white light and under far-red enhanced light. We will bring together for the first time experts in algae from Swansea University Biosciences with experts in microbial metabolism, genomics and bioinformatics from Swansea University’s Medical School. Our results will help us better understand how cyanobacteria can adjust their metabolism according to light conditions to maximise their growth under low light. The results could have application to growing other useful crops under low light conditions and for use in artificial photosynthesis, for example, in solar cells.

 


Development of an algal crop protection program through vaccination by novel viruses

Willie Wilson (Plymouth Marine Laboratory)


We will instigate a microalgal vaccination program exploiting naturally occurring persistent virus infections commonly found in a wide range of algae. The central theme of the project will be to maximize microalgal fitness by understanding and using viruses. We plan to improve microalgal growth performance and prevent further pathogen infections during industrial scale up.

Microalgae are grown globally at significant scale for high-value food, neutraceutical and cosmeceutical use, and biofuels. As technologies are being developed for their mass production, through either heterotrophic or phototrophic cultivation, little consideration has been given to algal crop protection. Good husbandry practices, akin to early agricultural practices (e.g. pond rotation; lying fallow; diversifying species; combining species) are used to try and limit infestation by autotrophic/heterotrophic contaminants or predation by a wide range of prokaryote, eukaryote, or viral parasites. It is a problem seldom admitted in public by algal biomass companies, but represents a serious bottleneck in high capacity production. There is even very little understanding on the nature of crop crashes, with a complete paucity of data on the role virus pathogens play, negatively and positively.

In this proof of concept project we plan to focus on the positive aspects of virus infection and their potential role in generating fitter strains of ‘immunized’ algae that confer resistance to further infection and improve growth yields. We propose that these fitter algae will have the potential to generate favourable phenotypes such as increased lipid production.


 

Round 2


A molecular toolbox to commercialise cyanobacteria: synthetic genetic sensor-regulator circuits for increased yields of phycobiliproteins

Alistair McCormick (University of Edinburgh)


Cyanobacteria are an established bioplatform for production of natural pigments, such as the phycobiliprotein C-phycocyanin (C-PC). Growing global interest has attracted considerable investment to bring cyanobacterial culturing technologies to commercial scale. However, investment in synthetic biology-based approaches to develop robust strains is still lacking. The market for cyanobacterial-based pigments is projected to reach £1 billion by 2019, at a CAGR of around 3.5% from 2014 (www.marketwatch.com). The FDA approved C-PC as a food colourant in 2013 and it is globally recognized as safe (GRAS) in nutritional supplements, cosmetics and pharmaceuticals. In the nutraceutical market C-PC is used as an anti-oxidant, while the use of C-PC as a natural blue food colourant (e.g. blue M&Ms) has experienced significant growth in the past five years (as confirmed by our industrial partner). Cyanobacterial pigments, such as C-PC, are also promising candidates for drug discovery, with applications in hepatic repair, cardiovascular disease, immune support, neurodegenerative diseases and next generation antibiotics. Exploitation of C-PC by the pharmaceutical, nutrition and cosmetic industries has given this biochemical a high market value; the £35 million market for it is expected to grow ten-fold by 2018, and demand has outstripped supply for C-PC.

Scottish Bioenergy designs, installs and operates microalgal photobioreactor systems for biochemical production and is seeking new approaches to meet this market demand. Our goal is to use a novel synthetic biology-based approach to develop robust strains of cyanobacteria that produce significantly increased yields of C-PC. The productivity of cyanobacterial cultures is restricted by limitations in control of growth and metabolism, often leading to large fluctuations during the biomass production process and downstream yields. We will design dynamic cellular gene control circuits that are able to sense and respond to the surrounding environment, and then co-ordinate cellular metabolism with C-PC production. This is a game-changing approach that goes beyond the simple use of genes driven by powerful promoters for bioproduction, with little consideration of the physiological consequences. Proof of the commercial viability of our concept will be demonstrated by modifying the output of the gene control circuit to regulate the production of C-PC, and then testing new strains in industrially relevant (up to 1,000 l) photobioreactor conditions. The modular approach of our system will allow future scalable circuit designs to direct metabolism in response to the input level of combinations of different environmental signals for optimising the production of other high value products.

 


Production of protein antibiotics in the algal chloroplast

Saul Purton (University College London)


Microalgae represent a promising biotechnology platform for the light-­driven synthesis of therapeutic proteins such as vaccines, antibodies and antimicrobials. Synthesis in the algal chloroplast is particularly attractive and in the last few years over 30 such proteins have been made in the chloroplast of the green alga Chlamydomonas reinhardtii. Although the work has highlighted the potential of this simple, low-­cost and benign host very few studies have gone on from small, lab-­scale production to investigate the biological and technical issues of scale-­up. Similarly, the industry-­scale extraction and purification of the recombinant proteins from C. reinhardtii yet to be seriously considered. A detailed study of these processes in combination with an analysis of the total costs of production in C. reinhardtii is essential in order to evaluate the feasibility and competitiveness of this microalga as a novel expression platform when compared to conventional systems.
The Purton group is currently exploring whether novel, protein-­based antibiotics can be produced efficiently in the C. reinhardtii chloroplast. The emergence of antibiotic resistance amongst bacterial pathogens is a major cause for concern, and there is a pressing need to develop new classes of anti-­microbials. We are focusing on endolysins – enzymes that are produced in bacteriophage-­infected bacteria and which lyse the cell at the end of the infection cycle by degrading the cell wall. These enzymes are highly specific for the cell wall of the target bacteria unlike broad-­spectrum antibiotic. Furthermore, the development of resistance is rare because endolysins evolved to target molecules that are essential for bacterial viability. We have created two transgenic lines that produce endolysins specific to the human pathogen Streptococcus pneumoniae. We have performed lab-­scale production and purification of these endolysins, and demonstrated their efficiency and specificity in killing the target bacterium (including antibiotic resistant clinical isolates). This project will investigate scale-­up of the cultivation using a commercial technology developed by our industrial partner, SBL, together with the development of a pilot scale method for cell harvesting, endolysin extraction and purification. This will allow a quantitative analysis of process economics and identify targets for process improvement. Such cost modeling of the algal platform will allow an informed evaluation of its commercial potential.

 


Engineering of supramolecular assemblies for enhanced high-value compound synthesis in the Chlamydomonas reinhardtii chloroplast

Colin Robinson (University of Kent)


There is intense interest in the exploitation of microalgae for biotechnological purposes because some microalgae contain high-value compounds (such as oils or colourants) while others can be developed as ‘cell factories’ for the production of recombinant proteins or high-value chemicals. However, microalgal cell factories have yet to enter mainstream industrial use, in part because they are insufficiently cost-effective when compared against traditional microbial or animal cell-based platforms. This application aims to improve the versatility and efficiency of a well-characterised green alga, Chlamydomonas reinhardtii, through the introduction of bacterial microcompartments (BMCs) in the chloroplast. Many bacterial species possess such BMCs and we have shown that these can be transferred to other bacteria with relative ease by expression of a small number of genes. Our work on the propanediol utilisation (Pdu) BMCs has shown that they can be produced heterologously in different bacteria. The key point is that BMCs allow the enzymes for specific reactions to be present in a concentrated form, where they are able to carry out metabolic reactions with exceptionally high efficiency. In this PoC we wish to investigate the use of BMCs in another platform chassis altogether, green algae.
BMCs will be produced in the C. reinhardtii chloroplast by introduction of the bacterial genes into the chloroplast genome using standard methods. Several variations will be generated. The simplest will involve expression of mutated forms of a single microcompartment ‘shell’ protein (PduA*) that forms huge lattices in the cell. These lattices will serve as stable scaffolds on which to attach large quantities of enzymes, and we will test the activities of attached enzymes in order to identify the most appropriate scaffolds. The second series of strains will generate transformants that will express 5 shell proteins (PduA-N) that are required to form an empty BMC, generating a structure with a defined external boundary. The empty BMCs will offer the potential for enclosing 1 or more enzymes in a protected environment for synthesis of high value chemicals. The net result will be the production of a suite of algal strains with unique properties and capabilities. These will be ready for use by industry for the efficient production of high-value chemicals and the size and stability of these scaffolds will offer considerable potential for a new generation of robust cell factories.

 


Tetramed: Spent media from Tetraselmis cultivation as a source of new bioactives

Michele Stanley (Scottish Association for Marine Science)


Microalgae are single celled plant-like organisms, which can be found living in marine and freshwater environments, and represent the first step in the aquatic food chain. They have long been studied in order to understand aquatic environmental interactions, as food sources for use in the aquaculture industry, as ingredients in nutritional products for human consumption eg. Spirulina, and more recently as possible new biofuels. To further understand the biology and potential use of these organisms, research has increasingly focused on their extensive biochemical diversity and the production of novel compounds.

One limitation to the commercial application of microalgal technology is the cost of production. A way to balance the economics is to generate multiple products from one source. This project will address this limitation by evaluating several microalgal strains that already have a role as aquaculture feeds, for their potential to produce higher value compounds of interest. Scientists at SAMS and GlycoMar Ltd are particularly interested in the production of carbohydrates/sugars by these microalgae, which are often very different to those from terrestrial plants, and can have novel and diverse biological activity. The selected target strains are known to secrete a range of carbohydrates into their growth media, potentially with useful activity, but they have never been fully characterised. In this project the carbohydrates produced by these microalgae will be isolated from culture media, biochemically analysed and tested for their biological effects, such as anti-microbial and anti-inflammatory activity. Culture conditions for the chosen strains will then be optimised experimentally, to generate the best yield of the target compounds.


 

Round 3


High value chemicals from algae: Developing a transgene-free genome editing toolbox to enhance carotenoid production

Attila Molnar (University of Edinburgh)


The products that we use every day and the foods that we eat are made up or a myriad of compounds, many of which are produced by the $3 trillion global chemicals industry. There is a significant burden of expectation placed on industrial biotechnology to produce more sustainable synthetic routes and to provide countries such as the UK a globally competitive position in a new manufacturing sector. New tools and technologies are needed to establish commercially competitive bioproduction in order to move away from unsustainable extraction from natural sources or environmentally costly synthetic chemistry production.
Astaxanthin is used as a feed supplement to enhance red pigments in salmon, crabs, shrimp and chicken products. In addition it is a dietary supplement for humans for antioxidant activity. The astaxanthin market was 280 metric tons valued at US$447 million in 2014 and is projected to reach 670 metric tons valued at US$1.1 billion by 2020 (source Global Astaxanthin Market – March 2015). The current market is dominated by synthetic astaxanthin (price is above $2500/kg, total market value is over $240M/year, Han et al., 2013).Because the synthetic version contains a mixture of stereoisomers with highly different biological activity/pigmentation, there is increasing demand for natural, more efficient products.
The goal of this application is to develop a transgene-free genome editing toolbox and to demonstrate its utility by enhancing astaxhantin biogenesis in microalgae with our partner, Scottish Bioenergy. The tools that currently exist rely on the expression of transgenes, which is not acceptable in many countries. Scottish Bioenergy designs, installs and operates microalgal photobioreactor systems for biochemical production and is seeking new approaches to meet this market demand. Its current business is focused on the production of the phycobiliprotein C-phycocyanin (C-PC). It is seeking to diversify its business through expansion into other high value pigments for food and cosmetic markets. Thus developing a highly customisable genome editing technology would pave the path to modify any biosynthetic pathways and to produce high value biochemical with transgene-free designer algae.


 

Round 4


Accelerating the biotechnological potential of marine microalgae using a molecular networking comparative metabolomics approach

Katherine Duncan (University of Strathclyde)


Often bacteria, especially the order actinomycetales are investigated for new drugs as they account for the production of over 45% of all bioactive microbial metabolites. Despite research into the biofuel potential of algal strains, the secondary metabolite potential of marine microalgae for novel antibiotics to combat antibiotic resistance is yet to be systematically investigated. Genome sequencing of microalgal strains has revealed that even well studied species (in terms of biofuels and primary metabolites) have the genetic potential to produce a wealth of secondary or specialized metabolites than discoveries to date would suggest. While this revelation has generated interest in the field of algal natural product discovery, there remains inefficiencies in the processes by which known natural products are detected and new compounds prioritized for isolation, structure elucidation and industrial bioprocessing. Taxonomically characterized strains will be fermented to induce chemically diverse metabolites from each strain. Through already developed protocols, the algal metabolites will be extracted using adsorbant resin, and metabolite profiles for each crude extract will be generated using Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS). The extracts will be further investigated using tandem mass spectrometry (HR-MS/MS) to generate fragmentation data of each parent ion in the crude extract. This HR-MS/MS data will be used to create a molecular network using Global Natural Products Social Molecular Networking. This will allow comparison of complex microalgal crude extracts based on similarities and differences of metabolite fragmentation across multiple strains (up to hundreds). This cutting-edge approach to chemical dereplication allows identification of known metabolites in addition to analogues of known chemicals and thus rapidly identifies areas of unique (to for example a particular strain) chemistry for isolation and chemical prioritization. Novel chemistry identified using this innovative dereplication strategy allows for streamlined antibiotic assay prioritisation and accelerates commercial targets for the IB sector based on chemical structural class information. Using this innovative approach will allow the unprecedented biological diversity of marine microalgae to be investigated as an exciting resource of chemical diversity across taxonomic and phylogenetic boundaries for biomedical applications in the comparative metabolomics era.

 


Identification of algal extracts with anti-cancer activity

Martin Michaelis (University of Kent)


Algae are a promising source of bioactive compounds including pharmaceuticals and nutraceuticals.
Examples of algal compounds/ extracts, which have shown biological activity, include those from a range of cyanobacteria, marine microalgae such as Nannochloropsis oculata and the commonly utilised alga Chlorella vulgaris. Less than 1% of the more than 30,000 algal species have been systematically analysed for biological activity. In this proof-of-concept study, we will screen a panel of algal extracts derived from N. oculata (previously described to have anticancer activity) plus Cylindotheca fusiformis, Tetraselmis suecica, Isochrysis galbana, Diacronema lutheri and Tisochrysis lutea for anti-cancer activity. Since there is a lack of information on how culturing conditions affect the levels of bioactivity observed, first standard operation procedures (SOPs) will be developed for the optimised production of algal extracts.
These extracts will then be tested in a unique, well-characterised panel of cancer cell lines that are resistant to clinically relevant anti-cancer drugs. Many tumours respond initially well to therapy (i.e. they shrink or even disappear). However, often cancer cells eventually become resistant to therapy resulting in tumour regrowth, therapy failure, and patient death. This ‘acquired’ drug resistance in cancer represents a major unmet medical need, and novel therapy options are needed.
To study resistance formation in cancer and to identify novel drug candidates, we have established the Resistant Cancer Cell Line (RCCL) collection, the only large collection of cancer cell lines that focuses on acquired drug resistance, currently, consisting of about 1,300 human drug-resistant cancer cell lines. Here, we will test the project algal extracts in a unique panel consisting of 36 breast cancer, lung cancer, ovarian cancer, and neuroblastoma cell lines for which whole exome profiles are available.
This project will provide the proof-of-concept for the discovery, development, and exploitation of novel anti-cancer drug candidates and lead compounds from algae. It will be followed by a multi-disciplinary bioindustrial approach enabled by the complementary expertise of the academic and industrial members of the PHYCONET consortium including the identification of the active ingredients, the modification of lead structures by chemical and/ or synthetic biology approaches, and the development of processing and production platforms. In addition, this project aims to develop and partially de-risk/ streamline the process of discovering novel pharmacologically active agents from algae by developing a practicable and effective biodiscovery pipeline. Whilst the focus here is targeted in the context of anticancer agents, we envisage that we will develop a template that will provide a robust platform for follow on bio-discovery projects to include investigation of extracts/ compounds from algae for further pharmacological/ biological activities.

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