Posters

Developing an enzymatic system for methacrylate intermediates and esters

Methyl methacrylate (MMA) is an organic compound used as a precursor in the manufacturing of plastics. MMA production has reached 4.8 million metric tonnes yearly in 2020, with no current equivalent on the market. Furthermore, it is currently produced chemically via half-dozen processes that use carbon fuel feedstocks. As a result, new sustainable manufacturing strategies are needed. The current biosynthetic pathway to MMA occurs not without limitations. Low conversion rates, namely too many side products, and difficulties in separating them, are just a few of the concerns that business is facing. Consequently, several enzymes have been exploited to try and solve these problems. Alcohol acyl transferases (AAT) are a group of enzymes with the ability of producing esters from acyl-donors (such as acyl-CoA) and fatty alcohols as substrates. They have been previously reported as source for biodiesels fuels from Saccharomyces cerevisiae. In the biotransformation to MMA, AATs can be used to produce key intermediates, such as acetate, isobutyrate and methacrylate esters. In this project, multiple acyl-donors and alcohols will be investigated as substrates among several transferases from various hosts to design the most optimal enzymatic system to yield methacrylate intermediates and esters.

Investigating Streptomyces clavuligerus Linear Replicons for Improved Clavulanic Acid Production

Streptomyces clavuligerus (Sclav) produces clavulanic acid and is composed of four giant linear plasmids (GLPs) and its chromosome. Various genes essential for the maintenance of linear replicons, such as tap and tpg which encode telomeric terminal proteins, are found on three out of four GLPs.

We will investigate plasmid-chromosome interactions to determine the role of tap-tpg and aim to cure GLPs for decreased metabolic burden and increased clavulanic acid production.

Previous work demonstrated a circularised chromosome and loss of plasmid after cutting the largest GLP, pSCL4, potentially due to the absence of tap-tpg. To determine the role of tap-tpg in chromosomal and plasmid linearity, we tested their inactivation using CRISPR-dcas9 multiplexing, targeting tap-tpg4 on pSCL4, tap-tpg3 on pSCL3 and tap-tpg2 on pSCL2. Sclav exconjugant colonies were patched to check for plasmid integration and colonies were screened for the loss of replicon telomeres by colony PCR.

PCRs established that the abolition of tap-tpg4 transcription led to chromosome circularity in some of the patched colonies Sclav DSM 738.

Future work will focus on pSCL3 and pSCL2 tap-tpg interaction with pSCL4 and the chromosome after knockdown of the genes through sgRNA multiplexing and elucidate the mechanisms of telomere replication in these multi-replicon organisms.

Escin tolerance of Saccharomyces cerevisiae sterol mutants

Escin is a mixture of glycosylated triterpenoids derived from Aesculus hippocastanum, predominantly containing the isomers escin 1a, escin 1b, isoescin 1a and isoescin 1b, which vary in their C21, C22 and C28 modifications. Glycosylated triterpenoids have industrial relevance in pharmaceuticals, food stuffs and home care items, but are unfortunately inhibitory to yeast in high concentrations. Therefore hindering the production of these products in yeast. We have been able to identify S. cerevisiae sterol mutants with enhanced tolerance to escin. We hypothesise that the interaction between the yeast membrane sterol, ergosterol and escin is the cause for this toxicity, and aim to demonstrate this experimentally. Findings would give new engineering strategies for producing industrially relevant glycosylated triterpenoids in microbial hosts.

Growth assays and propidium iodide experiments were conducted on wild-type and mutant strains, providing evidence of the toxicity of escin in wild-type S. cerevisiae, and resistance in two sterol mutants at the concentrations tested. Work in confocal microscopy and fluroenscene anisotropy is currently underway. The mutant strains have potential as a chassis for engineering the industrial production of escin-like compounds using biological techniques. Providing a solution to non-renewable methods that are currently used in pharmaceutical, food production and home care items.

From Seaweed to Next-Generation Anode

There is a growing demand for higher energy and power dense lithium-ion batteries, silicon has been proposed as a promising anode material due to its high energy capacity compared to current commercial graphite anodes. However, silicon undergoes large volume changes during its charging and discharging cycles. High-capacity electrochemical materials which exhibit these large volume changes require improved binder performances. Recent studies have shown that electrochemical stability is largely dependent on the characteristics of the binder, which ensures the electrode integrity during use. Current commercially accessible alginate-based binders can extend the longevity of high capacity batteries for >1000 cycles at 1200 mA g-1.The alginate binders used in this project are provided by MBL, who have developed a process to tune the alginate chemistry, allowing them to engineer bespoke alginates with tailored mechanical properties. In addition, nanostructuring of the silicon material can also guard against the substantial volume changes. The microwave synthesis of nanoporous silicon offers a new “green” method to make high surface area, mesoporous nanocomposites; this method will be exploited to enhance the capabilities of porous alginate/Si further.

Development of a Process to Valorise Lignin

The development of an economically feasible second generation biorefinery will be an essential step in shifting towards a more sustainable circular economy. As the building material of the plant cell wall, lignocellulose is the most abundant organic material on earth, and therefore will be a major feedstock for the industry. Sitka spruce is the most common forestry crop in Scotland so is well placed to be a significant and sustainable supply of lignocellulose

Lignin is an irregular and heterogeneous aromatic polymer that makes up around one third of lignocellulose. Its unique chemical properties confer a huge range of possible uses from material additives to fine platform chemicals. However, its natural recalcitrance inhibits degradation into specific homogeneous product by chemical or physical processes. This project aims to bring a combination of chemical and synthetic biology approaches to the valorisation of lignin, and in doing so contribute to the development of an economically viable biorefinery.

Commercial scale-up of porcine induced pluripotent stem cell lines for development of novel cultured meat products.

Meat production is rising faster than population growth. As livestock farming accounts for 14.5 % of greenhouse gas emissions, is detrimental to human health, and animal welfare, this represents monumental sustainability challenges. Cultivated meat, derived from induced pluripotent stem cells (iPSC) holds great promise to resolve these issues, however, the high costs and technical difficulties associated with iPSC culture and maintenance are major bottlenecks in commercialisation. This project aims to alleviate such challenges through developing an optimised iPSC culture medium, to maximise growth and drive down manufacturing costs. Production will subsequently be scaled up and validated under industrially relevant conditions using stirred tank bioreactors. Preliminary design of experiments guided optimisation of critical media components predicted a promising 60 % reduction in medium costs, whilst maintaining high cell densities and pluripotency characteristics. Furthermore, our carefully selected proprietary porcine iPS cell lines have favourable aggregation characteristics, allowing rapid adaptation to suspension culture. Cell densities of up to 820,000 cells/mL have been achieved in early Applikon MiniBio reactor cultivations. Future work will focus on single cell screening using the state-of-the-art Beacon optofluidic platform to select for clones with favourable characteristics and further maximise production of nutritionally viable cells at the lowest possible cost.

Exploring new applications for the brown seaweed Fucus serratus

In the UK, Ireland and France, the brown seaweed Fucus serratus is mainly harvested for production of liquid fertilisers and preparations for the cosmetics industry. Owing to the high amount of sugars and phenolics, extracts from this marine plant have the potential to be used in different sectors and various applications.

Ethanolic extracts were prepared from Fucus serratus (FS) harvested on three occasions over the years 2019-2021. Phenolic content, antioxidant and free radical scavenging properties were compared between extracts and neutral sugars and uronic acids content were also examined. Furthermore, the effect of the crude extract (crude FS) on lipid accumulation, energy metabolism and oxidative stress was evaluated in 3T3-L1 adipocytes and the nematode C. elegans.

Variations were observed in sugars and uronic acids content, as well as antioxidant activity between extracts produced in different years, however phenolic content and free radical scavenging activates were comparable. Crude FS was able to reduce lipid accumulation and oxidative stress in both 3T3-L1 adipocytes and C. elegans. In addition, it had a positive effect on glycerol release and altered the expression of genes linked to oxidative stress and lipid metabolism in the cell model.

This study highlights the potential use of extracts from Fucus serratus in the nutraceutical industry for treatment and/or prevention of obesity, as well as reduction of associated oxidative stress.

Development of a targeted genomic integration system for the comparison of expression vector improvements

The fed batch culture of Chinese Hamster Ovary (CHO) cells is used for the production of many biopharmaceuticals, particularly monoclonal antibodies (mAbs). Expression vector engineering strategies have contributed to monoclonal antibody titres reaching up to 10g/L. However, a growing number of next generation biologics, such as fusion proteins and bi-specific antibodies, are difficult to produce in CHO cells. Additionally, the loss of productivity during long term culture remains a problem in CHO cell bioproduction. To overcome such challenges, the next generation of expression vector optimization strategies will need to optimise existing components (such as promoters, untranslated regions and epigenetic regulatory elements) and generate novel components. Here, we are developing a targeted genomic integration system in Fujifilm’s Apollo X cell line, which allows expression vector components to be compared in identical genetic contexts. We first integrated a landing pad (flanked by Bxb1 attachment sites) into the Fer1L4 genomic locus of the CHO-DG44 cell line, using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones and used the Bxb1 recombinase to perform recombinase mediated cassette exchange with donor vectors containing mCherry expression cassettes. We will then use the system to compare promoters to improve the production of monoclonal antibodies.

Lignin-based metallic nanoparticle composites

Corrosion prevention and control remains a big challenge for the marine environment, from both an environmental and an economic point of view. It has an enormous impact on marine vessels and structures, reducing their integrity and performance, resulting in high global maintenance costs. Effective biocidal antifouling agents are strictly regulated and their use can be withdrawn if the environmental impacts are found to be severe. Therefore, there is an urgent need for efficient and sustainable anticorrosion coatings.

The aim of this project is to synthesise a novel lignin-metallic nanoparticle composite with anticorrosion and antifouling properties. Lignin is an abundant plant polymer and exhibit promising anticorrosion properties. Nanoparticles have unique antimicrobial activity and could therefore be synergized with lignin. Bacteria are one route to the green synthesis of nanoparticles and can be genetically engineered to express the proteins involved in the synthesis of nanoparticles that have been discovered in proteomic data from our lab. Results suggested the overexpression of these genes achieved higher rates of nanoparticle synthesis in addition to changes in nanoparticle morphology. These nanoparticles will be investigated for their antimicrobial activity and combined with lignin to produce the next generation of marine coatings.