Evolving novel yeast expression hosts for large scale biomaterial and biologic production

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Background:We previously reported(NT publications 1 & 3)recombinant spider silkproductionin E. colithat incorporates the un-natural bio-orthogonal amino acid L-azidohomoalanine (L-Aha) in place of methionine(Met),allowing the silk to besite-specificallyfunctionalised for a variety of healthcare roles. These newbiomaterialsshowsignificant promise due to a combination of intrinsic biocompatibility (low immunogenicity and pyrogenicity) and outstanding mechanical properties coupled tothe functionalities(cell anchors, antibiotics with controlled release)added. Translating this to an economical large-scale processis a major challenge for the recombinant silk sector,hampering it's development as a new sustainable material(Host Systems for the Production of Recombinant Spider Silk, Trends Biotechnol., 2021, 39, 560).Efficient incorporation of L-Aha into proteins using an E. coliMetauxotroph only occurs if the cells are extensively washed before the protein of Interest (PoI)is expressed in the presence of the L-Aha,whichisnot scalable. Similarly, expression in E.coliresults in the presence of endotoxin (LPS)contaminants in the PoI,causing inflammatory responses, septic shock and auto-immune diseases, again requiring extensive washing of the PoIbefore it can be used in contact with human cells.Baker's yeast (Sacchromyces cerevisiae) has a proven track-record in eukaryotic protein production.It offers a chassis suitable for overcoming the problemsidentified above.Itis already used on an industrial scale for protein productionanddoes not produce endotoxins. PoIs can be secreted for simple purificationby attachment of suitable leader sequences.Research Objectives/Milestones1: Optimise standard spidroin and apoF expression constructs forintracellular and secreted products. 2: Screen genetically diverse yeast libraries by FACS toimprove phenotypes, e.g. better yield, quality and L-Aha incorporation.3: Purify and characterise 4RepCT and apoF without the mCherry tag from fermentersusing 'click' chemistry with a 'turn-on' alkyne fluorophoreto confirm analogue incorporation.4: Comparison of E. coliand yeast-expressed proteins (with/without PTM's) interacting withmammalian cell systems.Methodology/Training: Optimisation of gene sequences for expression in yeast; bioinformatics to determine critical residues in PoI; cloning, expression and purification of proteins in E. coli&yeast; generation of yeast libraries and screening by FACS; un-natural amino acid mutagenesis; bioconjugation using 'click' chemistry; protein-conjugatecharacterization by CD, MS; mammalian cell culture, confocal fluorescence microscopy.