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

背景：我们以前报道过（NT出版物1和3）在E.大肠杆菌中掺入了非天然的生物正交氨基酸L-叠氮基高丙氨酸（L-Aha）代替甲硫氨酸（Met），使蚕丝能够位点特异性地功能化，用于各种医疗保健作用。这些新的生物材料由于内在的生物相容性（低免疫原性和致热原性）和与功能（细胞锚，控释抗生素）相结合的出色机械性能而显示出显著的前景。将其转化为经济的大规模工艺是重组丝部门的主要挑战，阻碍了其作为新的可持续材料的发展（Host Systems for the Production of Recombinant Spider Silk，Trends Biotechnol.，2021，39，560）。大肠杆菌间质营养型只有在L-Aha存在下表达目的蛋白（PoI）之前细胞被充分洗涤才能发生，这是不可扩展的。类似地，在大肠杆菌中的表达导致PoI中存在内毒素（LPS）污染物，引起炎症反应、败血性休克和自身免疫性疾病，同样，在与人体细胞接触之前，需要对PoI进行大量洗涤。（酿酒酵母）有一个被证明的轨迹-在真核生物蛋白质生产中的记录。它提供了一个适合于克服上述问题的底盘。它已经在工业规模上用于蛋白质生产，不产生内毒素。PoIs可以分泌用于简单的purificationby attachment of suitable leader sequences. Research Objectives/Milestones1：Optimise standard spidroin and apoF expression constructs for intracellular and secreted products.第二章：通过FACS筛选遗传多样性酵母文库以改善表型，例如更好的产量、质量和L-Aha纯化。3：使用带有"开启"炔荧光团的"点击"化学从发酵物中纯化和纯化不含mCherry标签的4RepCT和apoF以确认类似物纯化。方法学/培训：优化在酵母中表达的基因序列;确定PoI中关键残基的生物信息学;在大肠杆菌中克隆、表达和纯化蛋白质。大肠杆菌酵母文库的产生和通过FACS筛选;非天然氨基酸诱变;使用"点击"化学的生物缀合;通过CD、MS的蛋白缀合物表征;哺乳动物细胞培养，共聚焦荧光显微镜。&yeast;

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.