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

背景：我们之前报道（NT出版物1和3）大肠杆菌重组蜘蛛丝生产，其中包含非天然生物正交氨基酸l -叠氮多同丙氨酸（L-Aha）代替蛋氨酸（Met），使丝具有多种特定的功能。这些新的生物材料由于其固有的生物相容性（低免疫原性和热原性）和突出的机械性能与功能（细胞锚定，具有控释的抗生素）的结合而显示出巨大的前景。将其转化为经济的大规模过程是重组蛛丝部门面临的主要挑战，阻碍了它作为一种新的可持续材料的发展（重组蛛丝生产的宿主系统，趋势生物技术）。科学通报，2021,39,560)。只有在L-Aha存在的情况下表达目标蛋白（PoI）之前对细胞进行大量洗涤，才能使用大肠杆菌将L-Aha有效地结合到蛋白质中，这是不可扩展的。同样，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、感染性休克和自身免疫性疾病，在PoI与人体细胞接触之前，同样需要对PoI进行大量清洗。Baker's yeast （Sacchromyces cerevisiae）在真核蛋白生产方面有着良好的记录。它提供了一个适合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，并且不产生内毒素。通过连接合适的先导序列，可以分泌poi进行简单的纯化。研究目标/里程碑1：优化细胞内和分泌产物的标准spidroin和apoF表达构建。2：通过FACS筛选基因多样化的酵母文库，以改善表型，例如更好的产量，质量和L-Aha掺入。3：使用“点击”化学和“开启”炔荧光团来纯化和表征发酵罐中不含mCherry标签的4RepCT和apoF，以确认类似物的加入。4：大肠杆菌和酵母表达蛋白（含/不含PTM）与哺乳动物细胞系统相互作用的比较。方法/培训：酵母表达基因序列优化；生物信息学来确定PoI中的关键残留物；大肠杆菌和酵母蛋白的克隆、表达和纯化酵母文库的生成及FACS筛选；非天然氨基酸诱变；使用“点击”化学的生物偶联；蛋白偶联质谱鉴定；哺乳动物细胞培养，共聚焦荧光显微镜。

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.