Understanding and improving the specific productivity of pichia pastoris recombinant protein secretion

毕赤酵母分泌的大多数重组蛋白的比产率相对较差。高产重组菌株的鉴定在很大程度上是一个经验过程，对从摇瓶规模到高细胞密度工业发酵的大规模培养条件下的高生产率特征了解甚少。目前使用毕赤酵母表达蛋白质的做法是使用整合载体产生重组，从而实现与位置无关的表达。应用最广泛的系统是基于酒精氧化酶启动子（AOX），需要先抑制，然后用甲醇诱导。靶基因的拷贝数是一个重要的变量，但不一定与滴度相关。在摇瓶中获得高分泌蛋白滴度的克隆并不总是在高细胞密度发酵中获得高滴度，这使得选择“最佳”克隆用于扩大规模和生产变得复杂。这也受到宿主甲醇利用表型（mut+， mutS或mut-）的显著影响。研究（仅限于实验室过程）表明，毕赤酵母的低表达在某些情况下可能与内质网的折叠问题相关，从而诱导未折叠蛋白反应（UPR）。BRIC项目6/18 （David Leak, Imperial College (IC)）是(1)开发代谢组学方法，并将这些方法与转录组学相结合，以了解UPR应激、非应激和非表达培养的潜在生理学，(2)开发易于监测的UPR报告器，以便在小范围内确定理想的表达条件。该项目将使用工业相关蛋白质（用甲醇和非甲醇诱导）和蛋白质模型（低分泌滴度和高分泌滴度）以及工业发酵平台来表征和更好地理解上述参数与普遍定期审议（普遍定期审议工具由金砖四国06/18开发）诱导之间的相互作用。代谢组学信息（基于细胞外代谢物）也将被收集。学生将花费大量的时间在Avecia工作：2-3个模型蛋白，多种宿主的AOX重组，在摇瓶培养中建立拷贝数和滴度，建立克隆的研究细胞库以进行进一步的工作（mut/拷贝数/滴度矩阵），在15L补料批发酵中建立4-6个克隆的发酵性能-包括分析足迹代谢物和/或UPR相关的GFP表达。上述工作预计需要15个月，学生将在IC（见下文）和Avecia花费相同的时间。学生最初将在IC工作（ca.4个月），熟悉正在开发的普遍定期审议报告系统，并将构建必要的结构（例如各种不同背景的绿色荧光蛋白）整合到Avecia的工作中。前半部分（15-18个月）将定义一个模式/决策矩阵。根据这一结果，我们将：(1)扩大与绿色荧光蛋白的合作，它为我们提供了一个现成的变体分类系统，使其适合于高通量分析，以更基本地了解普遍定期审议的原因。结合在IC （BRIC 06/18）进行的转录组学分析，应该指出一些可以进一步开发的宿主特征，以改善分泌和蛋白质滴度。或者，(2)调查在线控制普遍定期审议的可能性，这将需要采取一个/多个表达不良的结构，并剖析表达条件，以确定特定生产力停滞/下降的条件，然后调查我们是否可以开发一种保持在这一水平的反馈控制制度。第二阶段的工作将主要在IC完成，但决定遵循哪条路线将涉及所有各方，考虑到学生的时间表和学术需求。

The specific productivity of the majority of recombinant proteins secreted by Pichia pastoris is relatively poor. Identification of high producing recombinant strains is largely an empirical process with little understanding of the characteristics that lead to high productivity on scale up of culture conditions from shake flask scale to high cell density industrial fermentations. Current practice when expressing proteins using Pichia pastoris is to produce recombinants using integrating vectors leading to position independant expression. The most widely used system is based on the alcohol oxidase promoter (AOX) which requires derepression and then induction by methanol. Copy number of the target gene is an important variable but does not necessarily correlate with titre. Selection of the 'best' clone for scale up and manufacturing is complicated by the fact that clones achieving high secreted protein titre in shake flasks do not always achieve high titre in high cell density fermentations. This is also significantly influenced by the impact of methanol utilisation phenotype (mut+, mutS or mut-) of the host. Studies (limited to laboratory processes) have shown that poor expression in Pichia pastoris can in some cases be correlated with folding problems in the ER, which induces an unfolded protein response (UPR). BRIC project 06/18 (David Leak, Imperial College (IC)) is (1) developing metabolomic methods and applying these with transcriptomics to understand the underlying physiology of UPR stressed, non-stressed and non-expressing cultures and, (2) developing easily monitored reporters of UPR to allow ideal expression conditions to be determined on a small scale. This project will use industrially relevant proteins (induced with methanol and non-methanol) and protein models (low and high secretion titres) with an industrial fermentation platform to characterise and better understand the inter-play between the parameters described above and induction of the UPR (UPR tools developed by BRIC 06/18). Metabolomic information (based on extracellular (footprint) metabolites) will also be collected. The student will spend a significant amount of time working at Avecia: 2-3 model proteins, AOX recombinants in range of mut hosts, establishing copy number and titre in shake flask culture, establishing research cell banks of clones for further work (matrix of mut/copy number/titre), establishing fermentation performance of 4-6 clones in 15L fed-batch fermentations - including analysis for footprint metabolites and/or UPR linked GFP expression. The above work is envisaged to take 15 mo with the student spending an equal amount of time at IC (see below) and Avecia. The student will initially work at IC (ca.4 mo) becoming familiarised with the UPR reporter systems being developed and will making constructs (e.g. GFP in various mut backgrounds) necessary for integration into the work at Avecia. The first half (15-18 mo) will define a pattern/decision matrix. Depending on the outcome of this we will either: (1) Expand work with GFP, which provides us with a ready made system for the sorting of variants, making it ammenable for high-throughput analysis to get a more fundamental understanding of the causes of UPR. This coupled with the transcriptomic analysis being done at IC (BRIC 06/18) should point to some host features that can be developed further to improve secretion and protein titre. Or, (2) Investigate the possibility of online control of UPR, which would require taking one/more of the poorly expressing constructs and dissecting the expression conditions to establish the conditions at which specific productivity plateaus/declines and then then investigating whether we can develop a feedback control regime which keeps at this level. The second phase of the work will be done mainly at IC, but decisions as to which route to follow will involve all parties taking into account the timescale and academic needs of the student.