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

巴斯德毕赤酵母分泌的大多数重组蛋白的比产率相对较低。高产重组菌的鉴定在很大程度上是一个经验过程，对导致从摇瓶规模到高细胞密度工业发酵的培养条件放大时高生产率的特征知之甚少。目前使用毕赤酵母表达蛋白质的实践是利用整合载体产生重组子，从而导致位置无关的表达。最广泛使用的系统是基于酒精氧化酶启动子(AOX)，它需要去阻遏，然后用甲醇诱导。目的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，这使得选择用于放大和生产的最佳克隆变得复杂。这也受宿主的甲醇利用表型(MUT+、MUT S或MUT-)的影响。研究(仅限于实验室过程)表明，在巴斯德毕赤酵母中的低表达在某些情况下与内质网中的折叠问题有关，内质网折叠问题导致未折叠蛋白反应(UPR)。金砖四国项目06/18(大卫·利克，帝国理工学院(IC))正在(1)开发代谢组学方法，并将这些方法与转录组学一起应用于了解UPR应激、非应激和非表达培养的潜在生理学，以及(2)开发易于监控的UPR报告，以便能够在小规模确定理想的表达条件。该项目将使用工业上相关的蛋白质(由甲醇和非甲醇诱导)和蛋白质模型(低和高分泌滴度)和工业发酵平台，以表征和更好地了解上述参数与UPR诱导之间的相互作用(由BRIC 06/18开发的UPR工具)。代谢组信息(基于细胞外(足迹)代谢物)也将被收集。学生将花费大量时间研究Avecia：2-3个模型蛋白质，MUT宿主范围内的AOX重组体，在摇瓶培养中建立拷贝数和滴度，建立用于进一步工作的克隆的研究细胞库(MUT/拷贝数/滴度矩阵)，在15L补料分批发酵中建立4-6个克隆的发酵性能-包括足迹代谢物和/或UPR连锁绿色荧光蛋白表达的分析。上述工作预计需要15个月的时间，学生在IC(见下文)和Avecia花同样多的时间。学生最初将在IC(约4个月)工作，熟悉正在开发的UPR报告系统，并将构建整合到Avecia工作中所需的结构(例如，各种MUT背景下的GFP)。前半部分(15-18个月)将定义模式/决策矩阵。根据结果，我们将：(1)扩大与GFP的合作，这为我们提供了一个现成的变种分类系统，使高通量分析能够更根本地了解UPR的原因。这一点，再加上IC(BRIC 06/18)所做的转录分析，应该会指出一些宿主特征，这些特征可以进一步发展，以改善分泌和蛋白质滴度。或者，(2)研究在线控制UPR的可能性，这将需要提取一个/多个低表达的构建体并剖析表达条件，以建立特定生产力停滞/下降的条件，然后调查我们是否可以开发出保持在这一水平的反馈控制机制。第二阶段的工作将主要在IC完成，但关于遵循哪条路线的决定将由各方参与，并考虑到学生的时间表和学业需要。

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.