Model-Driven Strain Engineering for Isoprenoid Drug Production

描述(由申请人提供)：设计具有卓越生产能力的微生物菌株是生物工艺开发中最具挑战性和最有趣的努力之一。然而，由于缺乏合理和系统的方法，菌株开发的时间和成本可能很大，有时对许多产品来说令人望而却步。在这项研究中，我们将利用一个以约束为基础的建模为中心的集成计算/实验平台来合理地设计微生物菌株，以提高类异戊二烯的生产。具体地说，第一阶段将侧重于提高阿莫法二烯在大肠杆菌中的生产，阿莫法二烯是强大的天然抗疟疾药物青蒿素的直接前体。首先，在基于约束的建模中，将使用最先进的计算方法来确定有前途的代谢工程目标。一种方法将依靠OptKnock框架来阐明一组基因缺失，这些基因缺失预计会导致碳在生长阶段强制进入类异戊二烯途径。另一种方法将集中在代谢调节最小化(MOMA)方法上，以寻找如果突变生物体中的代谢流经历了来自其亲代对应物的最小再分配，则导致阿莫二烯产量增加的缺失。已确定的缺失将在一株大肠杆菌中实施，该菌株被设计为生产阿莫二烯作为唯一的异戊二烯产品。此外，自适应进化将应用于OptKnock设计的菌株，以展示生长耦合生产。然后，将使用分批发酵对设计的菌株进行表征，并将测量各种工艺变量(例如，生长速度、氧气和底物吸收速度、产品/副产品产量)。最后，实验结果将与最初的预测相一致，以衡量组合建模/实验平台的总体成功。在该项目的后续阶段，我们将针对其他几个具有生物技术和生物医学重要性的异戊二烯类化合物，目标是产生至少一种具有工业竞争力的生产菌株。这项计划将导致开发代谢工程的系统方法，利用基因组信息和大量实验数据来合理设计生产宿主。所开发的技术将大大加快并降低生产多种治疗化合物的菌株开发成本。这项工作的最终目标是开发一个综合的计算/实验平台，以改进基于异戊二烯的药物和药物前体的微生物生产。微生物生产异戊二烯类化合物是化学提取或合成的一种有利的替代方案，因为这些化合物在自然界中通常含量极少，而且它们的合成往往昂贵且效率低下。该项目首先将重点放在改进阿莫二烯的生产上，阿莫二烯是一种强大的天然抗疟疾药物青蒿素的直接前体，然后在其他具有生物技术和生物医学重要性的异戊二烯类化合物方面取得进展。

DESCRIPTION (provided by applicant): Engineering microbial strains with superior production capabilities is one of the most challenging and intriguing endeavors in bioprocess development. However, due to the lack of rational and systematic approaches, the timelines and cost for strain development can be large and at times prohibitive for many products. In this STTR, we will utilize an integrated computational/experimental platform centered upon constraint-based modeling to rationally engineer microbial strains for enhanced isoprenoid production. Specifically, Phase I will focus on enhancing the production of amorphadiene, an immediate precursor to the powerful natural antimalarial drug artemisinin, in Escherichia coli. First, promising metabolic engineering targets will be identified using state-of-the-art computational approaches in constraint-based modeling. One approach will rely on the OptKnock framework to elucidate sets of gene deletions predicted to cause obligatory funneling of carbon into the isoprenoid pathway during the growth phase. Another approach will center upon the minimization of metabolic adjustment (MOMA) methodology to find deletions leading to increased amorphadiene production if the metabolic fluxes in the mutant organisms undergo a minimal redistribution from their parental counterpart. The identified deletions will be implemented in an E. coli strain engineered to produce amorphadiene as the sole isoprenoid product. In addition, adaptive evolution will be applied to strains designed by OptKnock to exhibit growth-coupled production. The designed strains will then be characterized using batch fermentations and various process variables (e.g., growth rate, oxygen and substrate uptake rates, product/byproduct production rates) will be measured. Lastly, the experimental findings will be reconciled with the original predictions to gauge the overall success of the combined modeling/experimental platform. In subsequent phases of the project, we will target several other isoprenoids of biotechnological and biomedical importance with the goal of generating at least one industrially competitive production strain. This program will lead to the development of a systematic approach to metabolic engineering that leverages genomic information and a host of experimental data for the rational design of production hosts. The developed technology will significantly expedite and lesson the cost of strain development for the production of multiple therapeutic compounds. The ultimate goal of this work is to develop an integrated computational/experimental platform for improving the microbial production of isoprenoid-based drugs and drug precursors. Microbial production of isoprenoids represents a favorable alternative to chemical extraction or synthesis as these compounds are typically found in extremely small quantities in nature and their synthesis is often expensive and inefficient. This project will first focus on improving the production of amorphadiene, an immediate precursor to the powerful natural antimalarial drug artemisinin, in Escherichia coli, and then progress towards other isoprenoids of biotechnological and biomedical importance.