Model-Driven Strain Engineering for Isoprenoid Drug Production

类异戊二烯药物生产的模型驱动应变工程

• 批准号: 7154823
• 负责人: ANTHONY P BURGARD
• 金额: $ 13.05万
• 依托单位: GENOMATICA, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7154823
• 关键词: drug design /synthesis /production; isoprenoid; model; motivation

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.

描述（由申请人提供）：工程改造具有上级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，菌株开发的时间表和成本可能很大，有时对许多产品来说是令人望而却步的。在这个STTR中，我们将利用基于约束的建模为中心的综合计算/实验平台，以合理地设计微生物菌株，以提高类异戊二烯的生产。具体而言，第一阶段将侧重于在大肠杆菌中提高紫穗槐二烯的生产，紫穗槐二烯是强效天然抗疟药物青蒿素的直接前体。首先，有前途的代谢工程目标将确定使用国家的最先进的计算方法在基于约束的建模。一种方法将依赖于OptKnock框架来阐明预测在生长阶段导致碳进入类异戊二烯途径的强制性功能的基因缺失集。另一种方法将集中在最小化代谢调节（MOMA）方法，以找到缺失，导致增加紫穗槐二烯生产，如果在突变体生物体中的代谢通量进行最小的再分配，从他们的父母对应。确定的删除将在E.大肠杆菌菌株工程化以产生紫穗槐二烯作为唯一的类异戊二烯产物。此外，适应性进化将应用于OptKnock设计的菌株，以展示生长耦合生产。然后将使用分批发酵和各种工艺变量（例如，生长速率、氧和底物吸收速率、产物/副产物生产速率）。最后，实验结果将与最初的预测相一致，以衡量综合建模/实验平台的整体成功。在该项目的后续阶段，我们将针对其他几种具有生物技术和生物医学重要性的类异戊二烯，目标是产生至少一种具有工业竞争力的生产菌株。该计划将导致代谢工程的系统方法的发展，该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。所开发的技术将显著加快并降低用于生产多种治疗性化合物的菌株开发成本。这项工作的最终目标是开发一个集成的计算/实验平台，用于改善基于类异戊二烯的药物和药物前体的微生物生产。类异戊二烯的微生物生产代表了化学提取或合成的有利替代方案，因为这些化合物通常在自然界中以极少量存在，并且它们的合成通常昂贵且低效。该项目将首先侧重于改进大肠杆菌中紫穗槐二烯的生产，紫穗槐二烯是强效天然抗疟药青蒿素的直接前体，然后再发展具有生物技术和生物医学重要性的其他类异戊二烯。