Collaborative Research: Continuous Biomanufacturing using Decoupled Growth and Production Stages for Efficient Production and Recovery

生物制造业务的范围和规模继续增长，可持续地在当地生产燃料、化学品、材料、食品、饮料和药品。目前，大多数生物制造设施以批处理工艺运行，这是一种70多年前开发的化学制造技术，在通过工艺优化实现的生产率提高和成本降低方面已经达到了其极限。在同一时期，在合成工程微生物细胞以增强其理想特征并最终提高其生产有价值生物产品的能力方面取得了重大进展。这促使人们重新审视当前分批和补料分批制造工艺的设计，这些技术已经落后于现代代谢工程进步的快节奏，限制了新重新连接的电池的真正转换潜力。该项目旨在建立一种新型的连续生物制造平台，克服传统(进料)间歇工艺的主要限制，导致反应器系统生产率的显著提高和制造成本的大幅降低。该项目产生的工程知识和科学发现将使目前大多数(FED)批量生物制造设施转变为具有成本效益的连续工艺，用于大规模生产燃料、化学品和其他高价值产品，巩固美国在生物制造领域的全球领先地位。这个跨学科、多大学的项目将吸引一支具有不同技能、经验和教育背景的研究团队，为高中生、本科生和研究生提供一个独特的研究环境。该项目整合了过程系统工程和合成生物学的先进技术，开发了一个新型的连续生物制造平台，用于从纤维素原料中生产脂类或脂类衍生的高价值产品。解脂Yarrowia的胞内脂肪和胞外游离脂肪酸(FFA)和酿酒酵母的脂肪酸乙酯(FAEE)将作为原型生物产品，开发连续生物制造平台。首先，将开发配备较小的生长生物反应器和较大的生产生物反应器的两级连续发酵工艺，以实现细胞生长和产品形成的分离，并可以独立优化工艺操作条件，以最大限度地同时生产生物量和目标产品。其次，两种具有代表性的酵母菌，解脂酵母和酿酒酵母，将被改造成能够通过环境和/或遗传切换控制不同的生长和生产阶段。解脂Y.lisoltica将被改造为使用从纤维素生物质中提取的C5和C6糖来生产细胞内脂肪或细胞外游离脂肪酸，干细胞重量(DCW)超过80%或等量。酿酒酵母也将被改造成生产高水平的FAEE。由于脂类产品的密度和浮力较低，通过简单的相分离就可以很容易地实现产品回收。将进行一项关于生物反应器中脂质分布的计算流体动力学(CFD)研究，以帮助进一步指导连续生物制造过程、耦合产品形成和现场产品移除的设计和优化。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The range and scale of biomanufacturing operations continue to grow, sustainably and locally producing fuels, chemicals, materials, foods, beverages, and medicines. Currently, most biomanufacturing facilities operate as batch processes, a chemical manufacturing technology developed over 70 years ago that has effectively reached its limits in productivity gains and cost reductions achievable through process optimization. Over this same period, significant progress has been made in the ability to synthetically engineer microbial cells to enhance their desirable traits and, ultimately, their ability to produce valuable bioproducts. This motivates revisiting the design of current batch and fed-batch manufacturing processes, technologies that have fallen behind the fast pace of modern metabolic engineering advances, limiting the true conversion potential of the newly rewired cells. This project aims to establish a novel continuous biomanufacturing platform that overcomes the major limitations of conventional (fed)-batch processes, leading to significant increases in reactor system productivity and a substantial reduction in manufacturing costs. The engineering knowledge and scientific discoveries generated from this project will enable the transformation of most current (fed)-batch biomanufacturing facilities to cost-effective continuous processes for large-scale production of fuels, chemicals, and other high-value products, strengthening the U.S. global lead in biomanufacturing. This interdisciplinary, multi-university project will engage a team of researchers with a diverse set of skills, experiences, and educational backgrounds, providing a unique research environment for high-school, undergraduate, and graduate students.This project integrates advanced technologies in process system engineering and synthetic biology to develop a novel continuous biomanufacturing platform for production of hydrophobic products such as lipids or lipid-derived high-value products from cellulosic feedstocks. Intracellular lipids and extracellular free fatty acids (FFAs) from Yarrowia lipolytica and fatty acid ethyl esters (FAEEs) from Saccharomyces cerevisiae will be used as archetype bioproducts to develop the continuous biomanufacturing platform. First, a two-stage continuous fermentation process equipped with a smaller growth bioreactor and a larger production bioreactor will be developed so that cell growth and product formation can be decoupled and where process operating conditions can be independently optimized to maximize the production of both biomass and target product simultaneously. Second, the two representative yeasts, Y. lipolytica and S. cerevisiae, will be engineered to enable distinct growth and production phases controlled by environmental and/or genetic switching. Y. lipolytica will be engineered to use both C5 and C6 sugars derived from cellulosic biomass to produce either intracellular lipids or extracellular FFAs with greater than 80% dry cell weight (DCW) or equivalent. S. cerevisiae will also be engineered to produce high levels of FAEEs. Due to the low density and buoyant force of the lipid products, the product recovery can be easily achieved via simple phase separation. A computational fluid dynamics (CFD) study on lipid distribution in bioreactors will be conducted to help further guide the design and optimization of the continuous biomanufacturing process, coupling product formation and in-situ product removal.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.