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持续增长，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都是批量生产，这是一种70多年前发展起来的化学制造技术，通过工艺优化，在提高生产率和降低成本方面已经达到了极限。在同一时期，在合成工程微生物细胞的能力方面取得了重大进展，以增强其理想特性，并最终提高其生产有价值生物产品的能力。这促使人们重新审视当前批次和补料批次制造工艺的设计，这些技术已经落后于现代代谢工程进步的快速步伐，限制了新重新连接的细胞的真正转化潜力。该项目旨在建立一种新型的连续生物制造平台，克服传统（喂料）批量工艺的主要局限性，从而显著提高反应器系统的生产率并大幅降低制造成本。该项目产生的工程知识和科学发现将使大多数当前（fed）批量生物制造设施转变为大规模生产燃料、化学品和其他高价值产品的成本效益连续过程，从而加强美国在生物制造领域的全球领先地位。这个跨学科的、多所大学参与的项目将吸引一组具有不同技能、经验和教育背景的研究人员，为高中、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，以开发一种新的连续生物制造平台，用于生产疏水产品，如脂质或脂质衍生的高价值纤维素原料产品。胞内脂质和胞外游离脂肪酸（FFAs）以及酿酒酵母（Saccharomyces cerevisiae）的脂肪酸乙酯（FAEEs）将被用作原型生物制品，以开发连续生物制造平台。首先，将开发一个两阶段连续发酵工艺，配备一个较小的生长生物反应器和一个较大的生产生物反应器，使细胞生长和产物形成可以解耦，并且可以独立优化工艺操作条件，以最大限度地同时生产生物质和目标产物。其次，将对两种具有代表性的酵母菌，脂肪酶酵母菌和酿酒酵母进行工程改造，使其能够通过环境和/或基因开关控制不同的生长和生产阶段。polylitica将被改造成使用来自纤维素生物质的C5和C6糖来生产细胞内脂质或细胞外FFAs，其干细胞重（DCW）大于80%或相当。此外，还将对葡萄球菌进行改造，使其产生高水平的faee。由于脂质产物的密度和浮力较低，通过简单的相分离可以很容易地实现产物的回收。对生物反应器中脂质分布进行计算流体动力学（CFD）研究，以进一步指导连续生物制造工艺的设计和优化，耦合产物形成和原位产物去除。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.