Engineered Consortia for Effective Utilization of a Cyanobacterial Carbohydrate Feedstock

目前，生物燃料的生产依赖于来自植物性作物的碳水化合物原料，这些作物与可食用作物争夺耕地和饮用水。替代的，光合作用衍生的碳水化合物原料来源可以将生物燃料生产与传统农业分离，并可能减少所需的土地足迹。该项目将利用一种经过基因工程改造的光合细菌（蓝藻），这种细菌将利用大气和光线中的二氧化碳制造蔗糖（食糖），蔗糖可以发酵成生物燃料和生物产品。这种微生物具有独特的能力，能够将其光合作用的大部分固定碳转向蔗糖的生产和排泄。这种工程蓝藻具有超过传统植物物种的碳水化合物生产力潜力，如果能够设计出有效的方法来扩大培养生长并将原料回收成本降至最低，则可用于替代碳水化合物原料。该项目将重点放在微生物财团的发展，作为一种手段，利用这些蔗糖分泌蓝藻的生产能力。在这种方法中，分泌糖的蓝藻将与酵母菌株共同培养，有效地利用碳水化合物，并在不需要蔗糖纯化或加工的情况下将其生物转化为商品。酵母染色稳健生长和利用蓝藻糖的能力的提高也将最终提高蔗糖转化为生物燃料和生物产品的能力。为此，将开发一个微生物联合体模型，其中工程酵母将利用光合作用产生的蔗糖生产脂肪醇，脂肪醇是一种有用的表面活性剂和潜在的生物燃料添加剂。该项目包括研究生的研究和教育经验，以及通过密歇根州立大学植物基因组学计划和Khorana学者计划的本科生。此外，该项目还包括参与Schoolcraft学院的一个本科生研究项目，并通过斯隆研究所赞助的德尔福政策研究，讨论将合成生物学用于生物技术应用的科学政策。技术描述本项目的总体目标是开发一种共培养系统，用于利用蓝藻菌和酵母的工程菌株从二氧化碳和光中过量生产蔗糖。该方法依赖于通过共培养的两步光生物生产蔗糖，并将为如何为此目的设计物种间相互作用提供基本的理解。该项目将利用一种工程蓝藻菌株，长聚球菌PCC 7942 （S. elongatus）最近在PI？它在基因上是稳定的，能够将高达85%的固定碳转化为排泄的蔗糖。该菌株具有较高的光合生产比活性，因此如果规模化培养可以缓解三个挑战，则具有相当大的潜力作为替代碳水化合物原料来源，这将由拟议的研究解决。这些挑战包括：1)将反应器的复杂性和成本降至与商品产品价值相一致的程度；2)降低净化、加工和向下游输送蓝藻碳水化合物的成本；3)降低光生物反应器污染的风险。为此，该项目将重点开发一种与酿酒酵母（酵母）直接共培养的长形葡萄球菌，它将灵活地将蔗糖转化为增值化合物。该项目将探索改善共培养酵母生长的具体策略，包括改善酵母的碳吸收，增加酵母对蓝藻光合代谢次级产物的耐受性，以及通过工程物理关联增强蓝藻对酵母的碳通量和特异性。这些策略将被评估，以提高酵母的稳健性和碳水化合物的转化效率，从S. elongatus在共培养中生长时，并提出最后的原理证明生产十六烷基醇通过使用已建立的脂肪酒精生产酵母菌株。总的来说，该项目通过提供代谢途径工程的替代方法或通过改进有吸引力的藻类来源的原料，有可能影响藻类生物燃料领域。该项目包括研究生的研究和教育经验，以及通过密歇根州立大学植物基因组学计划和Khorana学者计划的本科生。此外，该项目还包括参与Schoolcraft学院的一个本科生研究项目，并通过斯隆研究所赞助的德尔福政策研究，讨论将合成生物学用于生物技术应用的科学政策。

Principal Investigator: Daniel DucatNumber: 1437657Currently, the production of biofuels relies on carbohydrate feedstocks sourced from plant-based crop species that compete with edible crop species for arable landmass and potable water. Alternative, photosynthetically derived feedstock sources of carbohydrates can uncouple biofuel production from traditional agriculture and potentially decrease the land footprint required. This project will make use of a genetically engineered strain of a photosynthetic bacterium (cyanobacterium) that will use carbon dioxide in the atmosphere and light to make sucrose (table sugar), which can be fermented to biofuels and bio-products. This microorganism is uniquely capable of rerouting the majority of its photosynthetically fixed carbon towards the production and excretion of sucrose. This engineered cyanobacterium has carbohydrate productivity potential that exceeds that of traditional plant species, and could be used for an alternative carbohydrate feedstock if effective approaches can be designed to scale culture growth and minimize the cost of feedstock recovery. The project will focus on the development of microbial consortia as one means to capitalize upon the productive capacity of these sucrose-secreting cyanobacteria. In this approach, the sugar-secreting cyanobacterium with will be co-cultured with yeast strains that efficiently utilize the carbohydrates and biologically convert them into commodity products without the requirement of sucrose purification or processing. Improvements in the capacity for the yeast stain to robustly grow and utilize cyanobacterial sugars will also ultimately improve the conversion of sucrose into biofuels and bioproducts. Towards this end, a model microbial consortium will be developed whereby engineered yeast will utilize photosynthetically produced sucrose for the production of fatty alcohols, a useful surfactant and potential biofuel additive. This project includes research and educational experiences for a graduate student, as well as undergraduate students through the Plant Genomics Program and Khorana Scholars Program at Michigan State University. In addition, this project involves engagement with an undergraduate research program at Schoolcraft College, and science policy discussions on the use of synthetic biology for biotechnology applications through a Sloan Institute sponsored Delphi policy study. Technical DescriptionThe overall goal of this project is to develop a co-culture system for the overproduction of sucrose from carbon dioxide and light using engineered strains of cyanobacteria and yeast. The approach relies on a two-step photobiological production of sucrose through co-culture, and will provide fundamental understanding on how engineer of inter-species interactions for this purpose. This project will make use of an engineered cyanobacterial strain, Synechococcus elongatus PCC 7942 (S. elongatus) recently developed in the PI?s laboratory that is genetically stable and capable of rerouting up to 85% of its fixed carbon towards excreted sucrose. This strain possesses a high specific activity for photosynthetic production and therefore has considerable potential as an alternative carbohydrate feedstock source if scaled culturing can mitigate three challenges, which will be addressed by the proposed research. These challenges are to 1) minimize reactor complexity and cost to a degree consistent with the commodity product value, 2) reduce the costs of purifying, processing, and delivering cyanobacterial carbohydrates to downstream applications, and 3) to reduce the risk of photobioreactor contamination. Towards this end, the project will focus on the development of a direct co-culture of S. elongates with Saccharomyces cerevisiae (yeast) that will flexibly convert the sucrose into added-value compounds. The project will explore specific strategies to improve yeast growth in co-culture, including, improvement of yeast carbon uptake, increased yeast tolerance of cyanobacterial secondary products of photosynthetic metabolism, and enhancement of the flux and specificity of carbon from cyanobacteria to yeast through engineered physical association. These strategies will be evaluated to improve yeast robustness and efficiency of conversion of carbohydrates derived from S. elongatus when grown in co-culture, and a final proof-of-principle production of cetyl alcohol is proposed through the use of an established fatty-alcohol producing yeast strain. Overall, the project has potential to impact the field of algal biofuels by providing an alternative approach for metabolic pathway engineering or by improving upon an attractive algae-derived feedstock. This project includes research and educational experiences for a graduate student, as well as undergraduates through the Plant Genomics Program and Khorana Scholars Program at Michigan State University. In addition, this project involves engagement with an undergraduate research program at Schoolcraft College, and science policy discussions on the use of synthetic biology for biotechnology applications through a Sloan Institute sponsored Delphi policy study.