Leveraging Extreme Thermoacidophily for Bio-based Chemicals

硫化合物和二氧化碳作为天然气和石油精炼过程的副产品大量产生，这在其安全处理和经济处置方面产生了重大挑战。幸运的是，微生物可以为开发环保和节能的工艺来回收这些工业废物流提供很多帮助。硫和二氧化碳也恰好是一种叫做嗜热嗜酸古菌的微生物的潜在能量和碳源，这种微生物生长在极热的酸性环境中。处理过量工业硫和二氧化碳的一种潜在方法是设计这些古细菌的代谢途径，使它们利用副产物生产具有商业价值的化学品。该项目将应用最新开发的工具对极端嗜热嗜酸菌进行基因工程改造，以创造新的微生物。这项研究将检验这样一种假设，即将硫氧化基因从一种生物体插入另一种生物体将产生一种能够将二氧化碳和硫转化为工业相关有机化学品的新微生物。将硫氧化基因工程化到在极端条件下自然生长的微生物中具有许多潜在的好处。在热和酸性条件下生长的能力最大限度地减少了不需要的微生物污染的风险，并使生物过程能够像常规化学过程一样操作。微生物的高耐热性也有利于回收具有低沸点的生物化学产品。为了扩大这项研究的教育影响和包容性，北卡罗来纳州聋人学校（NCSD）的教师和学生将通过基于生物技术的教学模块参与。这些单元将包括闭路字幕和其他特点，以促进聋人和听力受损者的教育目标。将在NCSD现场演示模块。此外，NCSU生物技术计划将接待来自NCSD的访问初中和高中学生进行为期一天的实验室体验，这也将使他们熟悉高等教育机会。该项目的目标是对嗜热嗜酸古菌模型Sulfolobus acidocaldarius DSM 639菌株进行代谢工程改造，以从二氧化碳（CO2）和还原无机硫化合物（RISC）中产生化学物质。对极端嗜热嗜酸古菌的泛基因组学分析揭示了一种新的CO2固定循环和关键基因的存在，这些基因可以被招募来使S。酸热菌氧化硫。这些独特的生物学特性将在天然耐热和耐酸生物体的代谢工程中得到利用。代谢工程方法将通过对野生型和重组S.酸热生理学本项目的具体目标是：（1）构建和表征工程S。利用CO2作为碳源和RISC作为能源的嗜酸热菌DSM 639菌株。S. acidocaldarius编码一个CO2固定循环和一个不完整的硫氧化途径，可以用化能自养硫化叶菌的基因修复。(2)证明丙酮的功能性、生物合成、嗜热途径可以工程化到S.酸热菌DSM 639。已经鉴定了可以从其他嗜热微生物中募集的基因/酶，以使S.酸热菌从单糖中生产工业化学品。(3)证明S.酸热菌DSM 639可以从作为碳源的CO2和作为能源的RISC产生丙酮。目标是在化能自养条件下获得丙酮生产。(4)在生物反应器规模上，建立了工程化的S。嗜酸热菌DSM 639菌株可以产生丙酮，其可以通过原位蒸馏回收。T-X-Y信息表明，丙酮-水混合物在含水丙酮浓度下形成两相系统，这将促进作为工艺强化步骤的回收。该项目将展示极端嗜热嗜酸菌作为多功能代谢工程平台的实用性，展示从工业废物流中生产生物基化学品的独特方法。该奖项反映了NSF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。

Sulfur compounds and carbon dioxide are generated in copious amounts as byproducts of natural gas and petroleum refining processes, which creates significant challenges in their safe handling and economical disposal. Fortunately, microorganisms have much to offer toward the development of environmentally-sound and energy-efficient processes to recycle these industrial waste streams. Sulfur and carbon dioxide also happen to be potential sources of energy and carbon for microorganisms called thermoacidophilic archaea that grow in extremely hot, acidic environments. One potential approach to dealing with the excess industrial sulfur and carbon dioxide is to engineer the metabolic pathways of these archaea such that they use the byproducts to produce chemicals of commercial value. This project will apply recently developed tools for genetically engineering extreme thermoacidophiles to create novel microorganisms. The study will examine the hypothesis that inserting sulfur oxidation genes from one organism into another will result in a new microorganism capable of converting carbon dioxide and sulfur into industrially-relevant organic chemicals. Engineering sulfur oxidation genes into microorganisms that naturally thrive under extreme conditions has many potential benefits. The ability to grow at hot and acidic conditions minimize risk of contamination by undesirable microorganisms and enables the bioprocess to be operated much like a conventional chemical process. The high heat tolerance of the microorganisms is also advantageous in recovering biochemical products with low boiling points. To broaden the educational impact and inclusiveness of this study, faculty and students at the North Carolina School for the Deaf (NCSD) will participate through biotechnology-based teaching modules. The modules will incorporate closed-captioning and other features to facilitate educational objectives for the deaf and hearing-impaired. Modules will be demonstrated on-site at NCSD. Additionally, the NCSU Biotechnology Program will host visiting middle school and high school students from NCSD for a day-long laboratory experience that will also familiarize them with higher educational opportunities. The objective of this project is to metabolically engineer strains of a model thermoacidophilic archaeon, Sulfolobus acidocaldarius DSM639, to produce chemicals from carbon dioxide (CO2) and reduced, inorganic sulfur compounds (RISCs). Pangenomic analysis of the extremely thermoacidophilic archaea revealed the presence of a novel CO2 fixation cycle and key genes that can be recruited to enable S. acidocaldarius to oxidize sulfur. These unique biological characteristics will be exploited in the metabolic engineering of the naturally heat- and acid-tolerant organism. Metabolic engineering approaches will be informed by fundamental assessments of wild-type and recombinant S. acidocaldarius physiology. The specific objectives of this project are: (1) Create and characterize engineered S. acidocaldarius DSM639 strains that utilize CO2 as a carbon source and RISCs as energy sources. S. acidocaldarius encodes a cycle for CO2 fixation and an incomplete pathway for sulfur oxidation that can be repaired with genes recruited from chemolithoautotrophic Sulfolobales. (2) Demonstrate that a functional, biosynthetic, thermophilic pathway for acetone can be engineered into strains of S. acidocaldarius DSM639. Genes/enzymes have been identified that can be recruited from other thermophilic microorganisms to enable S. acidocaldarius to produce an industrial chemical from simple sugars. (3) Demonstrate that engineered strains of S. acidocaldarius DSM639 can produce acetone from CO2 as a carbon source and RISCs as energy sources. The goal is to obtain acetone production under chemolithoautotrophic conditions. (4) Establish, at bioreactor-scale, that engineered S. acidocaldarius DSM639 strains can produce acetone that can be recovered through in situ distillation. T-x-y information indicates that acetone-water mixtures form a two-phase system at aqueous acetone concentrations that will facilitate recovery as a process intensification step. The project will demonstrate the utility of extreme thermoacidophiles as versatile metabolic engineering platforms, presenting a unique approach toward bio-based chemical production from an industrial waste stream.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.