Synthetic biology of methane oxidising bacteria for the production of high-quality feed protein

背景：健康的家禽需要高质量的蛋白质和碳水化合物来源。现代家禽饲料主要由谷物和大豆等蛋白质来源组成，这可能与人类的食物消费直接竞争。加拿大和美国等国最近一直在积极生产用于动物饲料的高蛋白昆虫（Enterra饲料公司），并获得了国家监管部门的批准，可用于肉鸡饲料和宠物食品，这是同类成分中第一个获得批准的成分。同样，欧盟最近（2021年8月）批准在家禽和猪饲料中使用加工过的昆虫蛋白。为了满足家禽饲料市场的需求，同时解决环境和经济问题，我们提出了一种利用甲烷（CH4）固定细菌——甲烷氧化菌生产家禽饲料的新途径。这些细菌在单细胞蛋白质生产（用于鱼饲料）以及自然产生脂质方面有良好的商业记录。甲烷氧化菌还利用甲烷作为碳和能源的唯一来源，在工艺经济和可持续性方面具有显著优势。甲烷（CH4）是一种丰富而廉价的碳资源，但目前作为工业生物技术原料的利用不足，更多的是作为温室气体（GHG）污染物。甲烷也是厌氧消化大规模生产的沼气的主要成分，这项技术在英国已经很成熟。今天，大多数生物甲烷被用作能源燃烧，价值相对较小。作为一种低成本和可持续的原料，考虑到许多可用的来源、当前的浪费和相关的温室气体后果，甲烷为发酵和转化为高价值的生化代谢物（脂质、蛋白质和饲料）提供了一个令人兴奋的原料机会。作为世界领先的甲烷基蛋白质生产商，Calysta已经证明了甲烷氧化菌在甲烷营养成分和饲料的商业规模生产中的应用。荚膜甲球菌是他们的加工生物，已经在欧盟被批准用于养殖鱼类和猪等牲畜的饲料。大规模的甲烷发酵将减少对土地的需求，以种植牲畜所需的食物。然而，需要对以甲烷氧化菌为基础的家禽饲料和该过程的主力甲烷固定细菌进行更多的研究。目的：在本项目中，我们将探索以单细胞蛋白形式的甲烷固定菌作为鸡饲料中潜在的优质蛋白质来源的适用性。策略：我们的团队最近在荚膜分枝杆菌中建立了一个遗传工具库，包括CRISPR/Cas9， CRISPRi， Tn5转座子，这将使我们能够研究和提高荚膜分枝杆菌的甲烷固定率，并操纵细菌的生物量组成，以增强其作为鸡饲料成分的功能。主要项目任务将是：(i)鉴定具有修饰生物量组成和/或生长特征的Tn5转座子突变体，使用我们新开发的CRISPR技术研究修饰所涉及的基因和/或途径；（ii）对所选突变体在实验室规模的气体发酵生物反应器中的表现进行表征，以野生型和无糖原荚膜分枝杆菌菌株为基准；（iii）利用系统生物学和合成生物学相结合的方法研究荚膜分枝杆菌的非活性Calvin-Benson-Bassham循环；(v)对选用经改良的荚膜分枝杆菌制成的饲料的肉鸡进行小规模饲料试验，以确定其作为鸡饲料配方中一种替代的新型蛋白质来源的潜力。成果：与一家世界领先的C1公司合作，该学生将开发并展示荚膜分枝杆菌作为低成本甲烷气体全细胞蛋白质饲料的潜力。这将避免与粮食和土地资源的竞争，同时通过减少温室气体排放为环境和社会带来好处

BACKGROUND: Healthy poultry require high quality sources of protein and carbohydrates. Modern feeds for poultry consists largely of grain and protein sources such as soybean, which can be in direct competition with human food consumption. Countries like Canada and US have recently been active in producing high protein insects for animal feeds (Enterra Feed Corporation), and received national regulatory approval for use in broiler feed and pet food, the first ingredient of its kind to do so. Similarly, the EU recently (August 2021) approved the use of processed insect protein in poultry and pig feed. To meet poultry feed market demand in a way that tackles both environmental and economic concerns, we propose a novel route to poultry feed using methane (CH4) fixing bacteria, methanotrophs. These bacteria have a proven commercial track record for single cell protein production (for fish feed), as well as naturally producing lipids. Methanotrophs also utilize methane as the sole source of carbon and energy, providing significant advantages in both process economics and sustainability. Methane (CH4) is an abundant and cheap carbon resource, but is currently under-utilised as a feedstock for industrial biotechnology and is more associated as a greenhouse gas (GHG) pollutant. Methane is also a major component of biogas produced on a large scale by anaerobic digestion, technology that is well established in the UK. Today, most biogenic methane is burnt for energy and has relatively little value. Being a low-cost and sustainable feedstock, given the many sources available and current wastage and associated GHG ramifications, methane provides an exciting feedstock opportunity for fermentation and conversion into high value biochemical metabolites (lipids, proteins and feeds). As the world-leading methane-based protein producer, Calysta has demonstrated the use of methanotrophs in the commercial-scale production of nutritional ingredients and feed from methane. Methylococcus capsulatus is their process organism, which has already been approved in the EU for feeding to farmed fish and livestock such as pigs. Methane fermentations on a large scale would reduce the demand for land to grow food for livestock. However, more research is needed on methanotroph-based poultry feed and the workhorse of the process, methane-fixing bacteria. AIM: In this project, we will explore the suitability of methane-fixing bacteria in the form of single cell protein as a potential high quality protein source in chicken feed. STRATEGY: Our group recently established an arsenal of genetic tools in M. capsulatus, including CRISPR/Cas9, CRISPRi, Tn5 transposon, which will allow us to investigate and enhance the methane fixation rate in M. capsulatus and manipulate the biomass composition of bacteria to enhance their functionality as an ingredient of chicken feed.The main project tasks will be: (i) identify Tn5 transposon mutants with modified biomass composition and/or growth profile, investigate the gene and/or pathway involved in the modification using our newly developed CRISPR technology; (ii) characterize chosen mutants for its performance in lab scale gas fermentation bioreactors, using the wildtype and a glycogen minus M. capsulatus strains as benchmarks; (iii) investigate the inactive Calvin-Benson-Bassham cycle in M. capsulatus using a combined approach of system and synthetic biology; (v) carry out a small scale feed test on broiler chickens using feed made from a chosen improved M. capsulatus, to determine its potential as an alternative and novel source of protein in chicken feed formulation. OUTCOME: Working with one of the world's leading C1 companies, the student will develop and demonstrate the potential of M. capsulatus as whole cell protein feed from low cost methane gas. This will allow avoidance of competition with food and land resources while providing benefits to the environment and society through reduction in GHG emiss