Metabolic engineering of Halomonas for the utilisation of organic acids and CO2 as carbon sources

利用有机酸和二氧化碳作为碳源的卤单胞菌代谢工程

• 批准号: 2494710
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2494710
• 关键词: Metabolic; engineering; Halomonas; utilisation; organic

Halomonas is a robust microbial chassis currently in use in the industrial scale production of bioplastics (poly-3-hydroxybutyrate). New biotechnological applications for Halomonas currently under development include its use in the production of bio-LPG (propane and butane), monoterpenoid-based biofuel additives and mandelate. Effective sustainable and scalable microbial production strategies require considerable reductions in fermentation costs to enable scaled production to be competitive with existing biological and/or chemical production routes. Towards this end, Halomonas is a promising industrial chassis as it can be cultivated in seawater/waste water on renewable waste biomass under non-sterile conditions with minimal downstream processing, and (limited) use of fresh water. To improve the sustainability and low carbon strategy of Halomonas cultivation, a more efficient utilisation of mixed carbon sources is needed to maximise the biomass (and secondary product) yield per unit waste. A major waste product of of interest to C3 BIOTECH is acetic acid, which accumulates in some of its production processes. In this project, a synthetic biology approach will be used to engineer a genome integrated recombinant acetate utilisation route within Halomonas. Production and utilisation in E. coli are known to be carefully regulated via reversible acetylation by accessory proteins and other mechanisms. The comparable systems in Halomonas are unknown, so the E. coli system will be incorporated within Halomonas, including a variants of a key enzyme acetyl-CoA synthetase to allow the rerouting of acetate as a fermentation waste product into the central metabolite acetyl-CoA. This will increase overall carbon utilisation, both by recycling acetate generated during fermentation and the consumption of acetate naturally present in the waste biomass feed. The reduction in cytotoxic acetate accumulation within the culture should significantly improve Halomonas growth, which is likely to impact favourably on secondary product titres. A second target waste carbon source for Halomonas is atmospheric carbon dioxide. Prior studies with E. coli showed that chemolithoautotrophic CO2 fixation pathways could be constructed using native enzymes, which would function with the addition of an external energy source (e.g. hydrogen, formate and sulphur compounds). Multiple chemolithoautotrophic CO2 fixation pathways have been described, such as those based on the Calvin-Benson-Bassham cycle, reductive acetyl-CoA pathway, dicarboxylate/4-hydroxybutyrate cycle and the 3-hydroxypropionate/4-hydroxybutyrate cycle. This project will aim to incorporate one of the simpler designed CO2 fixation pathways, utilising thiosulphate as the energy source. This pathway will be initially tested for effectiveness on a plasmid-based system, before integration into the genome of an industrial strain of Halomonas. Successful implementation of these low carbon strategies will ultimately provide economic, sustainable, secure and clean alternatives to extant petrochemical LPG supplies and other industrially useful secondary products. This fits within the DTP stream of Industrial Biotechnology and Bioenergy by tackling the need for sustainable biotechnological solutions towards chemicals and biofuels production. At the same time, it addresses key climate control targets by envisioning a carbon neutral solution of generating an industrial carbon fixating strain of Halomonas, capable of delivering cost-competitive, non-fossil fuel derived biological routes towards useful compounds.

盐单胞菌是一种强大的微生物底盘，目前用于生物塑料（聚 3-羟基丁酸酯）的工业规模生产。目前正在开发的卤单胞菌的新生物技术应用包括其在生物液化石油气（丙烷和丁烷）、基于单萜的生物燃料添加剂和扁桃酸的生产中的用途。有效的可持续和可扩展的微生物生产策略需要大幅降低发酵成本，以使规模化生产能够与现有的生物和/或化学生产路线竞争。为此，盐单胞菌是一种很有前景的工业基础，因为它可以在非无菌条件下在海水/废水中的可再生废弃生物质上培养，下游加工最少，并且（有限）使用淡水。为了提高盐单胞菌培养的可持续性和低碳策略，需要更有效地利用混合碳源，以最大限度地提高每单位废物的生物量（和次级产品）产量。 C3 BIOTECH 感兴趣的主要废物是乙酸，它会在其一些生产过程中积累。在该项目中，将使用合成生物学方法在盐单胞菌内设计基因组整合的重组乙酸盐利用途径。众所周知，大肠杆菌的生产和利用是通过辅助蛋白和其他机制的可逆乙酰化来仔细调节的。盐单胞菌中的类似系统尚不清楚，因此大肠杆菌系统将被纳入盐单胞菌中，其中包括关键酶乙酰辅酶A合成酶的变体，以允许将作为发酵废物的乙酸盐重新路由到中心代谢物乙酰辅酶A中。这将通过回收发酵过程中产生的乙酸盐和消耗废弃生物质原料中天然存在的乙酸盐来增加总体碳利用率。培养物中细胞毒性乙酸盐积累的减少应显着改善盐单胞菌的生长，这可能对次级产物滴度产生有利影响。盐单胞菌的第二个目标废碳源是大气中的二氧化碳。先前对大肠杆菌的研究表明，可以使用天然酶构建化学自养型二氧化碳固定途径，该途径可以通过添加外部能源（例如氢、甲酸盐和硫化合物）来发挥作用。多种化学自养型 CO2 固定途径已被描述，例如基于 Calvin-Benson-Bassham 循环、还原乙酰辅酶 A 途径、二羧酸/4-羟基丁酸循环和 3-羟基丙酸/4-羟基丁酸循环的途径。该项目旨在采用设计更简单的二氧化碳固定途径之一，利用硫代硫酸盐作为能源。该途径将首先在基于质粒的系统上进行有效性测试，然后再整合到工业盐单胞菌菌株的基因组中。这些低碳战略的成功实施将最终为现有的石化液化石油气供应和其他工业上有用的次级产品提供经济、可持续、安全和清洁的替代品。通过解决化学品和生物燃料生产对可持续生物技术解决方案的需求，这符合工业生物技术和生物能源的 DTP 流程。与此同时，它通过设想一种碳中和解决方案来解决关键的气候控制目标，即产生工业固碳卤单胞菌菌株，该菌株能够提供具有成本竞争力的、非化石燃料衍生的生物途径来生产有用的化合物。