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

盐单胞菌是一种强大的微生物底盘，目前用于工业规模生产生物塑料（聚3-羟基丁酸酯）。目前正在开发的新的Halomonas生物技术应用包括将其用于生产生物液化石油气（丙烷和丁烷）、单萜类生物燃料添加剂和扁桃酸盐。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，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途的工业基质，因为它可以在非无菌条件下在可再生废生物质上的海水/废水中培养，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战略，需要更有效地利用混合碳源，以最大限度地提高单位废物的生物量（和二次产品）产量。C3生物技术公司感兴趣的主要废物是乙酸，它在一些生产过程中积累。在本项目中，将使用合成生物学方法来设计盐单胞菌基因组整合重组醋酸利用路线。已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精心调节的。盐单胞菌中类似的系统是未知的，因此大肠杆菌系统将被纳入盐单胞菌中，包括一个关键酶乙酰辅酶a合成酶的变体，以允许醋酸盐作为发酵废物进入中心代谢物乙酰辅酶a。通过回收发酵过程中产生的醋酸盐和消耗废弃生物质饲料中自然存在的醋酸盐，这将增加总体碳利用率。培养中细胞毒性醋酸盐积累的减少应显著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。嗜盐单胞菌的第二个目标废物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能自养CO2固定途径，这些酶将在添加外部能量源（例如氢，甲酸和硫化合物）的情况下发挥作用。多种化学岩石自养二氧化碳固定途径已被描述，如基于Calvin-Benson-Bassham循环、还原性乙酰辅酶a途径、二羧酸盐/4-羟基丁酸盐循环和3-羟基丙酸盐/4-羟基丁酸盐循环的途径。该项目旨在采用一种设计更简单的二氧化碳固定途径，利用硫代硫酸盐作为能源。在整合到嗜盐单胞菌的工业菌株基因组之前，该途径将首先在基于质粒的系统上进行有效性测试。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二次产品提供经济、可持续、安全和清洁的替代品。通过解决对化学品和生物燃料生产的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DTP流。同时，它解决了关键的气候控制目标，通过设想一种碳中性的解决方案，产生一种工业固定碳的盐单胞菌菌株，能够提供具有成本竞争力的，非化石燃料衍生的生物途径，以获得有用的化合物。

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.