甲烷营养细菌由于其使用CH4作为碳源和能源的独特能力,在生物地球化学碳循环中起着至关重要的作用。有证据表明一些甲烷污染菌,包括荚膜甲基球菌,也可以使用二氧化碳作为碳源,使这些细菌成为开发针对温室气体捕获和缓解的生物技术的有希望的候选者。然而,需要对CH4和CO2的双重代谢有更深入的了解,以指导甲烷菌菌株的改进并实现其工业实用性。在这项研究中,我们显示荚膜分枝杆菌表达五种碳酸酐酶(CA)亚型,一个α-CA,一个γ-CA,和三个β-CA,在其无机碳代谢和二氧化碳依赖性生长中起作用。CA同种型差异表达,并且所有同工型基因的转录都是响应CO2限制而诱导的。与等基因野生型对照相比,CA无效突变菌株表现出明显的生长受损,表明CA同工型具有独立的,在荚膜分枝杆菌代谢和生理学中的非冗余作用。一些过度表达,但不是全部,CA同工型改善了细菌生长动力学,并减少了消耗CH4的培养物中的CO2释放。值得注意的是,我们开发了一种过表达天然α-CA和β-CA的工程化甲烷营养生物催化剂,在CH4转化为生物质方面提高了2.5倍。鉴于产品产量是基于甲烷菌的生物过程的重要成本驱动因素,这里开发的工程菌株可以提高CH4生物催化的经济性,包括从天然气或厌氧消化衍生的沼气中生产单细胞蛋白。重要甲烷营养生物将CH4转化为CO2和多碳化合物,因此,它们在全球碳循环中起着至关重要的作用,并对生物技术应用感兴趣。一些甲烷营养生物,包括荚膜甲基球菌,也可以使用二氧化碳作为碳源,但是这种一碳的双重代谢还没有完全理解。在这项研究中,我们表明,荚膜分枝杆菌碳酸酐酶对于该细菌最佳利用CO2至关重要。我们开发了一种具有改善的CO2利用能力的工程菌株,该菌株增加了向细胞生物质的整体碳转化。本文观察到的对基于甲烷菌的产物产率的改进预期降低与CH4转化生物过程相关的成本。
Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH4 and CO2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO2-dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO2 evolution from CH4-consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH4 into CO2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus, can also use CO2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO2. We developed an engineered strain with improved CO2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH4 conversion bioprocesses.