Department of Biology and Biochemistry, Milner Centre for Evolution, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
Department of Architecture and Civil Engineering, BRE Centre for Innovative Construction Materials, University of Bath, Bath, BA2 7AY, United Kingdom.
Microb Cell Fact. 2021 Nov 18;20(1):214. doi: 10.1186/s12934-021-01704-1.
Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation.
In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity.
Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.
微生物诱导碳酸钙沉淀（MICP）是细菌的一种古老特性，最近因其在生物技术应用中的潜力而受到广泛关注。它是细菌代谢的副产物，涉及细胞外环境中的化学变化的组合，例如 pH 值升高，以及细胞表面上的成核位点或细菌产生的细胞外物质的存在。然而，支撑 MICP 的分子机制以及促成因素之间的相互作用仍知之甚少，从而阻碍了细菌生物矿化的全面生物技术和合成生物学开发。
在这项研究中，我们采用了自下而上的方法，系统地对枯草芽孢杆菌进行工程改造，枯草芽孢杆菌本身没有检测到内在的 MICP 活性，用于生物矿化。我们表明，异源产生的脲酶可以通过局部增加细胞外 pH 值来诱导 MICP，并且这可以通过共表达脲酶辅助基因来增强，用于尿素和镍的摄取，具体取决于环境条件。生物膜促进条件可以强烈增强 MICP，这似乎主要是由胞外多糖的产生驱动的，而生物膜基质的蛋白质成分是可有可无的。尝试调节枯草芽孢杆菌的细胞表面电荷出人意料地产生了较小的影响，我们的结果表明，该生物体可能固有地具有非常负的细胞表面，可能使其倾向于 MICP 活性。
我们的研究结果深入了解了在相关应用底盘生物体中驱动 MICP 的分子机制，以及可用于工程设计从头开始或增强生物矿化的遗传元件。这项研究还突出了遗传驱动因素与周围环境的化学成分之间的相互影响，这些因素决定了 MICP 的速度、空间分布和产生的矿物晶体。总而言之，这些数据为未来针对特定应用优化的理性设计合成沉淀剂菌株铺平了道路。
