利用II型CRISPA-Cas9系统改造锰氧化假单胞菌T34及用于生物模板法构建高势能电极材料的研究

Although increasingly more attention is being paid to the performance optimization of electrochemical materials based on the structural specificity of biomaterials, very limited reports exist on the fabrication of new biomaterials that are produced by biologically enzymatic reaction to improve material capabilities. The combinative biotemplate and bioconversion strategy that fabricates novel materials with a unique morphology and complicated structure has been recognized as a sustainable and environmentally friendly approach to material manufacturing. Previously, we have developed an electrochemical material using biomineralized manganese oxide aggregates from a Pseudomonas sp. T34 strain with Mn2+-oxidizing activity and the ability to form manganese oxide aggregates under Mn2+ induction, however, although the electrochemical performance was comparable to similar materials that were fabricated through chemical methods, the relatively low Mn content of the aggregates showed the necessity of improving Mn2+-oxidizing activity of T34 strain. In this proposal, we focus on the promotion of whole-cell Mn2+-oxidizing activity of T34 through genetic modification of this train. The whole-cell transcriptomics of T34 under Mn2+-inducing and Mn2+-lacking culture conditions will initially determined to allow probing the manganese oxidation network, substrate and energy metabolisms and metabolic influx, which will be referred to determine the target genes those are not relatively associated to manganese oxidation in its genome. A newly hot-spot genome-editing technology, namely Type II CRISPA_Cas9 system, will be employed to inactive total 50 different non-Mn oxidation-associated genes in T34’s genome that are selected above, and the activity-enhanced mutant strains with regard to both Mn2+-oxidation and aggregate formation with increased Mn content are screened through comparative assays. Consequently, one mutant strain with highest Mn2+-oxidizing activity and outstanding aggregate-formation capacity will be used as the parent strain as a precursor to synthesize a biocomposite that will be doped with Co, Ni and Al at different formula under mild shake-flask trial conditions, based on the biomineralization process of biogenic Mn oxides and the characteristics of metal ion subsiders through the distinctive biosorption/biooxidation activities of this train. The aggregates will be fabricated the hollow porous carbonaceous MnO-based cation-doped multiphasic biocomposites after high-temperature annealing of 800C and in the presence of Ar. Assays of HRTEM, pore size and porosity, BET specific surface area, thermogravimetric analysis, Raman spectrum, XRD (including XRD Rietveld analysis), EDS and XPS will performed to verify the phase composition and fine structures of the prepared materials. Furthermore, the cycling performance and rate performance of the fabricated materials used as the anode materials for lithium-ion batteries will compared by using different electrochemical analyses. The main aim of this proposal is at developing an anode material with the reversible discharge capacity after 100 cycles remains higher than 700 mAh g 1, at a current density of 0.1 Ag 1, which is a very good composite cycling stability and reversible specific capacity over the conventionally prepared similar materials by chemical methods. The proposed biotemplate strategy could be expanded to applications such as biocatalysis and sensor fields.

以一株具锰氧化和生物锰氧化物矿化活性的假单胞菌T34为对象，在测定该菌株在Mn2+诱导前后转录组活性的变化后，根据其锰氧化代谢网络和锰诱导下的物质与能量代谢流向，利用II型CRISPA-Cas9基因编辑技术沉默其基因组中与锰氧化非关联的50种基因，定向筛选锰氧化活性增强与锰氧化物矿物锰量增加的突变株，并利用其生物矿化活性和生物吸附掺杂作用，将Co2+/Ni2+/Al3+外源金属以不同配比掺杂于氧化物聚集体，和以其作为前体物在Ar气下于800度碳化，最终制备一种以MnO/C纳米晶体为基相和多相彼此掺杂并共同镶嵌于碳基质的、高势能中空多孔复合物材料。在进行扫描电镜、孔径分布与大小、比表面积、EDS、热重、拉曼光谱、XRD和XPS对聚集体进行多重表征后，测定其用于锂离子电池负极时的电池循环性能和倍率性能，使在0.1 A g-1电流密度下100个循环后的可逆放电容量大于700 mAh g-1。

生物锰氧化物是通过存在于淡水、海洋和土壤中的微生物活动产生的，被认为是环境中最丰富，反应性最强的锰氧化物。本项目以一株具有锰氧化和生物锰氧化物矿化活性的假单胞菌T34为对象，研究了一种以定向筛选锰氧化增效突变株为基础并利用其生物矿化活性和生物吸附掺杂作用制备高势能中空多孔复合材料的新技术。使用转录组测序、蛋白组测序、构建转座文库等复合筛选手段，并结合代谢通路分析和RT-qPCR实验，建立了T34中关键的碳代谢和芳香族代谢网络。通过对代谢网络中关键节点的筛选和锰氧化增效株转座突变位点的鉴定，确定需要进行编辑的基因。利用Ⅱ型CRISPR-Cas12a基因编辑系统和CRISPR-Cas9n单碱基编辑系统沉默了T34基因组中锰氧化非关联和代谢节点的多处基因，获得了锰氧化活性提高30%的增效突变株，对其进行XRD、XPS和EDS的表征，证明生物氧化锰结晶弱粒径小处于非晶体或弱结晶态，因此是良好的锂电池负极材料前驱体。设计了一种独特的生物矿化掺入方法，其中Ni2+和Co2+在温和条件下被结合到生物锰氧化物的晶体结构中,随后将聚集体在800°C高温下碳化，并制造出MnO/C/NiO和MnO/C/Co3O4两种高电化学性能的生物模板中空多孔复合材料。在0.1 A g–1的电流密度下，CMC-Co在50次循环后的剩余可逆放电容量为650 m Ah g–1，随着循环次数的增加效率稳定在99%，CMB-Ni在在50次循环后为379.29 m Ah g-1，放电比容量在第二次循环后基本不变，库仑效率始终稳定在100%左右。利用生物氧化锰和带有表面展示多铜氧化酶CotA的大肠杆菌细胞的复合物完全降解双酚A和壬基酚。构建了用于儿茶酚检测的表面展示漆酶的工程大肠杆菌细胞电化学微生物生物传感器。利用一株高活性的锰氧化活性的假单胞菌，通过用形成微/纳米结构的生物氧化锰(BMO)聚集体进行环丙沙星降解。制备了一种壳聚糖微球作为表面展示漆酶的恶臭假单胞菌的载体用于合成染料的脱色。对细菌锰氧化作用机制、尤其是细菌碳代谢对锰氧化作用和锰氧化矿物的引发与形成进行了研究分析。项目为深入了解细菌的生物锰氧化矿化作用的发生机制和建立方便实用的应用于电池电极、传感系统和生物环境修复等多领域生物模板技术平台系统提供了新的研究基础。