摘要
磷是植物生长发育必需的营养元素之一,然而植物从土壤中可获取的磷素十分有限,土壤中存在大量难利用的有机难降解磷,其中植酸(肌醇六磷酸)占据了较大部分。目的利用植酸酶对植酸的高效水解能力,对黑土土著细菌进行植酸酶基因修饰,提高土壤有效磷含量。方法利用锚定蛋白pGSA对细菌植酸酶AppA进行表面展示,以提高蛋白酶的稳定性和活性以及底物的接触效率。基于CRISPR靶向基因插入修饰技术,将表面展示的植酸酶融合蛋白基因靶向转座至一株从黑土分离的皮氏罗尔斯顿氏菌(Ralstonia pickettii)G3的基因组16S rRNA基因位点,以摆脱传统蛋白表达的载体依赖。结果细菌16S rRNA基因可以作为基因修饰的通用靶点,并且不会对该菌的增殖产生明显影响。植酸酶基因修饰菌对植酸的水解能力提高了8倍以上,并且可以在较宽的pH范围内发挥作用。将该基因修饰菌施用于黑土后,土壤植酸酶活性明显提升,且速效磷含量提高了近30%。结论植酸酶基因修饰的土著细菌能够促进土壤植酸水解,增加土壤有效磷含量,提高土壤磷素有效性。
Phosphorus is an essential nutrient element for plant growth and development.However,the available phosphorus in soil is extremely limited due to the presence of large amounts of organic phosphorus that is difficult to be degraded,with phytate(inositol hexaphosphate)accounting for a significant proportion.Phytases can efficiently hydrolyze phytate and release available phosphorus.Objective By taking advantage of the efficient hydrolysis ability of phytase,the phytase gene was gene modified in the indigenous bacteria of black soil to increase the available phosphorus content in the soil.Methods We employed the anchored protein pGSA for surface display of the bacterial phytase AppA,thereby enhancing the stability and enzymatic activity of the protein as well as improving the substrate contact efficiency.Furthermore,leveraging the CRISPR-targeted gene editing technology,we precisely integrated the surface-displayed phytase fusion protein into the 16S rRNA gene of Ralstonia pickettii G3 genome,isolated from black soil to overcome the dependence of protein expression on vectors.Results The 16S rRNA gene site could be used as a target for gene modification without significant effect on the proliferation of the bacteria.The phytase-modified engineered bacteria showed a more than 8-fold increase in the hydrolytic ability of phytate and functioned in a wide pH range.After this indigenous engineered bacterial strain was applied to black soil,the soil phytase activity significantly increased,and the available phosphorus content rose by nearly 30%.Conclusion Modifying phytase by gene editing can promote the hydrolysis of phytate,increase the content of available phosphorus,and improve the phosphorus availability in the soil.
作者
韩伟
解雨竹
陈琦
刘佳鑫
张博
张颖
HAN Wei;XIE Yuzhu;CHEN Qi;LIU Jiaxin;ZHANG Bo;ZHANG Ying(School of Resources and Environment,Northeast Agricultural University,Harbin,Heilongjiang,China)
出处
《微生物学报》
北大核心
2025年第8期3287-3300,共14页
Acta Microbiologica Sinica
基金
国家重点研发计划(2023YFD1501005)
国家自然科学基金(32101362)
黑龙江省自然科学基金(LH2022D005,LH2022D006)。
