Liang Yuqian, Motawaa Mohamed, Bu Xuying, Wei Junwei, Shao Yuan, Li Yingjun
National Key Laboratory of Agricultural Microbiology, College of Life Science & Technology, Huazhong Agricultural University, Wuhan, 430070, P.R. China.
Department of Agricultural Biotechnology, Faculty of Agriculture, Damietta University, Damietta, 34511, Egypt.
Microb Cell Fact. 2025 Jul 10;24(1):163. doi: 10.1186/s12934-025-02785-y.
Thermus thermophilus HB27 is a promising thermophilic chassis for recombinant thermostable protein production, owing to its high optimal growth temperature, which can simplify downstream processing and reduce contamination risks. However, maximizing its potential requires optimized genetic tools and host strains. Key limitations include a shortage of well-characterized strong constitutive promoters and potential degradation of recombinant proteins by proteases. To address these, we established a β-galactosidase reporter system (endogenous TTP0042) to screen for strong constitutive promoters and investigated the impact of deleting specific protease genes on protein expression.
Screening of 13 endogenous promoter regions identified P0984 as exhibiting significantly 13-fold higher activity than the control promoter driving the reporter gene. Constructing a plasmid-free strain (HB27ΔpTT27) successfully minimized 270 kb of the genome; it exhibited auxotrophy for cobalamin (requiring 0.1 µg/ml AdoCbl for growth) and a slightly reduced growth rate compared to the wild-type, while its transformation efficiency remained comparable. Notably, a CRISPR-deficient precursor strain (HB27ΔIII-ABΔI-CΔCRF3) showed a significant (~ 100-fold) increase in transformation efficiency compared to the wild-type, facilitating subsequent genetic manipulations. Systematic knockout of 16 predicted non-essential protease loci was performed. Characterization revealed that deletion of TTC0264 (putative ClpY/HslU) and TTC1905 (putative HhoB) significantly reduced extracellular proteolytic activity. Iterative deletion based on phenotypic analysis led to strain DSP9 (10 protease loci deletions), which maintained robust growth and exhibited enhanced accumulation of the β-galactosidase reporter protein compared to the parental strains.
This study provides foundational advancements for T. thermophilus HB27 chassis development, and genetic tools represent valuable resources for optimizing T. thermophilus as a platform for heterologous thermostable protein production and ideas for antibiotic-free systems.
嗜热栖热菌HB27因其较高的最适生长温度，是用于重组耐热蛋白生产的一种很有前景的嗜热底盘生物，这可以简化下游加工并降低污染风险。然而，要充分发挥其潜力需要优化的遗传工具和宿主菌株。关键限制包括缺乏充分表征的强组成型启动子以及蛋白酶对重组蛋白的潜在降解。为了解决这些问题，我们建立了一个β-半乳糖苷酶报告系统（内源性TTP0042）来筛选强组成型启动子，并研究删除特定蛋白酶基因对蛋白表达的影响。
对13个内源性启动子区域的筛选确定P0984驱动报告基因的活性比对照启动子显著高13倍。构建无质粒菌株（HB27ΔpTT27）成功使基因组减少了270 kb；它对钴胺素表现出营养缺陷型（生长需要0.1 μg/ml腺苷钴胺素），与野生型相比生长速率略有降低，但其转化效率仍相当。值得注意的是，与野生型相比，CRISPR缺陷型前体菌株（HB27ΔIII-ABΔI-CΔCRF3）的转化效率显著提高（约100倍），便于后续的基因操作。对16个预测的非必需蛋白酶基因座进行了系统敲除。表征显示，删除TTC0264（假定的ClpY/HslU）和TTC1905（假定的HhoB）显著降低了细胞外蛋白水解活性。基于表型分析的迭代删除产生了菌株DSP9（删除了10个蛋白酶基因座），与亲本菌株相比，该菌株保持了强劲的生长，并表现出β-半乳糖苷酶报告蛋白的积累增加。
本研究为嗜热栖热菌HB27底盘生物的开发提供了基础性进展，遗传工具是优化嗜热栖热菌作为异源耐热蛋白生产平台的宝贵资源，也为无抗生素系统提供了思路。
