副地衣芽孢杆菌合成甘氨酸甜菜碱对其耐盐性的影响

Synthesis of glycine betaine affects salt tolerance of Bacillus paralicheniformis

摘要:

盐渍化土地给生态和农业造成了极大影响。生物修复是盐渍化地治理的重要绿色手段，提高根际促生菌(plant growth-promoting rhizobacteria, PGPR)耐盐性是提升生物修复效果的关键。甘氨酸甜菜碱(简称甜菜碱，glycine betaine, GB)作为重要的渗透压调节剂，可增强微生物和植物的抗逆性。为提高PGPR在盐渍化土壤中的存活率，本研究采用生物法合成GB，使用新型PGPR副地衣芽孢杆菌(Bacillus paralicheniformis) Bp1为底盘菌株，改造GB合成途径。首先，敲除甘氨酸竞争/消耗途径中的相关基因maeA、aceB、iclR和gcvP，强化乙醛酸循环(引入大肠杆菌来源基因aceAK)，引入外源高效转氨酶基因agx1，得到菌株Bp1Z09，使GB前体甘氨酸在发酵30 h内最高产量达到34.27 mg/L，较野生型12 h时的最高产量提升293.9%。同时，为保证菌株稳定性，将aceAK整合至基因组得到菌株Bp1Z11。之后，在Bp1Z11中引入嗜盐隐杆藻(Aphanothece halophytica)甲基化酶基因gsmt和sdmt，构建GB合成工程菌Bp1Z12，发酵至36 h时GB产量为2.56 mg/L，较野生型提高326.7%。在0.3 mol/L NaCl (模拟中高度盐渍化土壤环境)胁迫下，工程菌株GB产量在发酵至36 h时达4.94 mg/L (较无盐对照提升93.7%)，生物量(OD₆₀₀)增至16.22 (较无盐对照提升20.9%)，凸显了其在盐渍化土壤微生物修复中的应用潜力。此外，Bp1Z12在盐渍化土中可有效缓解番茄受到的盐胁迫，提高番茄在盐渍化土中的生长能力，Bp1Z12处理组的根长较H₂O和Bp1处理组分别显著增加75.0%和27.3%，株高较H₂O和Bp1组分别增加76.9%和21.1%，叶面积较H₂O和Bp1组分别增加77.8%和45.0%。工程菌Bp1Z12能高效利用葡萄糖合成GB，兼具良好耐盐性与促植物耐盐能力，为后续以该菌株为基础开发抗逆微生物肥料或土壤处理剂及进行盐渍化土壤修复提供了新的思路。

Abstract:

Global salinized land greatly affects the ecology and agriculture. Biological remediation is an important green approach for managing salinized soil. Enhancing the salt tolerance of plant growth-promoting rhizobacteria (PGPR) is key to improving the effectiveness of biological remediation. Glycine betaine (GB), an important osmotic regulator, can enhance the stress resistance of microorganisms and plants. In order to improve the survival rate of PGPR in salinized soil, GB was synthesized by biological method. In this study, we used a novel PGPR strain Bacillus paralicheniformis Bp1 as the host to reconstruct the GB synthesis pathway. First, the genes maeA, aceB, iclR, and gcvP in the glycine competition/consumption pathway were knocked out, and the glyoxylate cycle was strengthened by introduction of aceAK from Escherichia coli. Additionally, the exogenous high-efficiency transaminase gene agx1 was introduced. The highest yield of GB precursor, glycine, reached 34.27 mg/L within 30 h of fermentation, representing a 293.9% increase compared with the highest yield of the wild-type at the time point of 12 h. Meanwhile, to ensure the stability of the strain, aceAK was integrated into the genome to obtain strain Bp1Z11. Next, the methyl transferase genes gsmt and sdmt from Aphanothece halophytica were introduced into Bp1Z11 to construct the engineered strain Bp1Z12, which achieved a GB yield of 2.56 mg/L (a 326.7% increase compared with the wild type) after 36 h of fermentation. Under 0.3 mol/L NaCl stress (simulating moderately salinized soil conditions), the engineered strain achieved a GB yield of 4.94 mg/L (a 93.7% increase compared with the salt-free control) after 36 h of fermentation, with the biomass (OD₆₀₀) increasing to 16.22 (a 20.9% increase compared with the salt-free control). Additionally, Bp1Z12 effectively alleviated salt stress of tomato plants and enhanced their growth in salinized soil. The root length of tomato plants in the Bp1Z12 treatment increased significantly by 75.0% and 27.3% compared with that in the water and Bp1 treatments, respectively. The plant height of the Bp1Z12 treatment increased by 76.9% and 21.1% compared with that in the water and Bp1 treatments, respectively. The leaf area of this treatment increased by 77.8% and 45.0% compared with that in the water and Bp1 treatments, respectively. The engineered strain Bp1Z12 can efficiently utilize glucose to synthesize GB, while exhibiting good salt tolerance and plant growth-promoting ability in salinized soils. This study provides new ideas for application of this strain in the development of stress-tolerant microbial fertilizers or the remediation of salinized soils in the future.