光合细菌耐铵突变株的构建与产氢特性研究

Construction and Hydrogen Production Characterization of Ammonium-Tolerant Photosynthetic Bacterial Mutants

摘要

为了缓解铵离子（NH⁺₄）对光发酵制氢的抑制作用，以荚膜红细菌（Rhodobacter capsulatus，R. capsulatus）SB1003为研究对象，通过基因工程构建耐铵突变株并评估其产氢性能。首先，采用重叠延伸聚合酶链式反应（SOE-PCR）对固氮调控基因（nifA）进行改造，获得nifA1 N端缺失突变株ZX01、nifA2 N端缺失突变株ZX02及双缺失突变株ZX03。然后，将上述菌株接种于以葡萄糖为碳源、不同浓度硫酸铵和谷氨酸钠为混合氮源的产氢液中，在光照、厌氧条件下进行光发酵产氢。通过实验比较不同突变株在含铵和无铵条件下的产氢性能及相关基因表达水平，结果表明，在铵态氮质量分数为20%时，ZX03的累计产氢量最高为（801.80±60.40）mL·L^-1，较野生型提高22.7%。Gompertz动力学拟合表明，ZX01的产氢迟滞期最短为（7.99±3.41）h，表明其产氢启动更快。实时定量聚合酶链式反应PCR（qPCR）分析结果表明，在高铵条件（NH⁺₄量浓度为8 mmol·L^-1）下，ZX03中氮信号转导蛋白编码基因glnB1和glnB2的转录水平较野生型上调220倍，电子传递相关基因rnfB及黄素氧化还原蛋白编码基因fldA的表达亦显著增强。研究结果表明，nifA1 N端缺失缩短产氢迟滞期，nifA2 N端缺失提升基础产氢能力；双缺失株耐铵性最优，且上调氮信号与电子传递相关基因表达，从而在高铵条件下实现最优产氢。该研究可为构建耐铵高效光发酵产氢工程菌株提供明确的分子改造策略与理论依据。

Abstract

To alleviate the inhibitory effect of ammonium ions (NH⁺ ₄) on photo-fermentative hydrogen production, Rhodobacter capsulatus SB1003 was selected as the model organism. Ammonium-tolerant mutant strains were constructed via genetic engineering, and their hydrogen production performance was evaluated. The nitrogen fixation regulatory gene (nifA) was modified using overlap extension PCR (SOE-PCR), generating three engineered strains: the nifA1 N-terminal deletion mutant ZX01, the nifA2 N-terminal deletion mutant ZX02, and the double-deletion mutant ZX03. These strains were inoculated into a hydrogen production medium with glucose as the carbon source and with different concentrations of ammonium sulfate and sodium glutamate as mixed nitrogen sources, and were cultivated under anaerobic and illuminated conditions for photo-fermentative hydrogen production. The hydrogen production performance, lag phase characteristics, and related gene expression profiles were comparatively analyzed under both ammonium-containing and ammonium-free conditions. The results show that at an ammonium nitrogen mass fraction of 20%, ZX03 achieves the highest cumulative hydrogen production of (801.80±60.40) mL·L^-1, representing a 22.7% increase compared with the wild-type strain. Gompertz kinetic modeling further shows that ZX01 exhibits the shortest lag phase (7.99±3.41) h, indicating significantly accelerated initiation of hydrogen production. Quantitative real-time polymerase chain reaction PCR (qPCR) analysis reveals that under high ammonium conditions (NH⁺ ₄ concentration of 8 mmol·L^-1), the transcription levels of nitrogen signal transduction genes (glnB1 and glnB2) in ZX03 are upregulated by approximately 220-fold relative to the wild type. In addition, genes associated with electron transport, including rnfB, and the flavodoxin-encoding gene fldA, are also markedly upregulated, indicating enhanced metabolic electron flux. Overall, these findings demonstrate that N-terminal deletion of nifA1 primarily shortens the hydrogen production lag phase, whereas nifA2 N-terminal deletion enhances basal hydrogen production capability. Notably, the double-deletion mutant ZX03 exhibits the strongest ammonium tolerance and optimal hydrogen production performance under high ammonium stress by simultaneously upregulating nitrogen signaling and electron transport-related genes. This study provides a clear and effective molecular engineering strategy, as well as a theoretical foundation, for the development of robust, ammonium-tolerant, high-efficiency photo-fermentative hydrogen-producing strains.

关键词

光发酵制氢 / 光合细菌 / 突变株 / 光发酵 / 产氢 / 基因编辑

Key words

photo-fermentative hydrogen production / photosynthetic bacteria / mutant / photo-fermentation / hydrogen production / gene editing

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基金资助

国家自然科学基金资助项目(52488201)