二氧化碳固定酶固碳效率及其对地衣芽孢杆菌代谢的影响

Carbon fixation efficiency of carbon dioxide fixation enzymes and its effect on the metabolism of Bacillus licheniformis

摘要:

【背景】随着代谢工程与合成生物学的快速发展，通过对异养微生物进行代谢改造，利用生物法进行二氧化碳固定成为一个新的趋势。生物代谢途径中存在着大量固碳酶，这些酶尚待挖掘与应用，不同的酶固碳效率之间也缺少比较。【目的】在体外和体内对固碳功能和效率进行评价。【方法】选取3种固碳酶，即核酮糖1,5-二磷酸羧化加氧酶(ribose 1,5-diphosphate carboxylation oxygenase, RuBisCo)、磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PCK)和乙酰辅酶A羧化酶(acetyl coenzyme A carboxylase, ACC)在大肠杆菌中异源表达并纯化。测定纯酶的酶活，并建立无细胞催化实验-液质联用评价酶固碳能力的方法。在厌氧发酵条件下检测代谢指标，比较过表达固碳酶的地衣芽孢杆菌相较于原始菌的代谢差异。【结果】3种酶均实现可溶性表达，纯酶的比酶活分别为66.43、1.16和12.52 U/mg。通过体外无细胞催化实验，ACC在3种酶中表现出最高的固碳效率。分别过表达了PCK、ACC的重组地衣芽孢杆菌，厌氧发酵主产物乳酸的转化率从48.6%分别提升至58.1%和59.7%。【结论】可以通过体外、体内结合的方式对固碳酶的效率进行评价，该研究可为固碳酶在微生物遗传改造中理性、精准地应用提供参考。

关键词:核酮糖1,5-二磷酸羧化加氧酶;磷酸烯醇式丙酮酸羧激酶;乙酰辅酶A羧化酶;二氧化碳固定;LC-MS/MS;地衣芽孢杆菌

Abstract:

[Background] With the rapid development of metabolic engineering and synthetic biology, carbon dioxide fixation by biological methods through metabolic modification of heterotrophic microorganisms has become a new trend. There are a large number of carbon fixation enzymes in biological metabolic pathways, which are yet to be explored and applied, and there is a lack of comparison among the carbon fixation efficiencies of different enzymes. [Objective] To evaluate the carbon fixation function and efficiency in vitro and in vivo. [Methods] Three carbon fixation enzymes, namely, ribulose 1,5-diphosphate carboxylation oxygenase (RuBisCo), phosphoenolpyruvate carboxykinase (PCK), and acetyl coenzyme A carboxylase (ACC), were heterologously expressed in Escherichia coli and then purified. The enzymatic activities of the pure enzymes were determined, and a cell-free catalytic assay-liquid mass spectrometry method was established to evaluate the carbon fixation capacity of the enzymes. Metabolic indicators were examined under anaerobic fermentation conditions and the differences between Bacillus licheniformis overexpressing carbon fixation enzymes compared to the original bacteria were compared. [Results] All three enzymes achieved soluble expression with specific enzyme activities of 66.43, 1.16, and 12.52 U/mg for pure enzymes, respectively. RuBisCo and ACC exhibited stronger carbon fixation efficiency in cell-free catalytic assays. The conversion of lactic acid, the main product of anaerobic fermentation, was increased from 48.6% to 58.1% and 59.7%, respectively, in B. licheniformis with the two expressed recombinant enzymes. [Conclusion] The efficiency of carbon fixation enzymes can be evaluated by in vitro and in vivo binding. This study may provide references for rational and precise application of carbon fixation enzymes in microbial genetic modification.

Key words:ribose 1,5-diphosphate carboxylation oxygenase;phosphoenolpyruvate carboxykinase;acetyl coenzyme A carboxylase;carbon dioxide fixation;LC-MS/MS;Bacillus licheniformis