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

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