| With the rapid development of synthetic biology and biotechnology,metabolic engineering of heterotrophic organisms for biocarbon fixation has become a trend.Existing biological fixation studies primarily focus on the analysis and research of the characteristics of natural carbon fixation pathways,lacking direct comparisons of the efficiency of carbon-fixing enzymes in different pathways and systems or conditions that establish their direct correlation.On the other hand,Bacillus licheniformis,when fermenting under oxygen-limited conditions,releases a significant amount of carbon source in the form of CO2,causing raw material waste,and research on carbon fixation in B.licheniformis is relatively limited.In this study,carbon-fixing enzymes were screened and functionally characterized,and an in vitro cell-free catalytic assay coupled with liquid chromatography-mass spectrometry（LC-MS/MS）was established to compare the carbon fixation efficiency of different carbon-fixing enzymes.Relevant carbon-fixing enzymes were overexpressed in B.licheniformis,and the metabolic characteristics of the recombinant strains were analyzed through fermentation experiments.The specific results are as follows:（1）Among the 20 identified carbon-fixing enzymes,ribulose-1,5-bisphosphate carboxylase/oxygenase（Ru Bis CO）,phosphoenolpyruvate carboxykinase（PCK）,and acetyl-Co A carboxylase（ACC）from different sources were heterologously expressed in Escherichia coli DE3.Soluble expression was achieved for at least one source of each enzyme.The purified enzymes exhibited specific activities of 66.43,1.16,and 12.52 U·mg-1,respectively,based on different enzymatic activity assay methods,demonstrating their biological activity.However,enzyme activity alone does not directly reflect the differences in carbon fixation efficiency among different carbon-fixing enzymes.（2）An in vitro cell-free catalytic assay coupled with LC-MS/MS was established to evaluate the carbon fixation efficiency of different carbon-fixing enzymes.The cell-free catalytic assay revealed that the carbon fixation rate of the enzymes was positively correlated with enzyme concentration and reaction time.All three enzymes effectively participated in the reaction with different forms of CO2 as their substrates.ACC exhibited a higher carbon fixation rate,consuming 0.124 m M of CO2 within 15 min of reaction.（3）Overexpression of carboxylase is the simplest way to enhance the fixation of carbon dioxide by heterotrophic organisms.PCK and ACC were overexpressed in B.licheniformis to study the metabolic characteristics of the recombinant strains.Fermentation results showed varying degrees of improvement in product conversion rate and product conversion rate per cell.For example,under anaerobic fermentation,BL2-p HY-P2-PCK increased the conversion rates of lactate,2,3-butanediol,and acetate from 48.6%,16.5%,and 15.3%to 58.1%,22.2%,and 23.2%,respectively,while BL2-p HY-P2-ACC increased the conversion rates of the three products to 59.7%,18.9%,and 26.8%,respectively.RT-q PCR results indicated that overexpression of carboxylases increased the overall transcription levels of key genes in the metabolic pathway,promoting intracellular metabolism and leading to more product formation.（4）Based on the identified ACC enzyme with higher carbon fixation efficiency,malonyl-Co A reductase（MCR）was introduced into the recombinant strain BL2-p HY-P2-ACC,constructing two theoretically 3-hydroxypropionic acid（3-HP）-producing strains,KWD1 and KWD2.Using fermentation medium containing cottonseed protein,trace amounts of 3-HP was detected in KWD2 after 120 hours of fermentation as confirmed by LC-MS/MS analysis.This study provides a research approach for comparing the efficiency of different carbon-fixing enzymes and offers a reference basis for the efficiency of key carboxylases in the rational design of artificial carbon fixation pathways.Furthermore,the study can serve as a reference for the rational and precise application of carbon-fixing enzymes in microbial genetic engineering. |
