The comprehensive profile of fermentation products during in situ CO2 recycling by Rubisco-based engineered Escherichia coli.

Background: In our previous study, the feasibility of Rubisco-based engineered E. coli (that contains heterologous phosphoribulokinase (PrkA) and Rubisco) for in situ CO2 recycling during the fermentation of pentoses or hexoses was demonstrated. Nevertheless, it is perplexing to see that only roughly 70 % of the carbon fed to the bacterial culture could be accounted for in the standard metabolic products. This low carbon recovery during fermentation occurred even though CO2 emission was effectively reduced by Rubisco-based engineered pathway.

Results: In this study, the heterologous expression of form I Rubisco was found to enhance the accumulation of pyruvate in Escherichia coli MZLF [E. coli BL21(DE3) Δzwf, Δldh, Δfrd]. This may be attributed to the enhanced glycolytic reaction supported by the increased biomass and the ethanol/acetate ratio. Besides, it was found that the transcription of arcA (encodes the redox-dependent transcriptional activators ArcA that positively regulates the transcription of pyruvate formate-lyase) was down-regulated in the presence of Rubisco. The enhanced accumulation of pyruvate also occurs when PrkA is co-expressed with Rubisco in E. coli MZLF. Furthermore, E. coli containing Rubisco-based engineered pathway has a distinct profile of the fermentation products, indicating CO2 was converted into fermentation products. By analyzing the ratio of total C-2 (2-carbon fermentation products) to total C-1 (1-carbon fermentation product) of MZLFB (MZLF containing Rubisco-based engineered pathway), it is estimated that 9 % of carbon is directed into Rubisco-based engineered pathway.

Conclusions: Here, we report for the first time the complete profile of fermentation products using E. coli MZLF and its derived strains. It has been shown that the expression of Rubisco alone in MZLF enhances the accumulation of pyruvate. By including the contribution of pyruvate accumulation, the perplexing problem of low carbon recovery during fermentation by E. coli containing Rubisco-based engineered pathway has been solved. 9 % of glucose consumption is directed from glycolysis to Rubisco-based engineered pathway in MZLFB. The principle characteristics of mixotroph MZLFB are the high bacterial growth and the low CO2 emission.