Abstract
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a key enzyme responsible for biological CO2 assimilation. RuBisCO can be heterologously expressed in Escherichia coli so that glucose and CO2 are co-metabolized to achieve high mixotrophic metabolite production, where the theoretical yield of mixotrophic metabolite production is 2.4 mol(ethanol+acetate+pyruvate)/molglucose. However, RuBisCO is known for its low kcat and for forming inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates, yet the inhibited form of RuBisCO can be reversed by RuBisCO activase (Rca). In this study, RuBisCO forms I and II were cloned and expressed in Escherichia coli for in situ CO2 recycling, where CO2 produced during glucose fermentation was recycled and co-metabolized with the glucose. In addition, forms I and II RuBisCO activases were co-expressed with RuBisCO in E. coli to determine their in vivo effects on in situ CO2 recycling. Form I RuBisCO activase (Rca1) was co-expressed with form I RuBisCO and form II RuBisCO activase (Rca2) was co-expressed with form II RuBisCO. The results showed that both form I and form II RuBisCO exhibit comparable activities in E. coli and generated similar levels of in situ CO2 recycling. A significant increase in the total metabolite yield from 1.5 ± 0.1 to 2.2 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose occurred when Rca2 was co-expressed with form II RuBisCO. Meanwhile, the total metabolite yield increased from 1.7 ± 0.1 to 2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose when Rca1 was co-expressed with form I RuBisCO. This data suggests that both forms I and II RuBisCO are subject to in vivo RuBP inhibition yet can be relieved by the co-expression of Rca. Interestingly, it is suggested that the in vivo RuBP inhibition of form II RuBisCO can be more easily reversed compared to form I. When the catalytic power of RuBisCO is maintained by Rca, the high activity of phosphoribulokinase (Prk) plays an important role in directing glucose to the RuBisCO-based engineered pathway and fermentation yields of 2.1–2.3 mol(ethanol+acetate+pyruvate)/molglucose can be obtained. This study is the first to demonstrate that in vivo RuBP inhibition of RuBisCO can be a bottleneck for in situ CO2 recycling in E. coli.
Highlights
The Calvin-Benson-Bassham (CBB) cycle is one of the main metabolic pathways for converting inorganic CO2 to organic carbon during photosynthesis (Tabita et al, 2008; Nielsen et al, 2012; François et al, 2020)
This study demonstrated that both form I and form II RuBisCO can be functionally expressed in E. coli (Table 2), which is consistent with previous studies which showed that the functional expression of bacterial Rubisco in E. coli is not of concern (Parikh et al, 2006; Tsai et al, 2015; Antonovsky et al, 2016; Tseng et al, 2018)
The enzymatic assay of RuBisCO indicates that both form I and form II RuBisCO are subject to RuBP inhibition, but the RuBP inhibition can be relieved in the presence of RuBisCO activase (Rca) (Table 2)
Summary
The Calvin-Benson-Bassham (CBB) cycle is one of the main metabolic pathways for converting inorganic CO2 to organic carbon during photosynthesis (Tabita et al, 2008; Nielsen et al, 2012; François et al, 2020). The first RuBisCO structure determined by X-ray crystallography was from the bacterium Rhodospirillum rubrum, which is categorized as form II (Tabita et al, 2008) This enzyme is composed of only two large subunits (L2) which share 25–30% identity to the L subunits of form I RuBisCO. Based Pathway Elevates Both the Performance of the in situ CO2 Recycling and Glucose Consumption of the E. coli Strain Harboring Form I RuBisCO. In addition to increasing the total metabolite yields for the two cases with form I RuBisCO, the introduction of the Pdc-based pathway recovered the glucose consumption back to 40 mM (Figure 2B) and increased the carbon recovery (Figure 2C) for the two cases with form I RuBisCO. When Rca was co-expressed with the RuBisCO-based engineered pathway that was equipped with a matching form of RuBisCO, the total metabolite yields of the strains with form I RuBisCO and Rca were found to be close to 2 (2.0 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose for rbcLS and 1.9 ± 0.1 mol(ethanol+acetate+pyruvate)/molglucose for AfcbbLS), while a strain with form II RuBisCO with Rca exceeded 2 and reached 2.2 ± 0.2 mol(ethanol+acetate+pyruvate)/molglucose (p-values are less than 0.05 for three pairs regarding the Rca co-expression)
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