BackgroundIn 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.ResultsIn 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.ConclusionsHere, 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0530-7) contains supplementary material, which is available to authorized users.

Phosphoribulokinase (PrkA) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been proposed to create a heterologous Rubisco-based engineered pathway in Escherichia coli for in situ CO2 recycling. While the feasibility of a Rubisco-based engineered pathway has been shown, heterologous expressions of PrkA and Rubisco also induced physiological responses in E. coli that may compete with CO2 recycling. In this study, the metabolic shifts caused by PrkA and Rubisco were investigated in recombinant strains where ppc and pta genes (encodes phosphoenolpyruvate carboxylase and phosphate acetyltransferase, respectively) were deleted from E. coli MZLF (E. coli BL21(DE3) Δzwf, ΔldhA, Δfrd). It has been shown that the demand for ATP created by the expression of PrkA significantly enhanced the glucose consumptions of E. coli CC (MZLF Δppc) and E. coli CA (MZLF Δppc, Δpta). The accompanying metabolic shift is suggested to be the mgsA route (the methylglyoxal pathway) which results in the lactate production for reaching the redox balance. The overexpression of Rubisco not only enhanced glucose consumption but also bacterial growth. Instead of the mgsA route, the overproduction of the reducing power was balanced by the ethanol production. It is suggested that Rubisco induces a high demand for acetyl-CoA which is subsequently used by the glyoxylate shunt. Therefore, Rubisco can enhance bacterial growth. This study suggests that responses induced by the expression of PrkA and Rubisco will reach a new energy balance profile inside the cell. The new profile results in a new distribution of the carbon flow and thus carbons cannot be majorly directed to the Rubisco-based engineered pathway.

Whole-cell degradation of polyesters not only avoids the tedious process of enzyme separation, but also allows the degraded product to be reused as a carbon source. In this study, Escherichia coli BL21(DE3) harboring phaZ , a gene encoding poly(3-hydroxybutyrate) (PHB) depolymerase from Caldimonas manganoxidans, is constructed. The extra-cellular fraction of E. coli/pPHAZ exhibits a fast PHB degradation rate where it only took 35 h to completely degrade PHB films, while C. manganoxidans takes 81 h to do the same. The co-expression of ORF (a putative periplasmic substrate binding protein that is within the same operon of phaZ ) further improves the PHB degradation. While 28 h is needed for E. coli/pPHAZ to cause an 80% weight loss in PHB films, E. coli/pORFPHAZ needs only 21 h. Furthermore, it is able to degrade at-least four different polyesters, PHB, poly(lactic acid) (PLA), polycaprolactone (PCL), and poly(butylene succinate-co-adipate) (PBSA). Testing of the time course of 3-hydroxybutyrate concentration and the turbidity of the degradation solutions over time shows that PhaZ has both exo- and endo-enzymatic activity. The whole-cell E. coli/pORFPHAZ can be used for recycling various polyesters while ORF can potentially be a universal element for enhancing the secretion of recombinant protein.