Design and analysis of synthetic carbon fixation pathways

Abstract

Carbon fixation is the process by which CO 2 is incorporated into organic compounds. In modern agriculture in which water, light, and nutrients can be abundant, carbon fixation could become a significant growth-limiting factor. Hence, increasing the fixation rate is of major importance in the road toward sustainability in food and energy production. There have been recent attempts to improve the rate and specificity of Rubisco, the carboxylating enzyme operating in the Calvin-Benson cycle; however, they have achieved only limited success. Nature employs several alternative carbon fixation pathways, which prompted us to ask whether more efficient novel synthetic cycles could be devised. Using the entire repertoire of approximately 5,000 metabolic enzymes known to occur in nature, we computationally identified alternative carbon fixation pathways that combine existing metabolic building blocks from various organisms. We compared the natural and synthetic pathways based on physicochemical criteria that include kinetics, energetics, and topology. Our study suggests that some of the proposed synthetic pathways could have significant quantitative advantages over their natural counterparts, such as the overall kinetic rate. One such cycle, which is predicted to be two to three times faster than the Calvin-Benson cycle, employs the most effective carboxylating enzyme, phosphoenolpyruvate carboxylase, using the core of the naturally evolved C4 cycle. Although implementing such alternative cycles presents daunting challenges related to expression levels, activity, stability, localization, and regulation, we believe our findings suggest exciting avenues of exploration in the grand challenge of enhancing food and renewable fuel production via metabolic engineering and synthetic biology. metabolic engineering | synthetic biology | photosynthesis | carboxylation | biological optimization I n the process of transforming sunlight into stored chemical energy, plants absorb approximately 10 times more CO 2 from the atmosphere than the total amount emitted by human activities globally (1). Moreover, agriculture, which is effectively a massive carbon fixation industry, makes use of the majority of cultivatable land on earth and accounts for most of the fresh water used by humanity (2). These figures point to the central role that carbon fixation by plants plays in our global ecological footprint. In nature, the factors limiting the growth of photosynthetic organisms vary among habitats and often include the availability of water, light, fixed nitrogen, iron, and phosphorus (e.g., ref.3). However, in agriculture today, the use of fertilizers and irrigation can make carbon fixation a rate-limiting factor. For example, many C3 plants have shown a significant increase in biomass when exposed to twice the atmospheric CO 2 concentration (4). Impressive growth enhancements have been demonstrated by addressing several biochemical limiting factors, related both to the light-dependent and light-independent reactions (5-9). For instance, tran...

A warm welcome for alternative CO2 fixation pathways in microbial biotechnology

“…(A) The ‘inefficient’ Calvin–Benson–Bassham cycle with the ‘slow’ carboxylase Rubis CO as key enzyme, the cycle has a consumption of seven ATP per pyruvate or acetyl‐CoA produced, which does not include additional costs for photorespiration in aerobic conditions (Bar‐Even et al ., 2010). (B) An example of a proposed, promising synthetic MOG ‐pathway, based on the ‘faster’ carboxylase pyruvate carboxylase (Bar‐Even et al ., 2010), estimates for its ATP consumption are based on assimilation via the bacterial glycerate pathway for pyruvate and the reverse glyoxylate shunt for acetyl‐CoA.…”

“…(B) An example of a proposed, promising synthetic MOG ‐pathway, based on the ‘faster’ carboxylase pyruvate carboxylase (Bar‐Even et al ., 2010), estimates for its ATP consumption are based on assimilation via the bacterial glycerate pathway for pyruvate and the reverse glyoxylate shunt for acetyl‐CoA. Catalytic turnover rates indicated for both carboxylases are based on ambient CO 2 concentrations (Bar‐Even et al ., 2010). …”

“…This side‐activity results in the formation of 2‐phoshpoglycoylate that has to be re‐assimilated to the Calvin cycle through photorespiration pathways. These photorespiration pathways partly counteract CO 2 fixation by releasing some CO 2 and typically add ~40–50% extra NADPH and ATP to the costs of CO 2 fixation (Bar‐Even et al ., 2010). …”

“…Moreover, attractive carboxylases can be embedded in synthetic CO 2 fixation pathways, which have been extensively explored by computational analyses (e.g. Bar‐Even et al ., 2010; Volpers et al ., 2016). Several of the alternative natural and synthetic CO 2 fixation pathways have lower ATP costs than the Calvin cycle, however, some trade‐offs exist.…”

“…As an example, a promising group of synthetic pathways are the malonyl‐CoA–oxaloacetate–glyoxylate (MOG) pathways (Fig. 1B) (Bar‐Even et al ., 2010). These pathways include the catalytically fast phosphoenol pyruvate carboxylase or pyruvate carboxylase and are expected to have fast overall pathway kinetics.…”