Original Research ArticleReaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea
Introduction
Concerns over sustainability and global climate change have generated interest in developing biological systems for industrial production of fuels and chemicals, with particular interest in using inorganic carbon feed stocks, such as CO2 (Conrado et al., 2013). To do so a CO2 fixation pathway is needed, six of which are currently known: the 3-hydroxypropionate (3HP) bicycle, the dicarboxylate/4-hydroxybutyrate (DC/4HB) cycle, the reductive citric acid cycle, the reductive acetyl-CoA (Wood-Ljungdahl) pathway, the Calvin-Benson-Bassham cycle, and the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle (Berg, 2011, Berg et al., 2010, Herter et al., 2002, Huber et al., 2008). There have been efforts to produce chemicals and fuels based on several of these pathways, including the 3HP bicycle, Calvin-Benson-Bassham cycle, reductive acetyl-CoA pathway, and, of interest here, the 3HP/4HB cycle (Keller et al., 2013, Li et al., 2012, Mattozzi et al., 2013, Muller et al., 2013, Ueki et al., 2014, Yuzawa et al., 2012) (see Table 1).
The 3HP/4HB cycle is a promising candidate for microbial production of chemicals from CO2 for several reasons. First, it functions at high temperatures, allowing it to be used in an extremely thermophilic host with concomitant minimal risk of contamination and reduced cooling costs (Keller et al., 2015, Zeldes et al., 2015). Second, the 3HP/4HB cycle can function in either an aerobic or anaerobic host, unlike the DC/4HB and reductive acetyl-CoA pathways, which are found exclusively in anaerobic organisms (Fast and Papoutsakis, 2012). Third, it was shown that the 3HP/4HB cycle can drive rapid autotrophic growth with a doubling time of less than five hours (Hawkins et al., 2013), suggesting the potential for fast pathway kinetics. Components of the 3HP/4HB cycle can be identified in genomes within the crenarchaeal order Sulfolobales (Kockelkorn and Fuchs, 2009), and has been studied most intensively in the extremely thermoacidophilic archaeon Metallosphaera sedula (Topt=73 °C; pHopt=2.0) (Auernik and Kelly, 2010, Berg et al., 2010, Hawkins et al., 2014). The cycle can be divided into three sub-pathways to track the reduction of CO2 into acetyl-CoA. In the first sub-pathway, acetyl-CoA is carboxylated by acetyl-CoA/propionyl-CoA carboxylase (ACC) and subsequently reduced to the stable intermediate 3HP (Fig. 1, reactions 1–3). In the second sub-pathway, 3HP is ligated to coenzyme A (CoA), reduced to propionyl-CoA, carboxylated by ACC, converted to succinyl-CoA, which is further reduced to the second stable intermediate 4HB (Fig. 1, reactions 4–12). In the third sub-pathway, 4HB is ligated to CoA and cleaved to regenerate the starting substrate and produce an additional molecule of acetyl-CoA (Fig. 1, reactions 13–17). Cellular intermediates used for biomass generation are drawn from the cycle through the intermediate acetyl-CoA, as well as through succinic semialdehyde via oxidation to succinate by succinic semialdehyde dehydrogenase (Fig. 1, reactions 18–22) (Estelmann et al., 2011). Isotopic labeling studies in M. sedula have shown that 65% of cellular intermediates are formed via succinate, while the remaining 35% of carbon enters cellular metabolism via acetyl-CoA (Estelmann et al., 2011). Putative cycle enzymes have been previously characterized to various extents, although some remain to be verified and characterized in purified form (Alber et al., 2006, Alber et al., 2008, Han et al., 2012, Hawkins et al., 2013, Hawkins et al., 2014, Hugler et al., 2003, Kockelkorn and Fuchs, 2009, Ramos-Vera et al., 2011, Teufel et al., 2009).
Outside of its natural context, there are many opportunities to use 3HP/4HB cycle as a route to renewable production of chemicals. The complete cycle for metabolic engineering could enable the production of chemicals directly from CO2, while an alternative is to use portions of the complete cycle for production of chemicals from sugars via the intermediate acetyl-CoA. Prior to introducing all or parts of the 3HP/4HB cycle into a metabolically engineered host organism, it is useful to identify and address potential bottlenecks. To this end, genome-scale flux balance modeling has been used extensively to assist metabolic engineering of model microorganisms, such as Escherichia coli and Saccharomyces cerevisiae (Kerkhoven et al., 2014, McCloskey et al., 2013). However, enzyme kinetics-based models can account for features such that the interplay of biochemical reaction pathways can be considered, making them valuable for testing metabolic engineering strategies (Kerkhoven et al., 2014, Loder et al., 2015). Here, we describe the development of such a model to explore aspects of the 3H/4HB cycle first as it operates in its native form in the model extreme thermoacidophile Metallosphaera sedula and then as a basis for production of bio-based fuels and chemicals, focusing on the cycle intermediates acetyl-CoA, 3HP, and succinate.
Section snippets
Materials
Growth conditions for M. sedula (DSM 5348) and genomic DNA purification were conducted, as reported previously (Auernik and Kelly, 2010). Strains and vectors used for cloning included the pET-46b EK/LIC cloning Kit, pRSF-2 Ek/LIC Vector Kit, pCDF-2 Ek/LIC Vector Kit, Novablue GigaSingles™ E. coli competent cells (Novagen, San Diego, CA), and Rosetta™ (DE3) E. coli competent cells (Stratagene, La Jolla, CA). The reagents and devices used include: n-propionyl-Coenzyme A lithium salt, succinyl-CoA
Characterization of recombinant cycle enzymes involved in 3HP to 4HB conversion
While enzymes of the 3HP/4HB cycle in M. sedula had been characterized to some extent prior to this work, detailed kinetics information was not available in many cases for the purified proteins. This was especially the case for the segment of the cycle converting 3HP to 4HB. To this end, the gene identities of HPCS, HPCD, ACR, MCR, and SSR were confirmed via recombinant expression of active enzymes in E. coli (characterization of MCE and MCM was reported previously (Han et al., 2012)). Kinetic
Metabolic engineering analysis of the 3HP/4HB cycle
To examine ways in which the 3HP/4HB cycle could be used for metabolic engineering, different pathways to three products were analyzed using the reaction kinetics model. These pathways, composed of subsets of the cycle enzymes, were for the production of acetyl-CoA from CO2 (autotrophic growth), succinate from CO2 (autotrophic growth), 3HP from acetyl-CoA (heterotrophic growth), and succinate from acetyl-CoA (heterotrophic growth). The first two routes require all of the carbon to be derived
Concluding remarks
Assessment of potential strategies for in vivo applications of the 3HP/4HB cycle indicates advantages and disadvantages relative to previously described efforts (Table 5). The autotrophic acetyl-CoA and heterotrophic 3HP pathways have the most promise, the former because it offers the opportunity to produce chemicals directly from CO2, and the latter because of its high flux potential along with the potential for reduced CO2 emissions. Both pathways for succinate production (autotrophic and
Author contributions
R.M.K. and M.W.W.A. conceived of and managed the research. A.J.L. developed the mathematical model. A.J.L., Y.H., H.L., A.B.H. performed enzyme analysis, G.J.S. and G.L.L. provided advice on biochemical and genetic experiments. A.J.L., Y.H., A.B.H, M.W.W.A., and R.M.K. wrote the manuscript.
Acknowledgments
This work was supported by grants to RMK and MWWA by the US Department of Energy Research ARPA-E Electrofuels Program (DE-AR0000081), the US National Science Foundation (CBET-1264052, CBET-1264053), and the US Air Force Office of Scientific Research (AFOSR) (FA9550-13-1-0236). ABH acknowledges support from a US Department of Education (P200A140020) GAANN Fellowship and AJL acknowledges support from an NIH Biotechnology Traineeship (2T32GM008776).
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- 1
Contributed equally to this work.
- 2
Current address: Novozymes North America Inc., Franklinton, NC 27525.
- 3
Current address: Institute of Process Engineering, Chinese Academy of Sciences, 1 Beiertiao, Zhongguancun, Haidan, Beijing, 100190, China.
- 4
Current address: School of Public Health, Xiamen University, South Xinag-An Road, Xiang-An District, Xiamen, Fujan Province, 361102, China.