Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum

Abstract

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO₂ and H₂). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (t_D~4 h) compared to growth on yeast extract (t_D~9 h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.

Introduction

Gas fermentation has emerged as an attractive alternative for renewable and sustainable production of fuels and chemicals from abundant, inexpensive and non-food-based feedstocks e.g. industrial waste gases, syngas (CO/CO₂ and H₂) and methane (Bertsch and Müller, 2015, Daniell et al., 2012, Dürre and Eikmanns, 2015, Köpke et al., 2011, Latif et al., 2014, Liew et al., 2016b). Production of fuels and chemicals via gas fermentation does not compete for arable land and food resources, in contrast to using farmed sugars as feedstock. Furthermore, gas fermentation offers high product versatility while capturing carbon that would otherwise be released into the atmosphere. Syngas fermentation is particularly interesting as CO and H₂ are energy-rich, and many low-cost syngas sources are available. Since gases can be derived from a variety of sources, including industrial processes and gasification of carbonaceous waste, gas fermentation provides a robust platform for the production of a wide variety of fuels and chemicals. As the technology advances, gas fermentation could potentially be used to produce many of the chemicals identified by the US Department of Energy as important for the transition to a bio-based economy (Cueto-Rojas et al., 2015).

Acetogenic clostridia are the ideal biocatalysts for gas fermentation since they can autotrophically grow on gas as their sole carbon and energy source using the Wood-Ljungdahl pathway (WLP) (Fuchs, 2011, Ragsdale and Pierce, 2008, Wood, 1991). From the many acetogens, Clostridium autoethanogenum is a promising gas-fermenting biocatalyst used at industrial scale (Liew et al., 2016b). C. autoethanogenum natively produces acetate, ethanol, 2,3-butanediol and lactate as by-products of growth (Abrini et al., 1994, Köpke et al., 2011, Marcellin et al., 2016). In most cases, the majority of carbon (both during hetero- and autotrophic growth) is excreted as acetate to restore ATP balance in the WLP, which is an unwanted carbon loss in a bioprocess. However, it seems that acetate excretion per se is not hard-wired into acetogenic metabolism as both process optimization (Abubackar et al., 2016, Abubackar et al., 2015, Richter et al., 2013) and genetic engineering (Huang et al., 2016, Liew et al., 2016a) approaches have successfully abolished acetate excretion.

Future biorefineries need to be versatile and rely on the production of several products. Expanding the product spectrum of acetogens through rational designs is therefore essential. In particular, for production of energy-intensive molecules from gas, limitation in ATP availability is a challenge in the design of new strains, as acetogenic metabolism operates at the thermodynamic edge of life (Bertsch and Müller, 2015, Ragsdale and Pierce, 2008, Schuchmann and Müller, 2014). Thus, to accelerate engineering of efficient biocatalysts that avoid by-products such as acetate, alternative ATP-generating pathways need to be explored.

Genome-scale metabolic models (GEMs) have become an essential part of metabolic engineering and systems biology analysis of cells. GEMs combine a general metabolic reconstruction capturing the stoichiometry of all known metabolic reactions in a given organism with appropriate boundary values and an optimality criterion for a specific question of interest. GEMs can, for instance, predict phenotypes, explain metabolic switches and estimate intracellular flux patterns (Bordbar et al., 2014, Dash et al., 2016, O’Brien et al., 2015). In conjunction with shadow price analysis (Hillier and Lieberman, 2010), GEMs have been used to determine substrates, metabolites or products having the biggest effect on ATP production (Teusink et al., 2006) or cell growth (Varma et al., 1993). The shadow price is the incremental change in the objective function when a constraining boundary flux is incrementally changed.

In this work, we refined a previous C. autoethanogenum GEM (Marcellin et al., 2016) and used it to identify in silico alternative ATP-generating pathways and growth-boosting nutrients for C. autoethanogenum. In silico simulations were validated experimentally and arginine (ARG) in particular was found to significantly boosts both hetero- and autotrophic growth of C. autoethanogenum. Importantly, ARG addition almost abolishes acetate production due to the stoichiometric generation of ATP from ARG catabolism through the arginine deiminase (ADI) pathway, as demonstrated by RNA-sequencing. Growth on CDM also enabled analysis of carbon, energy and redox balances using the refined GEM.