The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum.

The Wood-Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald-Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh-Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these 'stored assets' for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.

Metabolic engineering of Clostridium autoethanogenum for ethyl acetate production from CO.

BACKGROUND: Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. RESULTS: We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. CONCLUSIONS: This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future.

Functional expression of a bacterial α-ketoglutarate dehydrogenase in the cytosol of Saccharomyces cerevisiae.

Efficient production of fuels and chemicals by metabolically engineered micro-organisms requires availability of precursor molecules for product pathways. In eukaryotic cell factories, heterologous product pathways are usually expressed in the cytosol, which may limit availability of precursors that are generated in other cellular compartments. In Saccharomyces cerevisiae, synthesis of the precursor molecule succinyl-Coenzyme A is confined to the mitochondrial matrix. To enable cytosolic synthesis of succinyl-CoA, we expressed the structural genes for all three subunits of the Escherichia coli α-ketoglutarate dehydrogenase (αKGDH) complex in S. cerevisiae. The E. coli lipoic-acid scavenging enzyme was co-expressed to enable cytosolic lipoylation of the αKGDH complex, which is required for its enzymatic activity. Size-exclusion chromatography and mass spectrometry indicated that the heterologously expressed αKGDH complex contained all subunits and that its size was the same as in E. coli. Functional expression of the heterologous complex was evident from increased αKGDH activity in the cytosolic fraction of yeast cell homogenates. In vivo cytosolic activity of the αKGDH complex was tested by constructing a reporter strain in which the essential metabolite 5-aminolevulinic acid could only be synthetized from cytosolic, and not mitochondrial, succinyl-CoA. To this end HEM1, which encodes the succinyl-CoA-converting mitochondrial enzyme 5-aminolevulinic acid (ALA) synthase, was deleted and a bacterial ALA synthase was expressed in the cytosol. In the resulting strain, complementation of ALA auxotrophy depended on activation of the αKGDH complex by lipoic acid addition. Functional expression of a bacterial αKGDH complex in yeast represents a vital step towards efficient yeast-based production of compounds such as 1,4-butanediol and 4-aminobutyrate, whose product pathways use succinyl-CoA as a precursor.

Requirements for Carnitine Shuttle-Mediated Translocation of Mitochondrial Acetyl Moieties to the Yeast Cytosol.

UNLABELLED: In many eukaryotes, the carnitine shuttle plays a key role in intracellular transport of acyl moieties. Fatty acid-grown Saccharomyces cerevisiae cells employ this shuttle to translocate acetyl units into their mitochondria. Mechanistically, the carnitine shuttle should be reversible, but previous studies indicate that carnitine shuttle-mediated export of mitochondrial acetyl units to the yeast cytosol does not occur in vivo This apparent unidirectionality was investigated by constitutively expressing genes encoding carnitine shuttle-related proteins in an engineered S. cerevisiae strain, in which cytosolic acetyl coenzyme A (acetyl-CoA) synthesis could be switched off by omitting lipoic acid from growth media. Laboratory evolution of this strain yielded mutants whose growth on glucose, in the absence of lipoic acid, was l-carnitine dependent, indicating that in vivo export of mitochondrial acetyl units to the cytosol occurred via the carnitine shuttle. The mitochondrial pyruvate dehydrogenase complex was identified as the predominant source of acetyl-CoA in the evolved strains. Whole-genome sequencing revealed mutations in genes involved in mitochondrial fatty acid synthesis (MCT1), nuclear-mitochondrial communication (RTG2), and encoding a carnitine acetyltransferase (YAT2). Introduction of these mutations into the nonevolved parental strain enabled l-carnitine-dependent growth on glucose. This study indicates intramitochondrial acetyl-CoA concentration and constitutive expression of carnitine shuttle genes as key factors in enabling in vivo export of mitochondrial acetyl units via the carnitine shuttle. IMPORTANCE: This study demonstrates, for the first time, that Saccharomyces cerevisiae can be engineered to employ the carnitine shuttle for export of acetyl moieties from the mitochondria and, thereby, to act as the sole source of cytosolic acetyl-CoA. Further optimization of this ATP-independent mechanism for cytosolic acetyl-CoA provision can contribute to efficient, yeast-based production of industrially relevant compounds derived from this precursor. The strains constructed in this study, whose growth on glucose depends on a functional carnitine shuttle, provide valuable models for further functional analysis and engineering of this shuttle in yeast and other eukaryotes.