Two propanediol utilization-like proteins of Moorella thermoacetica with phosphotransacetylase activity

Abstract

Moorella thermoacetica is one of the model acetogenic bacteria for the resolution of the Wood-Ljungdahl (acetyl-CoA) pathway in which CO2 is autotrophically assimilated yielding acetyl-CoA as central intermediate. Its further conversion into acetate relies on subsequent phosphotransacetylase (PTA) and acetate kinase reactions. However, the genome of M. thermoacetica contains no pta homologous gene. It has been speculated that the moth_0864 and moth_1181 gene products sharing similarities with an evolutionarily distinct phosphotransacylase involved in 1,2-propanediol utilization (PDUL) of Salmonella enterica act as PTAs in M. thermoacetica. Here, we demonstrate specific PTA activities with acetyl-CoA as substrate of 9.05 and 2.03 U/mg for the recombinant enzymes PDUL1 (Moth_1181) and PDUL2 (Moth_0864), respectively. Both showed maximal activity at 65 °C and pH 7.6. Native proteins (90 kDa) are homotetramers composed of four subunits with apparent molecular masses of about 23 kDa. Thus, one or both PDULs of M. thermoacetica might act as PTAs in vivo catalyzing the penultimate step of the Wood-Ljungdahl pathway toward the formation of acetate. In silico analysis underlined that up to now beside of M. thermoacetica, only Sporomusa ovata contains only PDUL like class(III)-PTAs but no other phosphotransacetylases or phosphotransbutyrylases (PTBs).

Acetic acid stress response of the acidophilic sulfate reducer Acididesulfobacillus acetoxydans

“…The Pta‐AckA pathway can work in reverse and the flux is thermodynamically controlled by the extracellular acetate concentration (Enjalbert et al, 2017). Interestingly, a phosphate propanoyl transferase ( pdul , 2860), was 9.2‐fold upregulated in Ac‐Sp1 (compared to the control) potentially acting as alternative Pta in response to perturbation in the Pta‐Ack pathway (Breitkopf et al, 2016; Liu, Leal, et al, 2007). This flux perturbation could be a mechanism for growth inhibition due to ATP expenditure next to proton and anion stress (Pinhal et al, 2019).…”

Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production

“…The differences could be derived from the responsible enzymatic activities, levels of gene expression, or metabolic redox state, in addition to the original metabolic features of M. thermoacetica Y72. For example, both key enzymatic activities, PduL1 for acetate and bsALDC for acetoin, were higher at 60°C than at 55°C in vitro ( Breitkopf et al, 2016 ; Jia et al, 2017 ). Furthermore, PduL1 activity was more enhanced to a greater extent by a temperature shift ( Breitkopf et al, 2016 ).…”

“…For example, both key enzymatic activities, PduL1 for acetate and bsALDC for acetoin, were higher at 60°C than at 55°C in vitro ( Breitkopf et al, 2016 ; Jia et al, 2017 ). Furthermore, PduL1 activity was more enhanced to a greater extent by a temperature shift ( Breitkopf et al, 2016 ). These differences may have contributed to different culture profiles at different temperatures.…”

Isopropanol production via the thermophilic bioconversion of sugars and syngas using metabolically engineered Moorella thermoacetica

“…However, no ethanol production was observed in the pduL2 -deleted strain, which slowed the carbon flow to acetate. The reported activity of Sadh for acetyl-CoA reduction is 5.5 U/mg [ 18 ], whereas that of PduL1, which is responsible for the remaining acetate production, is 265 U/mg [ 28 ]. The difference in the activity of these competing enzymes might explain no ethanol production.…”