RESUMO
One of seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl Pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.
RESUMO
Clostridium autoethanogenum is a model gas-fermenting acetogen for commercial ethanol production. It is also a platform organism being developed for the carbon-negative production of acetone and isopropanol by gas fermentation. We have assembled a 5.5 kb pCA plasmid for type strain DSM10061 (JA1-1) using three genome sequence datasets. pCA is predicted to encode seven open-reading frames and estimated to be a low-copy number plasmid present at approximately 12 copies per chromosome. RNA-seq analyses indicate that pCA genes are transcribed at low levels and two proteins, CAETHG_05090 (putative replication protein) and CAETHG_05115 (hypothetical, a possible Mob protein), were detected at low levels during batch gas fermentations. Thiolase (thlA), CoA-transferase (ctfAB), and acetoacetate decarboxylase (adc) genes were introduced into a vector for isopropanol production in C. autoethanogenum using the native plasmid origin of replication. The availability of the pCA sequence will facilitate studies into its physiological role and could form the basis for genetic tool optimization.
RESUMO
Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.
