Relative abundance indexes (values 1 and 2), changes in protein expression ratios (value 3), and associated V diff values (value 4) indicating confidence levels of changes in expression ratios for enzymes involved in (A) conversion of phosphoenolpyruvate to pyruvate (B) catabolism of pyruvate into end-products, and (C) electron transfer pathways between ferredoxin (Fd), NAD-(P)H, and H2. PEP, phosphoenol pyruvate; OAA, oxaloacetate; Fd, ferredoxin. Pyruvate Catabolism and end-product synthesis Synthesis of organic end-products from pyruvate is mediated by enzymes comprising two major branchpoints, namely the pyruvate/acetyl-CoA/lactate branchpoint and
the acetyl-CoA/ethanol/acetate branchpoint, while H2 can be generated from reduced ferredoxin selleck inhibitor (Fdr), NADH, or NADPH using multiple hydrogenase
(H2ase) complexes (Figure 3). While the functionality of these pathways has been verified using enzyme assays [4, 55], and transcriptional expression of the genes involved in these pathways has recently been elucidated [22, 36, 37], there have been no reports regarding the expression levels of these genes at the protein level. Given that click here there are apparent redundancies in genes encoding enzymes with analogous functions (e.g. pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, hydrogenases) according to the current annotation, it is important that protein abundances and their expression profiles under physiological conditions be determined for the effective application of metabolic engineering strategies to improve rates and/or yields of H2, ethanol, and other desired end-products. Pyruvate/acetyl-CoA/lactate branchpoint C. thermocellum may convert pyruvate into (i) CO2, Fdr, and acetyl-CoA, (ii) formate and acetyl-CoA, and (iii) lactate Urease via pyruvate:ferredoxin oxidoreductase (POR), pyruvate:formate lyase (PFL), and lactate deATM signaling pathway hydrogenase (LDH), respectively . Based on end-product profiles (Figure 1), carbon flux is preferentially channelled through POR. C. thermocellum encodes two 4-subunit PORs. While the γ, δ, α, and β subunits encoded by the gene cluster Cthe_2390-2393 are highly expressed, proteins encoded
by Cthe_2794-2797 are not detected by 2D-HPLC-MS/MS, in agreement with mRNA profiles reported by Raman et al. and Fong et al.. This contrasted with RT-PCR experiments performed by Carere et al., who reported high expression of subunit Cthe_2796 and low expression of subunit Cthe_2392 in exponential phase cultures grown on cellulose . Three putative single subunit POR-like oxidoreductases, including Cthe_3120, a putative pyruvate:flavodoxin oixidoreductase, Cthe_0866, a putative 2-oxogluterate synthase, and Cthe_0614, a putative indolepyruvate:fd oxidoreuctase, were also detected at high levels using 2D-HPLC-MS/MS. In agreement with our relative protein abundance profiles, RT-PCR experiments have confirmed high expression levels of Cthe_3120 .