Identification and characterization of proteins of unknown function (PUFs) in Clostridium thermocellum DSM 1313 strains as potential genetic engineering targets

Abstract

Background Mass spectrometry-based proteomics can identify and quantify thousands of proteins from individual microbial species, but a significant percentage of these proteins are unannotated and hence classified as proteins of unknown function (PUFs). Due to the difficulty in extracting meaningful metabolic information, PUFs are often overlooked or discarded during data analysis, even though they might be critically important in functional activities, in particular for metabolic engineering research. Results We optimized and employed a pipeline integrating various “guilt-by-association” (GBA) metrics, including differential expression and co-expression analyses of high-throughput mass spectrometry proteome data and phylogenetic coevolution analysis, and sequence homology-based approaches to determine putative functions for PUFs in Clostridium thermocellum. Our various analyses provided putative functional information for over 95% of the PUFs detected by mass spectrometry in a wild-type and/or an engineered strain of C. thermocellum. In particular, we validated a predicted acyltransferase PUF (WP_003519433.1) with functional activity towards 2-phenylethyl alcohol, consistent with our GBA and sequence homology-based predictions. Conclusions This work demonstrates the value of leveraging sequence homology-based annotations with empirical evidence based on the concept of GBA to broadly predict putative functions for PUFs, opening avenues to further interrogation via targeted experiments.

Functional Analysis of H + -Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum

“…optStoic results are inherently dependent upon the quality of the genome annotation. Given that bioinformatically predicted annotations are not always correct, as seen for clo1313_1686 , and that approximately 20% of the C. thermocellum genome encodes proteins of unknown function (also called hypothetical proteins) ( 53 ), it is possible that important PP i -supplying mechanisms are currently missed. In an attempt to address this possibility, the KEGG database was probed for PP i -generating cycles carrying out the net conversion, , which would be feasible in C. thermocellum ( 26 ) with the addition of one reaction currently absent from iCBI655.…”