Proteiniphilum saccharofermentans str. M3/6T isolated from a laboratory biogas reactor is versatile in polysaccharide and oligopeptide utilization as deduced from genome-based metabolic reconstructions

Abstract

Proteiniphilum saccharofermentans str. M3/6^T is a recently described species within the family Porphyromonadaceae (phylum Bacteroidetes), which was isolated from a mesophilic laboratory-scale biogas reactor. The genome of the strain was completely sequenced and manually annotated to reconstruct its metabolic potential regarding biomass degradation and fermentation pathways. The P. saccharofermentans str. M3/6^T genome consists of a 4,414,963 bp chromosome featuring an average GC-content of 43.63%. Genome analyses revealed that the strain possesses 3396 protein-coding sequences. Among them are 158 genes assigned to the carbohydrate-active-enzyme families as defined by the CAZy database, including 116 genes encoding glycosyl hydrolases (GHs) involved in pectin, arabinogalactan, hemicellulose (arabinan, xylan, mannan, β-glucans), starch, fructan and chitin degradation. The strain also features several transporter genes, some of which are located in polysaccharide utilization loci (PUL). PUL gene products are involved in glycan binding, transport and utilization at the cell surface. In the genome of strain M3/6^T, 64 PUL are present and most of them in association with genes encoding carbohydrate-active enzymes. Accordingly, the strain was predicted to metabolize several sugars yielding carbon dioxide, hydrogen, acetate, formate, propionate and isovalerate as end-products of the fermentation process. Moreover, P. saccharofermentans str. M3/6^T encodes extracellular and intracellular proteases and transporters predicted to be involved in protein and oligopeptide degradation. Comparative analyses between P. saccharofermentans str. M3/6^T and its closest described relative P. acetatigenes str. DSM 18083^T indicate that both strains share a similar metabolism regarding decomposition of complex carbohydrates and fermentation of sugars.

Keywords: Anaerobic digestion; Bioconversion; Biomethanation; Carbohydrate-active enzymes; Metabolic pathway reconstruction; Polysaccharide utilization loci.

Fig. 1

Genome plot of P. saccharofermentans str. M3/6^T. The first outer circle represents the genome scale in kb. The origin of replication was determined based on GC skew analyses. Second and third circle: predicted protein-coding sequences (CDS) on the forward and the reverse strands colored according to the assigned COG categories. Fourth and fifth inner circles represent the GC-content and GC-skew, respectively. Red squares on the outer circle indicate CRISPR-cas systems.

Fig. 2

Schematic overview of PUL (Polysaccharide Utilization Loci) predicted in the P. saccharofermentans str. M3/6^T genome. To facilitate visualization of gene arrangements, these are colored according to the function of the encoded proteins SusC (blue), SusD (purple), SusE (light blue), HP (hypothetical protein, gray), GHs (Glycoside Hydrolase, pink), CBM (Carbohydrate-Binding Module, green), PL (Polysaccharide Lyases, light orange), CE (Carbohydrate Esterase, dark orange), TonB (TonB-linked outer membrane protein, light pink), Pept (Peptidase, light green), MFS (Major Facilitator Superfamily, light pink), regulators (AraC, LacI, HTCS, ECF, Gntr, Anti-sigma, yellow). Genes that do not encode PUL components were marked with’ non-PUL genes’ (nPULg, dark brown).

Fig. 3

Schematic overview on carbohydrate and protein degradation pathways based on enzymes predicted from the P. saccharofermentans str. M3/6^T genome sequence. Intracellular and extracellular reactions are separated by the cell envelope. Names in blue denote the hemicellulose polysaccharides. The following intracellular metabolic pathways are shown: glycolysis, pentose phosphate pathway, central pyruvate metabolism and the tricarboxylic acid cycle (TCA). Arrows symbolize enzymatic reactions. Crossed red arrows mark enzymatic reactions for which corresponding enzymes were not predicted in the P. saccharofermentans str. M3/6^T genome. Green metabolites represent fermentation pathway end-products (acetate, propionate, isovalerate, formate, and molecular hydrogen). Abbreviations: Asp, aspartic acid; Gln, glutamine; His, histidine; Ile, isoleucine; Leu, leucine; Thr, threonine; Val, valine; OPT, Oligopeptide Transporter Family proteins; ABC, ABC transporter; SSS, Solute-Sodium Symporter Family proteins; MFS, Major Facilitator Superfamily proteins; NP, no prediction.

Fig. 4

CRISPR-cas systems of the P. saccharofermentans str. M3/6^T genome. CRISPR-cas system type II-C: cas operon, eight genes (7460 bp); Leader, 109 bp; CRISPR array (2967 bp), 39 47-bp-direct repeats and 38 spacers of 29–30 bp. CRISPR-cas system type I-B: cas operon, eight genes (9131 bp); Leader, 211 bp; CRISPR array (7307 bp), 114 29-bp-direct repeats and 113 spacers of 34–39 bp; Coding sequences (CDS) colored in grey encode hypothetical proteins. The coordinates and sequences of the CRISPR-cas systems are provided as Supplemental material (File S2).