Genomics of aerobic cellulose utilization systems in actinobacteria

Abstract

Cellulose degrading enzymes have important functions in the biotechnology industry, including the production of biofuels from lignocellulosic biomass. Anaerobes including Clostridium species organize cellulases and other glycosyl hydrolases into large complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems comprised of independently acting enzymes, often with carbohydrate binding domains. Numerous actinobacterial genomes have become available through the Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. We identified putative cellulose-degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms were tested in several assays for cellulose degradation, and eight of the organisms showed evidence of cellulase activity. The three with the highest cellulase activity were Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica. Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter are regulated by the transcriptional regulator CelR. Nine organisms appear to use the CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR also regulates cellulases, while cellulases are controlled by different regulatory sites in three organisms. Mining of genome data for cellulose degradative enzymes followed by experimental verification successfully identified several actinobacteria species which were not previously known to degrade cellulose as cellulolytic organisms.

Figure 1. Phylogenetic analysis of family GH9 and GH6 proteins from actinobacterial genomes and experimentally characterized proteins.

Only the glycosyl hydrolase domains were included in the alignment. a. GH9 family proteins; branch including “Theme B" proteins is in red. C_cellulolyticum: Clostridium cellulolyticum; C_thermocellum: Clostridium thermocellum; C_fimi: Cellulomonas fimi; S_reticuli: Streptomyces reticuli. b. GH6 family proteins; branch including predicted exocellulases is colored blue, branch including predicted endocellulases is colored green. KNP414_06149 – exoglucanase from Paenibacillus mucilaginosus KNP414, XCC3160 and XCC3534 – exoglucanases from Xanthomonas campestris pv. campestris ATCC 33913; e_gww2_1_557_1_1 – cbhII from Phanerochaete chrysosporium; RSp0583 – exoglucanase from Ralstonia solanacearum GMI1000.

Figure 2. Operons with ABC transporters, beta-glucosidases, and LacI family regulators in actinobacteria.

Red: ABC transporter substrate binding protein; orange: ABC transporter membrane protein; grey: ABC transporter ATPase protein; blue: beta-glucosidase; green: LacI family transcriptional regulator; yellow: cellulase; purple: aldose 1-epimerase; white: cellobiose phosphorylase. Reg: regulatory site. Numbers refer to the locus tags of the proteins; for example, 0130 indicates the gene with locus tag Acel_0130.

Figure 3. AZCL-HEC assays of eleven actinobacteria.

Plates contained 1.5% agar, 0.5% yeast extract, and 0.2% AZCL-HEC. Photographs were taken after seven days. The N. dassonvillei plate shown here had 0.01% cellobiose added. The C. acidiphila and A. robiniae plates were at pH 5.5; others were at pH 7.0.