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. 2020 Oct 1;86(20):e00909-20.
doi: 10.1128/AEM.00909-20. Print 2020 Oct 1.

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

Jordan F Russell  1   2 , Matthew L Russo  1   2 , Xuewen Wang  3 , Neal Hengge  4   2 , Daehwan Chung  4   2 , Lance Wells  5 , Yannick J Bomble  4   2 , Janet Westpheling  6   2
Affiliations

Deletion of a Peptidylprolyl Isomerase Gene Results in the Inability of Caldicellulosiruptor bescii To Grow on Crystalline Cellulose without Affecting Protein Glycosylation or Growth on Soluble Substrates

Jordan F Russell et al. Appl Environ Microbiol. .

Abstract

Caldicellulosiruptor bescii secretes a large number of complementary multifunctional enzymes with unique activities for biomass deconstruction. The most abundant enzymes in the C. bescii secretome are found in a unique gene cluster containing a glycosyl transferase (GT39) and a putative peptidyl prolyl cis-trans isomerase. Deletion of the glycosyl transferase in this cluster resulted in loss of detectable protein glycosylation in C. bescii, and its activity has been shown to be responsible for the glycosylation of the proline-threonine rich linkers found in many of the multifunctional cellulases. The presence of a putative peptidyl prolyl cis-trans isomerase within this gene cluster suggested that it might also play a role in cellulase modification. Here, we identify this gene as a putative prsA prolyl cis-trans isomerase. Deletion of prsA2 leads to the inability of C. bescii to grow on insoluble substrates such as Avicel, the model cellulose substrate, while exhibiting no differences in phenotype with the wild-type strain on soluble substrates. Finally, we provide evidence that the prsA2 gene is likely needed to increase solubility of multifunctional cellulases and that this unique gene cluster was likely acquired by members of the Caldicellulosiruptor genus with a group of genes to optimize the production and activity of multifunctional cellulases.IMPORTANCECaldicellulosiruptor has the ability to digest complex plant biomass without pretreatment and have been engineered to convert biomass, a sustainable, carbon neutral substrate, to fuels. Their strategy for deconstructing plant cell walls relies on an interesting class of cellulases consisting of multiple catalytic modules connected by linker regions and carbohydrate binding modules. The best studied of these enzymes, CelA, has a unique deconstruction mechanism. CelA is located in a cluster of genes that likely allows for optimal expression, secretion, and activity. One of the genes in this cluster is a putative isomerase that modifies the CelA protein. In higher eukaryotes, these isomerases are essential for the proper folding of glycoproteins in the endoplasmic reticulum, but little is known about the role of isomerization in cellulase activity. We show that the stability and activity of CelA is dependent on the activity of this isomerase.

Keywords: biomass deconstruction; cellulase; prolyl isomerases.

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Figures

FIG 1
FIG 1
Bioinformatic analysis of PrsA proteins. (A) Sequences of PrsA proteins from C. bescii were aligned with PrsA proteins from B. subtilis and L. monocytogenes using the ClustalW2 alignment tool in the Genious (v.11.1.5) software platform. Shaded residues indicate conserved amino acids: black, identity; gray, similar amino acids; unshaded, nonconserved residues. A consensus sequence is displayed above the alignment. The colored bar above each residue represents the mean pairwise identity score between all pairs in the column with green indicating 100% identity, yellow indicating <100 to 30% identity, and red indicating <30% identity. The B. subtilis PrsA shares 28.7 and 22.1% overall identity with C. bescii PrsA1 and PrsA2, respectively. The two C. bescii proteins share 37.7% identity with each other. (B) Phylogenetic tree of previously described PrsA proteins.
FIG 2
FIG 2
Deletion of prsA2 in C. bescii. (A) Chromosome map of gene cluster that includes celA and prsA2. (B) Depiction of the deletion cassette consisting of a fused 5′ and 3′ flanking region in a nonreplicating plasmid, pJRW016, with a copy of the pyrF gene from Clostridium thermocellum (Clo1313_1266) for selection of uracil prototrophic transformants of a ΔpyrF background strain. Counterselection with 5-FOA selected strains that had undergone a second recombination event resulted in strain JWCB161 (ΔpyrF ΔcbeI ΔprsA2). (C) Agarose gel showing PCR products amplified with primers JR050 and JR051 on gDNA templates from the C. bescii parent strain JWCB018, (ΔpyrF ΔcbeI) (lane 2), JWCB161, the ΔprsA2 (lane 3), and no-template control (lane 1). MW, DNA molecular weight standards. The expected band for the wild-type locus is at 3.2 kb and that for the ΔprsA2 mutant is 2.2 kb.
FIG 3
FIG 3
Effect of the prsA2 deletion on protein production. Panels A and B show the same SDS-PAGE gel with extracellular and intracellular fractions (as labeled) for the following strains: JWCB018 (ΔpyrF ΔcbeI parent strain) (lane 1), JWCB143 (ΔpyrF ΔcbeI ΔAthe_1864, glycosyltransferase deletion) (lane 2), JWCB161 (ΔpyrF ΔcbeI ΔprsA2) (lane 3), and JWCB162 (ΔpyrF ΔcbeI ΔAthe_1862) (lane 4). Lanes M, molecular weight standards. (A) Stained with glycoprotein stain; (B) counterstained with RAPIDstain to visualize protein.
FIG 4
FIG 4
Growth of the ΔprsA2 mutant on soluble and insoluble substrates. (A) Growth of wild-type (DSMZ6725; blue), JWCB018 (ΔpyrF ΔcbeI) parent (orange), and JWCB161 (ΔpyrF ΔprsA2) (gray) strains at 65°C on cellobiose as measured by determining the OD680. (B) Growth of the same strains, with the addition of JWCB029 (ΔpyrF ΔcbeI ΔcelA strain), on Avicel reported as CFU/ml after plating and after incubation at 24 h (yellow) and 36 h (green).
FIG 5
FIG 5
Analysis of extracellular soluble protein activity and stability from stationary-phase cultures of the ΔprsA2 mutant. (A) Coomassie blue-stained SDS-PAGE gel of the extracellular fractions of JWCB018 (ΔpyrF ΔcbeI) parent strain (lane 1) and JWCB161 (ΔpyrF ΔprsA2) (lane 2) using 25-μg loadings. The same protein fractions are shown before and after centrifugation and filtration with a 0.45-μm filter were carried out to remove precipitation. Protein from both strains were filtered despite only JWCB161 protein exhibiting visible precipitation. (B) Western blot with the CBM3b antibody to probe the same unfiltered protein fractions from panel A here using 10-μg loadings. (C) Glucose release from an Avicel digestion assay carried out at 75°C with the filtered extracellular protein (15 mg/g substrate) from JWCB018 (orange) and JWCB161 (gray) expressed as a percentage of maximum theoretical conversion.
FIG 6
FIG 6
Precipitated protein fraction from the ΔprsA2 mutant. (A) Coomassie blue-stained SDS-PAGE gel of the extracellular fraction of JWCB018 (lane 1) using a 25-μg loading and the pelleted protein from JWCB161 cell supernatant samples (lanes 2) using loadings of 50, 25, 10, and 2 μg. These fractions were made by pelleting the particulate matter in JWCB161 ECP samples and washing the pellet twice in Tris-HCl to remove soluble protein before a final resuspension in SEC buffer. (B) Western blot (αCBM3b) of the same protein fractions with JWCB018 extracellular protein (lane 1) using a 10-μg loading and the JWCB161 pelleted fraction (lanes 2) using loadings of 50, 25, 10, and 2 μg.
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