Molecular Engineering of Fungal GH5 and GH26 Beta-(1,4)-Mannanases toward Improvement of Enzyme Activity

<div><p>Microbial mannanases are biotechnologically important enzymes since they target the hydrolysis of hemicellulosic polysaccharides of softwood biomass into simple molecules like manno-oligosaccharides and mannose. In this study, we have implemented a strategy of molecular engineering in the yeast <i>Yarrowia lipolytica</i> to improve the specific activity of two fungal endo-mannanases, <i>Pa</i>Man5A and <i>Pa</i>Man26A, which belong to the glycoside hydrolase (GH) families GH5 and GH26, respectively. Following random mutagenesis and two steps of high-throughput enzymatic screening, we identified several <i>Pa</i>Man5A and <i>Pa</i>Man26A mutants that displayed improved kinetic constants for the hydrolysis of galactomannan. Examination of the three-dimensional structures of <i>Pa</i>Man5A and <i>Pa</i>Man26A revealed which of the mutated residues are potentially important for enzyme function. Among them, the <i>Pa</i>Man5A-G311S single mutant, which displayed an impressive 8.2-fold increase in <i>k_cat</i>/K_M due to a significant decrease of K_M, is located within the core of the enzyme. The <i>Pa</i>Man5A-K139R/Y223H double mutant revealed modification of hydrolysis products probably in relation to an amino-acid substitution located nearby one of the positive subsites. The <i>Pa</i>Man26A-P140L/D416G double mutant yielded a 30% increase in <i>k_cat</i>/K_M compared to the parental enzyme. It displayed a mutation in the linker region (P140L) that may confer more flexibility to the linker and another mutation (D416G) located at the entrance of the catalytic cleft that may promote the entrance of the substrate into the active site. Taken together, these results show that the directed evolution strategy implemented in this study was very pertinent since a straightforward round of random mutagenesis yielded significantly improved variants, in terms of catalytic efiiciency (k_cat/K_M).</p></div

Structural view of <i>Pa</i>Man5A (PDB 3ZIZ) exhibiting substituted amino-acids.

<p>A. Surface view of the catalytic cleft of <i>Pa</i>Man5A with mannotriose modelled in the −2 and −3 subsites and mannobiose modelled in the +1 and +2 subsites. The structures of GH5 from <i>T. reesei</i> and <i>T. fusca</i> in complex with mannobiose and mannotriose, respectively, were superimposed on the top of the structure of <i>Pa</i>Man5A to map the substrate-binding subsites. The two catalytic glutamate residues, E177 and E283, are coloured in red. The substituted amino-acids are labelled and coloured in yellow. B. Structural based sequence alignment of the region around position 311 (according to <i>Pa</i>Man5A numbering) from <i>Podospora anserina</i> (<i>Pa</i>Man5A), <i>Aplysia kurodai</i> (<i>Ak</i>Man, PDB 3VUP), <i>Mytilus edulis</i> (<i>Me</i>Man5A, PDB 2C0H), <i>Cellvibrio mixtus</i> (<i>Cm</i>Man5A, PDB 1UUQ), <i>Trichoderma reesei</i> (<i>Tr</i>Man5A, PDB 1QNR), <i>Lycopersicon esculentum</i> (<i>Le</i>Man4A, PDB 1RH9) and <i>Thermomonospora fusca</i> (<i>Tf</i>Man5, PDB 2MAN). Secondary structure elements, α-helix α7 and β-strand β8, are indicated below the sequences as a cylinder and an arrow, respectively. Strictly conserved residues, G311 and W315 (according to <i>Pa</i>Man5A numbering), are shown with a yellow and a grey background, respectively. C. Surface view of <i>Pa</i>Man5A rotated of about 90° along the horizontal axis. The front clipping plane has been moved in order to visualize the location of G311 inside the molecule. The zoom shows a compact hydrophobic core in the vicinity of G311.</p

Mannanase activity of selected <i>Y. lipolytica</i> variants.

<p>The mannanase activity was measured at 40°C in sodium acetate buffer 50 mM, pH 5.2 using 1% (w/v) galactomannan. The coefficient of variation (CV) was defined as the ratio of the standard deviation to the mean and was calculated for each of the wild-type enzymes. wt, wild type.</p

List of primers used in the study.

<p>List of primers used in the study.</p

Kinetic constants of wild-type enzymes and selected variants toward galactomannan, mannohexaose (M₆) and mannopentaose (M₅).

<p>The kinetic parameters were determined at 40°C in sodium acetate buffer 50 mM, pH 5.2 as described in the Methods section. Paired t test was used to compare the kinetic parameters of mutants versus native enzyme. The difference was considered statistically significant when <i>p</i><0.05 (*). wt, wild type.ND: not determined.</p

Progress curves of the manno-oligosaccharides generated by the wild-type <i>Pa</i>Man5A and the <i>Pa</i>Man5A-K139R/Y223H variant upon hydrolysis of mannohexaose.

<p>18.2 nM of the wild-type <i>Pa</i>Man5A (A) and the <i>Pa</i>Man5A-K139R/Y223H variant (B) were incubated with 1 mM of mannohexaose in acetate buffer pH 5.2 at 40°C. The amount of each manno-oligosaccharide, i.e., mannobiose (full circles), mannotriose (full squares), mannotetraose (crosses), and mannohexaose (full diamonds), is indicated during the course of the reaction.</p

Error-prone PCR strategy used in the study.

<p>PCR1: error prone-PCR performed on <i>paman5a</i> (HM357135) and <i>paman26a</i> (HM357136); PCR2: PCR without mutation performed on Ura3d1 (selection marker), pPOX2 (inducible promotor of acyl-coA oxidase 2) and prepro Lip2 (secretion signal sequence); PCR3: overlapping PCR to reconstruct the entire sequence between zeta platforms. Primers used are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079800#pone-0079800-t003" target="_blank">Table 3</a>.</p

Screening strategy and mutant selection.

<p>The number of variants screened at each step is indicated at the top (<i>Pa</i>Man5A) and at the bottom (<i>Pa</i>Man26A) of the diagram.</p

Structural view of <i>Pa</i>Man26A (PDB 3ZM8) exhibiting substituted amino-acids.

<p>The central panel shows a surface view of the entire <i>Pa</i>Man26A structure, which is composed of a carbohydrate binding module (CBM) belonging to the CBM35 family in cyan, a linker in violet and a catalytic domain belonging to the GH26 family in green. The two catalytic glutamate residues, E300 and E390, are coloured in red. The two substituted amino-acids, P140 and D416, are labelled and coloured in yellow. The top view represents the surface view of the catalytic cleft of <i>Pa</i>Man26A rotated about 90° along the horizontal axis with mannotriose modelled into the −2 to −4 subsites. The structure of GH26 from <i>C. fimi</i> in complex with mannotriose was superimposed on the top of the structure of <i>Pa</i>Man26A to map the substrate-binding subsites. The bottom view displays the <i>Pa</i>Man26A linker (from residue 131 to residue 141) in stick representation. The molecule has been rotated of about 90° along the horizontal axis and in the opposite direction compared to the top view. The proline residues of the linker are labelled.</p

DataSheet_1_Taxonomic composition and carbohydrate-active enzyme content in microbial enrichments from pulp mill anaerobic granules after cultivation on lignocellulosic substrates.zip

Metagenomes of lignocellulose-degrading microbial communities are reservoirs of carbohydrate-active enzymes relevant to biomass processing. Whereas several metagenomes of natural digestive systems have been sequenced, the current study analyses metagenomes originating from an industrial anaerobic digester that processes effluent from a cellulose pulp mill. Both 16S ribosomal DNA and metagenome sequences were obtained following anaerobic cultivation of the digester inoculum on cellulose and pretreated (steam exploded) poplar wood chips. The community composition and profile of predicted carbohydrate-active enzymes were then analyzed in detail. Recognized lignocellulose degraders were abundant in the resulting cultures, including populations belonging to Clostridiales and Bacteroidales orders. Poorly defined taxonomic lineages previously identified in other lignocellulose-degrading communities were also detected, including the uncultivated Firmicutes lineage OPB54 which represented nearly 10% of the cellulose-fed enrichment even though it was not detected in the bioreactor inoculum. In total, 3580 genes encoding carbohydrate-active enzymes were identified through metagenome sequencing. Similar to earlier enrichments of animal digestive systems, the profile encoded by the bioreactor inoculum following enrichment on pretreated wood was distinguished from the cellulose counterpart by a higher occurrence of enzymes predicted to act on pectin. The majority (> 93%) of carbohydrate-active enzymes predicted to act on plant polysaccharides were identified in the metagenome assembled genomes, permitting taxonomic assignment. The taxonomic assignment revealed that only a small selection of organisms directly participates in plant polysaccharide deconstruction and supports the rest of the community.</p