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

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

List of primers used in the study.

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

Schematic representation of the constructions used for production of free and anchored xylanase in <i>Y. lipolytica</i>.

<p>Constructions used for production of free TxXYN A) TxXYN fused with YlCWP110 C-terminal amino acids B), YlPir 100 C-terminal amino acids C) and YlCBM 87 C-terminal amino acids D). ppLIP2, preproLIP2 used as secretion signal peptide; H, 6 histidines tag; LK, 10 amino acids linker peptide; relevant restriction sites in the encoding genes are indicated above the resulting proteins, while relevant amino acids are indicated below (diamonds for the 4 N-Glycosylation sites and stars for the 2 catalytic glutamic acids in TxXYN, 4 cysteines in Pir100 and the asparagine as acceptor of the GPI anchor in CWP110).</p

Xylanase activities as determined in cell walls (lyophilised cell pellets) and growth medium when producing the three fusion proteins in <i>Y. lipolytica</i> JMY1212.

<p>Xylanase activities determined for strain <i>Y. lipolytica</i> JMY1212 transformed with JMP62-TEF-ppLIP2-CBM87-TxXYN, JMP62-TEF-ppLIP2-Pir100-TxXYN, JMP62-TEF-ppLIP2-TxXYN-CWP110 and cultivated overnight in 10 g/L oleic acid. Xylanase units per gram dried cells in culture supernatant and lyophilised cell pellets are displayed in grey and spotted bars, respectively. Units of bound xylanase per gram dried cells and corresponding percentages are indicated for each construction. The % activity anchored on cells is the ratio between total units in cell pellet and total units in the whole culture (94 mg cell pellet and 10 mL supernatant). Mean and standard deviation of three experiments are presented.</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

Immobilisation efficiencies of different anchoring systems depending on the carbon source.

<p>Total xylanase activities obtained for strain <i>Y. lipolytica</i> JMY1212 transformed with JMP62-TEF-ppLIP2-Pir100-TxXYN, JMP62-TEF-ppLIP2-CBM87-TxXYN, JMP62-TEF-ppLIP2-TxXYN-CWP110 cultivated 4 days with 3 different carbon sources; strain JMY1212 transformed with JMP61-POX2-ppLIP2-TxXYN was used as control. Xylanase units per gram dried cells in culture supernatant (25 mL) and lyophilised cell pellets (0.7, 0.89 and 1 g in glycerol, glucose and oleic acid, respectively) are displayed in grey and spotted bars, respectively. Units of bound xylanase per gram dried cells and corresponding percentages are indicated for each construction. Mean and standard deviation of four experiments are presented.</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