Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution

Abstract Background β-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass. Results We have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2. Conclusions D2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

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Production of l-Ribose from l-Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

Applied and Environmental Microbiology ◽

10.1128/aem.07012-11 ◽

2012 ◽

Vol 78 (11) ◽

pp. 3880-3884 ◽

Cited By ~ 17

Author(s):

Yu-Ri Lim ◽

Soo-Jin Yeom ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Content Type ◽

Mannose 6 Phosphate

ABSTRACTA triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificanswas obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+. The triple-site variant produced 213 g/literl-ribose from 300 g/literl-ribulose for 60 min, with a volumetric productivity of 213 g liter−1h−1, which was 4.5-fold higher than that of the wild-type enzyme. Thekcat/Kmand productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilusR142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.

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Characterization of a Mannose-6-Phosphate Isomerase fromThermus thermophilusand Increased l-Ribose Production by Its R142N Mutant

Applied and Environmental Microbiology ◽

10.1128/aem.01793-10 ◽

2010 ◽

Vol 77 (3) ◽

pp. 762-767 ◽

Cited By ~ 17

Author(s):

Soo-Jin Yeom ◽

Eun-Sun Seo ◽

Bi-Na Kim ◽

Yeong-Su Kim ◽

Deok-Kun Oh

Keyword(s):

Specific Activity ◽

Thermus Thermophilus ◽

Catalytic Efficiency ◽

Maximal Activity ◽

Wild Type ◽

Active Site Residues ◽

Geobacillus Thermodenitrificans ◽

Wild Type Enzyme ◽

Mannose 6 Phosphate

ABSTRACTAn uncharacterized gene fromThermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed inEscherichia coli. The maximal activity of the recombinant enzyme forl-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu2+. Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion ofl-ribulose tol-ribose, a potential starting material for manyl-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase inl-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (kcat/Km) forl-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. Thekcat/Kmof the R142N mutant was 3.8-fold higher than that ofGeobacillus thermodenitrificansmannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reportedkcat/Km. The R142N mutant enzyme produced 213 g/literl-ribose from 300 g/literl-ribulose for 2 h, with a volumetric productivity of 107 g liter−1h−1, which was 1.5-fold higher than that of the wild-type enzyme.

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Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula×tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris

Biochemical Journal ◽

10.1042/bj20041749 ◽

2005 ◽

Vol 390 (1) ◽

pp. 105-113 ◽

Cited By ~ 47

Author(s):

Åsa M. Kallas ◽

Kathleen Piens ◽

Stuart E. Denman ◽

Hongbin Henriksson ◽

Jenny Fäldt ◽

...

Keyword(s):

Pichia Pastoris ◽

Specific Activity ◽

Glycosylation Site ◽

Populus Tremula ◽

Site Directed Mutagenesis ◽

Glycoside Hydrolase Family ◽

Sequence Motif ◽

Hybrid Aspen ◽

Xyloglucan Endotransglycosylase ◽

Wild Type

The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula×tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the α-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn93 to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn93→Ser) mutant.

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Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

Biotechnology for Biofuels ◽

10.1186/s13068-021-01936-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Laura Navone ◽

Thomas Vogl ◽

Pawarisa Luangthongkam ◽

Jo-Anne Blinco ◽

Carlos H. Luna-Flores ◽

...

Keyword(s):

Pichia Pastoris ◽

Phytic Acid ◽

Disulfide Bond ◽

Wild Type ◽

Wild Type Enzyme ◽

E Coli ◽

Biochemical Characterisation ◽

Disulfide Bond Isomerase ◽

Microbial Systems ◽

Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

Author(s):

F Jacob ◽

B Joris ◽

J M Frère

Keyword(s):

Active Site ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Serine Residue ◽

Wild Type Enzyme ◽

Ph Values ◽

Streptomyces Albus ◽

Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

Biochemistry and Cell Biology ◽

10.1139/o90-153 ◽

1990 ◽

Vol 68 (7-8) ◽

pp. 1037-1044 ◽

Cited By ~ 14

Author(s):

Peter C. Loewen ◽

Jacek Switala ◽

Mark Smolenski ◽

Barbara L. Triggs-Raine

Keyword(s):

Escherichia Coli ◽

Specific Activity ◽

Polymerase Chain Reaction Amplification ◽

Protein A ◽

Wild Type ◽

Wild Type Enzyme ◽

Gene Encoding ◽

Reaction Amplification ◽

Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

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Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2790035 ◽

1991 ◽

Vol 279 (1) ◽

pp. 35-41 ◽

Cited By ~ 86

Author(s):

R Chambert ◽

M F Petit-Glatron

Keyword(s):

Bacillus Subtilis ◽

Intermediate Formation ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Chain Elongation ◽

Directed Mutagenesis ◽

Water Mixture ◽

Wild Type ◽

Wild Type Enzyme ◽

Sucrose Hydrolysis

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

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Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris

Biological Procedures Online ◽

10.1186/s12575-020-00138-0 ◽

2020 ◽

Vol 22 (1) ◽

Author(s):

Roghayyeh Baghban ◽

Safar Farajnia ◽

Younes Ghasemi ◽

Reyhaneh Hoseinpoor ◽

Azam Safary ◽

...

Keyword(s):

Pichia Pastoris ◽

Proteolytic Activity ◽

Mutational Analysis ◽

Expression System ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Vitreomacular Adhesion ◽

Autolytic Activity

Abstract Background Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. Results The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. Conclusions The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

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Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

Journal of Biological Chemistry ◽

10.1074/jbc.m111.220517 ◽

2011 ◽

Vol 286 (27) ◽

pp. 24417-24425 ◽

Cited By ~ 14

Author(s):

Chi-Yuan Chou ◽

Liang Tong

Keyword(s):

Binding Sites ◽

Analytical Ultracentrifugation ◽

Substrate Inhibition ◽

Kinetic Studies ◽

The Other ◽

Biotin Carboxylase ◽

Binding Modes ◽

Wild Type ◽

Wild Type Enzyme ◽

Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

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