scholarly journals Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

2014 ◽  
Vol 80 (11) ◽  
pp. 3496-3507 ◽  
Author(s):  
Patricia Molina-Espeja ◽  
Eva Garcia-Ruiz ◽  
David Gonzalez-Perez ◽  
René Ullrich ◽  
Martin Hofrichter ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Signal Peptide ◽  
Oxygen Transfer ◽  
Colorimetric Assay ◽  
Total Activity ◽  
Functional Expression ◽  
Hydrophobic Core ◽  
Content Type ◽  
Agrocybe Aegerita ◽  
Access Channel

ABSTRACTUnspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected theAgrocybe aegeritaUPO1-encoding gene to directed evolution inSaccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using anad hocdual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in thekcat/Kmvalue for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

scholarly journals Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

2011 ◽  
Vol 78 (5) ◽  
pp. 1370-1384 ◽  
Author(s):  
S. Camarero ◽  
I. Pardo ◽  
A. I. Cañas ◽  
P. Molina ◽  
E. Record ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Fusion Gene ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Redox Mediators ◽  
Pycnoporus Cinnabarinus ◽  
Content Type ◽  
Potential Use

ABSTRACTWhile thePycnoporus cinnabarinuslaccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression inSaccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase inkcat. While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted byAspergillus niger(∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

scholarly journals Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

2003 ◽  
Vol 69 (2) ◽  
pp. 987-995 ◽  
Author(s):  
Thomas Bulter ◽  
Miguel Alcalde ◽  
Volker Sieber ◽  
Peter Meinhold ◽  
Christian Schlachtbauer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Enzyme Stability ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Glycosylation Site ◽  
C Terminus ◽  
Improved Stability

ABSTRACT Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

scholarly journals Design of an improved universal signal peptide based on the α-factor mating secretion signal for enzyme production in yeast

2021 ◽  
Author(s):  
Pablo Aza ◽  
Gonzalo Molpeceres ◽  
Felipe de Salas ◽  
Susana Camarero
Keyword(s):  
Directed Evolution ◽  
Signal Peptide ◽  
Enzyme Production ◽  
Signal Sequence ◽  
Functional Expression ◽  
Saturation Mutagenesis ◽  
Foreign Protein ◽  
Heterologous Proteins ◽  
Post Translational Modifications ◽  
Secretion Signal

AbstractSaccharomyces cerevisiae plays an important role in the heterologous expression of an array of proteins due to its easy manipulation, low requirements and ability for protein post-translational modifications. The implementation of the preproleader secretion signal of the α-factor mating pheromone from this yeast contributes to increase the production yields by targeting the foreign protein to the extracellular environment. The use of this signal peptide combined with enzyme-directed evolution allowed us to achieve the otherwise difficult functional expression of fungal laccases in S. cerevisiae, obtaining different evolved α-factor preproleader sequences that enhance laccase secretion. However, the design of a universal signal peptide to enhance the production of heterologous proteins in S. cerevisiae is a pending challenge. We describe here the optimisation of the α-factor preproleader to improve recombinant enzyme production in S. cerevisiae through two parallel engineering strategies: a bottom-up design over the native α-factor preproleader (αnat) and a top-down design over the fittest evolved signal peptide obtained in our lab (α9H2 leader). The goal was to analyse the effect of mutations accumulated in the signal sequence throughout iterations of directed evolution, or of other reported mutations, and their possible epistatic interactions. Both approaches agreed in the positive synergism of four mutations (Aα9D, Aα20T, Lα42S, Dα83E) contained in the final optimised leader (αOPT), which notably enhanced the secretion of several fungal oxidoreductases and hydrolases. Additionally, we suggest a guideline to further drive the heterologous production of a particular enzyme based on combinatorial saturation mutagenesis of positions 86th and 87th of the αOPT leader fused to the target protein.

scholarly journals Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

2021 ◽  
Vol 22 (3) ◽  
pp. 1157
Author(s):  
Pablo Aza ◽  
Felipe de Salas ◽  
Gonzalo Molpeceres ◽  
David Rodríguez-Escribano ◽  
Iñigo de la Fuente ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Signal Peptide ◽  
Laccase Activity ◽  
Laccase Production ◽  
Wide Range ◽  
Mating Factor ◽  
Conserved Amino Acids ◽  
Basidiomycete Fungi

Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

scholarly journals The Putative Amino-terminal Signal Peptide of the Cloned Rat Brain Na+-Ca2+Exchanger Gene (RBE-1) Is Not Mandatory for Functional Expression

1995 ◽  
Vol 270 (32) ◽  
pp. 19120-19127 ◽  
Author(s):  
Ian Furman ◽  
Orna Cook ◽  
Judith Kasir ◽  
Walter Low ◽  
Hannah Rahamimoff
Keyword(s):  
Rat Brain ◽  
Signal Peptide ◽  
Functional Expression ◽  
Amino Terminal

scholarly journals Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

2015 ◽  
Vol 198 (4) ◽  
pp. 720-730 ◽  
Author(s):  
Stephanie Swanson ◽  
Thomas R. Ioerger ◽  
Nathan W. Rigel ◽  
Brittany K. Miller ◽  
Miriam Braunstein ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Peptide ◽  
Protein A ◽  
Structural Difference ◽  
Structural Similarity ◽  
Structural Features ◽  
Dominant Negative ◽  
Content Type ◽  
Functional Regions ◽  
Suppressor Mutations

ABSTRACTWhile SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure ofMycobacterium tuberculosisSecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in howMtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negativesecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG.IMPORTANCESecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 ofMycobacterium tuberculosis(the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of asecA2mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG.

scholarly journals Xylanase Attachment to the Cell Wall of the Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 190 (4) ◽  
pp. 1350-1358 ◽  
Author(s):  
Wolfgang Liebl ◽  
Christoph Winterhalter ◽  
Wolfgang Baumeister ◽  
Martin Armbrecht ◽  
Michael Valdez
Keyword(s):  
Outer Membrane ◽  
Signal Peptide ◽  
Cellular Localization ◽  
Culture Supernatant ◽  
Thermotoga Maritima ◽  
Surrounding Medium ◽  
Hydrophobic Core ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Amino Terminal

ABSTRACT The cellular localization and processing of the endo-xylanases (1,4-β-d-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane (“toga”) of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.

scholarly journals Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

2018 ◽  
Vol 84 (16) ◽  
Author(s):  
Wei Zhou ◽  
Rui Huang ◽  
Zhiguang Zhu ◽  
Yi-Heng P. Job Zhang
Keyword(s):  
High Activity ◽  
Double Layer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Specific Activity ◽  
Colorimetric Assay ◽  
Petri Dish ◽  
Rapid Identification ◽  
Thermobifida Fusca ◽  
Content Type

ABSTRACT Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

Cassette mutagenic analysis of the yeast invertase signal peptide: effects on protein translocation

1989 ◽  
Vol 9 (8) ◽  
pp. 3400-3410
Author(s):  
J K Ngsee ◽  
W Hansen ◽  
P Walter ◽  
M Smith
Keyword(s):  
Signal Peptide ◽  
Protein Translocation ◽  
Plasmid Vector ◽  
Peptide Sequence ◽  
Secretory Process ◽  
Hydrophobic Core ◽  
Class Iii ◽  
Yeast Plasmid ◽  
Signal Peptide Sequence ◽  
Transcription Terminator

The coding sequence of the SUC2 locus was placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere-based yeast plasmid vector from which invertase is expressed in a Suc- strain of Saccharomyces cerevisiae. Mutants in the signal peptide sequence were produced by replacing this region of the gene with synthetic oligonucleotide cassettes containing mixtures of nucleotides at several positions. The mutants could be divided into three classes on the basis of the ability to secrete invertase. Class I mutants produced secreted invertase but in reduced amount. The class II mutant, 4-55B, also exhibited reduced a level of invertase, but a significant fraction of the enzyme was intracellular. Class III mutants were partially defective in translocation from the cytoplasm to the endoplasmic reticulum and produced enzymatically active, unglycosylated preinvertase in the cytoplasm. Class III mutant preinvertases were also defective in translocation across canine pancreas microsomes. These results suggested that the reduced level of invertase resulted from proteolytic degradation of inefficiently transported intermediates. Comparison of the sequences of the mutant signal peptides indicated that amino acids at the extreme amino terminus and adjacent to the cleavage site play a crucial role in the secretory process when combined with a mutation within the hydrophobic core.

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