Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution

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.     

Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution

Abstract

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs₆₁₀T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs₆₁₀T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs₆₁₀T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.     

Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound.

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3, 4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both kcat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect kcat and the oxidation rate of dimer I.     

Screening mutant libraries of fungal laccases in the presence of organic solvents

Reliable screening methods are being demanded by biocatalysts' engineers, especially when some features such as activity or stability are targets to improve under non-natural conditions (i.e., in the presence of organic solvents). The current work describes a protocol for the design of a fungal laccase-expressed in Saccharomyces cerevisiae-highly active in organic cosolvents. A high-throughput screening assay based on ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) oxidation was validated. The stability of the ABTS radical cation was not significantly altered in the presence of acetonitrile, ethanol, or DMSO. With a coefficient of variance below 10% and a sensitivity limit of 15 pg laccase/microL, the assay was reproducible and sensitive. The expression system of Myceliophthora thermophila laccase variant T2 in S. cerevisiae was highly dependent on the presence of Cu2+. Copper concentration was limited up to 10 microM CuSO4 where expression levels (approximately 14-18 mg/L) were acceptable without compromising the reliability of the assay. A mutant library was created by error-prone PCR with 1.1 to 3.5 mutations per kb. After only 1 generation of directed evolution, mutant 6C9 displayed about 3.5-fold higher activities than parent type in the presence of 20% acetonitrile or 30% ethanol. The method provided here should be generally useful to improve the activity of other redox enzymes in mixtures of water/cosolvents.     

Directed evolution of a temperature-, peroxide- and alkaline pH-tolerant versatile peroxidase

The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In the present study, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution, achieving a level of secretion in Saccharomyces cerevisiae (21?mg/l) as yet unseen for any ligninolytic peroxidase. The evolved variant for expression harboured four mutations and increased its total VP activity 129-fold. The signal leader processing by the STE13 protease at the Golgi compartment changed as a consequence of overexpression, retaining the additional N-terminal sequence Glu-Ala-Glu-Ala that enhanced secretion. The engineered N-terminally truncated variant displayed similar biochemical properties to those of the non-truncated counterpart in terms of kinetics, stability and spectroscopic features. Additional cycles of evolution raised the T50 8°C and significantly increased the enzyme's stability at alkaline pHs. In addition, the Km for H2O2 was enhanced up to 15-fold while the catalytic efficiency was maintained, and there was an improvement in peroxide stability (with half-lives for H2O2 of 43?min at a H2O2/enzyme molar ratio of 4000:1). Overall, the directed evolution approach described provides a set of strategies for selecting VPs with improvements in secretion, activity and stability.     

A Semi-Rational Approach to Engineering Laccase Enzymes

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t _1/2 at pH 5 = 35 days), neutral (t _1/2 at pH 7 = 38 days) and alkaline (t _1/2 at pH 10 = 62 days) pH values.     

Development of chimeric laccases by directed evolution

DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high‐redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α‐factor prepro‐leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high‐throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 2978–2986. © 2012 Wiley Periodicals, Inc.     

Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-alcohol oxidase

Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-alcohol oxidase (AAO) and 5-hydroxymethylfurfural oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.     

Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris

The ability to engineer proteins by directed evolution requires functional expression of the target polypeptide in a recombinant host suitable for construction and screening libraries of enzyme variants. Bacteria and yeast are preferred, but eukaryotic proteins often fail to express in active form in these cells. We have attempted to resolve this problem by identifying mutations in the target gene that facilitate its functional expression in a given recombinant host. Here we examined expression of HRP in Saccharomyces cerevisiae. Through three rounds of directed evolution by random point mutagenesis and screening, we obtained a 40-fold increase in total HRP activity in the S.cerevisiae culture supernatant compared with wild-type, as measured on ABTS ?2, 2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (260 units/l/OD(600)). Genes from wild-type and two high-activity clones were expressed in Pichia pastoris, where the total ABTS activity reached 600 units/l/OD(600) in shake flasks. The mutants show up to 5.4-fold higher specific activity towards ABTS and 2.3-fold higher specific activity towards guaiacol.     

Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a nonmutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S. cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with approximately 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC- and E615K -GAG/AAG-) because of the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.     

Comparison of Fungal Laccases and Redox Mediators in Oxidation of a Nonphenolic Lignin Model Compound

Several fungal laccases have been compared for the oxidation of a nonphenolic lignin dimer, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propan-1,3-diol (I), and a phenolic lignin model compound, phenol red, in the presence of the redox mediators 1-hydroxybenzotriazole (1-HBT) or violuric acid. The oxidation rates of dimer I by the laccases were in the following order: Trametes villosa laccase (TvL) > Pycnoporus cinnabarinus laccase (PcL) > Botrytis cinerea laccase (BcL) > Myceliophthora thermophila laccase (MtL) in the presence of either 1-HBT or violuric acid. The order is the same if the laccases are used at the same molar concentration or added to the same activity (with ABTS [2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] as a substrate). During the oxidation of dimer I, both 1-HBT and violuric acid were to some extent consumed. Their consumption rates also follow the above order of laccases, i.e., TvL > PcL > BcL > MtL. Violuric acid allowed TvL and PcL to oxidize dimer I much faster than 1-HBT, while BcL and violuric acid oxidized dimer I more slowly than BcL and 1-HBT. The oxidation rate of dimer I is dependent upon both k_cat and the stability of the laccase. Both 1-HBT and violuric acid inactivated the laccases, violuric acid to a greater extent than 1-HBT. The presence of dimer I or phenol red in the reaction mixture slowed down this inactivation. The inactivation is mainly due to the reaction of the redox mediator free radical with the laccases. We did not find any relationship between the carbohydrate content of the laccases and their inactivation. When the redox potential of the laccases is in the range of 750 to 800 mV, i.e., above that of the redox mediator, it does not affect k_cat and the oxidation rate of dimer I.     

Predicting the emergence of antibiotic resistance by directed evolution and structural analysis

Directed evolution can be a powerful tool to predict antibiotic resistance. Resistance involves the accumulation of mutations beneficial to the pathogen while maintaining residue interactions and core packing that are critical for preserving function. The constraint of maintaining stability, while increasing activity, drastically reduces the number of possible mutational combination pathways. To test this theory, TEM-1 beta-lactamase was evolved using a hypermutator E. coli-based directed evolution technique with cefotaxime selection. The selected mutants were compared to two previous directed evolution studies and a database of clinical isolates. In all cases, evolution resulted in the generation of the E104K/M182T/G238S combination of mutations ( approximately 500-fold increased resistance), which is equivalent to clinical isolate TEM-52. The structure of TEM-52 was determined to 2.4 A. G238S widens access to the active site by 2.8 A whereas E104K stabilizes the reorganized topology. The M182T mutation is located 17 A from the active site and appears to be a global suppressor mutation that acts to stabilize the new enzyme structure. Our results demonstrate that directed evolution coupled with structural analysis can be used to predict future mutations that lead to increased antibiotic resistance.     

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ～65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.     

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

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.     

Adaptive laboratory evolution--harnessing the power of biology for metabolic engineering

Adaptive laboratory evolution (ALE) strategies allow for the metabolic engineering of microorganisms by combining genetic variation with the selection of beneficial mutations in an unbiased fashion. These ALE strategies have been proven highly effective in the optimization of production strains. In contrast to rational engineering strategies and directed modification of specific enzymes, ALE has the advantage of letting nonintuitive beneficial mutations occur in many different genes and regulatory regions in parallel. So far, the majority of applications of ALE in metabolic engineering have used well-characterized platform organisms such as Saccharomyces cerevisiae and Escherichia coli; however, applications for other microorganisms are on the rise. This review will focus on current applications of ALE as a tool for metabolic engineering and discuss advancements and achievements that have been made in this field.     

Characterization of α-factor pheromone and pheromone receptor genes of Ashbya gossypii

The genome of Ashbya gossypii contains homologs of most of the genes that are part of the Saccharomyces cerevisiae pheromone-signal transduction cascade. However, we currently lack understanding of a potential sexual cycle for this pre-whole genome duplication hemiascomycete. The sequenced strain bears three identical copies encoding MATa. We show that the syntenic A. gossypii homolog of MFα1 (AFL062w) does not encode a mature α-factor peptide, but identified another gene, AAR163c, which encodes a candidate α-specific mating pheromone and is thus reannotated as AgMFα2. The expression of the AgSTE2α-factor receptor in an Scste2 S. cerevisiae MATa strain resulted in dosage-dependent growth arrest upon exposure to A. gossypiiα-factor, which indicated that the pheromone response was effectively coupled to the S. cerevisiae signal transduction cascade. Comparison of α-pheromones and α-pheromone receptors showed greater conservation between Eremothecium cymbalariae and S. cerevisiae than between A. gossypii and E. cymbalariae. We constructed A. gossypii strains deleted for the STE2 and STE3 pheromone receptors. These strains showed no phenotypic abnormalities and an ste2, ste3 double mutant is still able to sporulate. The deletion of STE12 as the downstream target of pheromone signalling, however, led to a hypersporulation phenotype.     

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Unspecific 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 the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces 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 an ad hoc dual-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 the k_cat/K_m value 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.     

Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.     

Engineered NADH-dependent GRE2 from Saccharomyces cerevisiae by directed enzyme evolution enhances HMF reduction using additional cofactor NADPH

Furfural and 5-hydroxymethylfurfural (HMF) are inhibitors generated by lignocellulosic biomass pretreatment such as dilute acid hydrolysis that inhibit microbial growth and interfere with subsequent fermentation. It is possible to in situ detoxify these inhibitory compounds by aldehyde reductions using tolerant Saccharomyces cerevisiae. YOL151W (GRE2) is a commonly recognized up-regulated gene expressed under stress conditions that encodes reductase activities toward furfural and HMF using cofactor NADH. Applying a directed enzyme evolution approach, we altered the genetic code of GRE2 yielding two mutants with amino acid substitutions of Gln261 to Arg261 and Phe283 to Leu283; and Ile107 to Val107, Gln261 to Arg261, and Val285 to Asp285 for strain Y62-C11 and Y62-G6, respectively. Clones of these mutants showed faster growth rates and were able to establish viable cultures under 30 mM HMF challenges when compared with a wild type GRE2 clone when inoculated into synthetic medium containing this inhibitor. Compared with the wild type control, crude cell extracts of the two mutants showed 3- to 4-fold and 3- to 9-fold increased specific enzyme activity using NADH toward HMF and furfural reduction, respectively. While retaining its aldehyde reductase activities using the cofactor NADH, mutant Y62-G6 displayed significantly greater reductase activities using NADPH as the cofactor with 13- and 15-fold increase toward furfural and HMF, respectively, as measured by its partially purified protein. Using reverse engineering and site directed mutagenesis methods, we were able to confirm that the amino acid substitution of the Asp285 is responsible for the increased aldehyde reductase activities by utilizing the additional cofactor NADPH.     

A High-Throughput Screening Method to Reengineer DNA Polymerases for Random Mutagenesis

A screening system for directed evolution of DNA polymerases employing a fluorescent Scorpion probe as a reporter has been developed. The screening system has been validated in a directed evolution experiment of a distributive polymerase from the Y-polymerase family (Dpo4 from Sulfolobus solfataricus) which was improved in elongation efficiency of consecutive mismatches. The engineering campaign yielded improved Dpo4 polymerase variants one of which was successfully benchmarked in a sequence saturation mutagenesis experiment especially with regard to the desirable consecutive transversion mutations (>2.5-fold increase in frequency relative to a reference library prepared with Dpo4 WT). The Scorpion probe screening system enables to reengineer polymerases with low processivity and fidelity, and no secondary activities (i.e. exonuclease activity or strand displacement activity) to match demands in diversity generation for directed protein evolution.