Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein.     

Enhanced expression and purification of fungal galactose oxidase in Escherichia coli and use for analysis of a saturation mutagenesis library

Galactose oxidase (GO) displays broad primary alcohol substrate specificity and so offers potential for engineering new substrate specificity by directed evolution. Producing variant libraries of sufficient complexity ideally requires expression of functional protein in a host such as Escherichia coli. Wild-type GO is produced by the fungus Fusarium graminiarum and is expressed poorly in E. coli. We introduced silent mutations within codons 2-7 of the mature GO coding sequence to enhance GO translation and have combined these with other expression-enhancing mutations. We selected the best E. coli host strain, autoinduction medium, induction temperature, harvest time and cell lysis procedure to produce active recombinant GO. Although normally secreted by the fungus, we have expressed GO in the cytoplasm of E. coli and have used a C-terminal Streptag II sequences for single-step affinity purification. This resulted in purification of 240 mg of functional GO per litre of shake flask culture. We have generated a saturation mutagenesis library at residue Cys383, known to influence substrate binding, and have used the optimised expression conditions to purify and characterise the resulting enzymes.     

Functional expression of a plant hydroxynitrile lyase in Escherichia coli by directed evolution: creation and characterization of highly in vivo soluble mutants

Low protein solubility of recombinantly expressed proteins in Escherichia coli is a major factor hindering their application and analysis. We generated highly in vivo soluble mutants of a hydroxynitrile lyase in E.coli using protein engineering. Structure-guided saturation mutagenesis caused high solubility of single Lys-Pro mutations at positions 176, 199 and 224 of this low soluble wild-type enzyme. The triple Lys-Pro mutant generated at these surface conserved residues showed up to 8-fold increase in specific activity in the cell-free extract. Random mutagenesis also created a mutant of His103Met with 18.5-fold increase. The main expression form was reversed from insoluble to the soluble fraction following both types of above-mentioned mutations in E.coli at 37°C. The findings challenge the rationale of producing recombinant proteins in this host at 37°C. Formerly wild type low soluble protein was then present as soluble protein by these mutations, which also elevated the total soluble protein fraction in E.coli. Saturation mutagenesis of His103 provided other highly soluble mutants with hydrophobic substitutions. These mutations caused only minor secondary structural changes as determined by circular dichroism and Fourier-transform infrared spectroscopy and affected catalytic efficiency slightly for the purified mutants (0.82-1.6-fold for benzaldehyde and 0.9-1.9-fold for mandelonitrile). The stability of the mutants was differed from that of the wild type at high temperatures and at pH >8. Exchanging the buried basic-polar residue His103 with hydrophobic amino acids is in line with the overall structure of the enzyme, i.e. having hydrophilic residues in solvent-exposed areas and hydrophobic residues in the core.     

多铜氧化酶在大肠杆菌中的分泌表达

生物胺是存在于发酵食品中的一类有机物,过量摄入会危害人体健康。多铜氧化酶中的某些酶具有降解多种生物胺的活性,在减控发酵食品中的氨(胺)类危害物方面具有良好的应用前景。研究多铜氧化酶的分泌表达,对酶的特性改造和工业化生产与应用具有重要意义。本研究通过在解淀粉芽孢杆菌来源的多铜氧化酶N端融合信号肽PhoA实现了多铜氧化酶在大肠杆菌中的分泌表达,胞外酶活为69.8 U/L。通过优化诱导条件和酶的分泌确定了多铜氧化酶最优发酵条件为诱导温度25℃、IPTG浓度0.05 mmol/L、诱导时菌体OD600=1.0、诱导6 h后添加150 mmol/L甘氨酸;发酵40 h时胞外多铜氧化酶酶活达到238.1 U/L,是优化前的3.4倍。     

Enhanced fructose oxidase activity in a galactose oxidase variant

Galactose oxidase (GO; EC 1.1.3.9) catalyses the oxidation of a wide range of primary alcohols including mono-, oligo- and polysaccharides. High-resolution structures have been determined for GO, but no structural information is available for the enzyme with bound substrate or inhibitor. Previously, computer-aided docking experiments have been used to develop a plausible model for interactions between GO and the D-galactose substrate. Residues implicated in such interactions include Arg330, Gln406, Phe464, Phe194 and Trp290. In the present study we describe an improved expression system for recombinant GO in the methylotrophic yeast Pichia pastoris. We use this system to express variant proteins mutated at Arg330 and Phe464 to explore the substrate binding model. We also demonstrate that the Arg330 variants display greater fructose oxidase activity than does wild-type GO.     

Biosystems design by directed evolution

Through iterative rounds of genetic diversification and screening or selection, directed evolution has been widely used to engineer relatively simple biosystems such as nucleic acids and proteins with desired functions. In addition, directed evolution has played an important role in engineering more complex biosystems such as pathways and genomes. Since 2013, directed evolution has been further explored for biosystems design with numerous newly developed techniques that have enabled design and engineering of proteins, pathways, and genomes in a much more effective manner. In this review, we will highlight the abiotic biotransformations arisen from directed evolution and novel strategies for continuous evolution in vivo and ultrahigh-throughput screening. We will also discuss the future challenges and opportunities of applying directed evolution for biosystems design.     

Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state

Phosphotriesterase from Pseudomonas diminuta (PTE) is an extremely efficient metalloenzyme that hydrolyses a variety of compounds including organophosphorus nerve agents. Study of PTE has been hampered by difficulties with efficient expression of the recombinant form of this highly interesting and potentially useful enzyme. We identified a low-level esterolytic activity of PTE and then screened PTE gene libraries for improvements in 2-naphthyl acetate hydrolysis. However, the attempt to evolve this promiscuous esterase activity led to a variant (S5) containing three point mutations that resulted in a 20-fold increase in functional expression. Interestingly, the zinc holoenzyme form of S5 appears to be more sensitive than wild-type PTE to both thermal denaturation and addition of metal chelators. Higher functional expression of the S5 variant seems to lie in a higher stability of the metal-free apoenzyme. The results obtained in this work point out another-and often overlooked-possible determinant of protein expression and purification yields, i.e. the stability of intermediates during protein folding and processing.     

High-level Expression of Cecropin X in Escherichia coli

Cecropin X is a short cationic peptide with a broad antibacterial and antitumor spectrum. Here, we report the production of a tumor necrosis factor (TNFα)-cecropin X fusion protein under the control of a temperature-inducible P_R promoter in the bacterial expression vector pRC. During fermentation, we studied and optimized essential parameters including the type of host cells, medium, timing of induction, post-induction time and dissolved oxygen level. Using the suitable conditions in the fermentation, up to 20 % ~ 23 % of the total cellular proteins is produced as the fusion protein, mostly in the form of inclusion bodies. After washing, on average about 5.27 g dried inclusion bodies could be collected from 1 L broth and the purity of inclusion bodies reached 80 %. Cecropin X obtained by cleaving the fusion protein with cyanogen bromide showed remarkable tumorcidal activity against mouse Lewis lung carcinoma 3LL in vivo.     

Engineering aggregation-resistant proteins by directed evolution

A method was recently described for selecting aggregation-resistant antibody domains. A repertoire of domains displayed on filamentous bacteriophage were heated/cooled and selected for binding to the affinity ligand protein A specific for the folded domains. Here we describe a generalization of the method based on the selection for retained phage infectivity and for the binding of an appended sequence tag, and applicable to any protein displayable in multivalent form on phage.     

Thermostabilization of firefly luciferase by in vivo directed evolution

Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four?of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity.     

组成型天冬氨酸转氨酶基因工程菌的构建与高效表达

天冬氨酸转氨酶AspAT是苯丙酮酸转氨制备L-苯丙氨酸的关键酶. 本研究将大肠杆菌中天冬氨酸转氨酶基因aspC克隆到3种不同质粒中,构建组成型表达质粒pUC/P-aspC, pSE/P-aspC, pET/P-aspC,并分别转化至6种常用的大肠杆菌宿主中. 通过对18种重组子的生长及产酶情况的分析,比较了各种重组子生长压力、质粒稳定性与表达酶活的关系,并经SDS-PAGE电泳分析AspAT的表达量,筛选出高产AspAT的重组子BL21(pET/P-aspC),以该工程菌发酵液直接作为酶液,以天冬氨酸和苯丙酮酸(20 g/L)为底物,发酵液与底物以1:3的体积比转化生成L-苯丙氨酸16.2 g/L,转化率高达80.1%. 该体系表达无需诱导,转化无需添加辅酶PLP,展现了良好的产业化前景.     

Expression characteristics of the maeA and maeB genes by extracellular malate and pyruvate in Escherichia coli

The malate-pyruvate conversion pathway is catalyzed by two malic enzyme isomers, MaeA and MaeB. qRT-PCR was carried out under malate and pyruvate supplemented conditions to understand the dynamics of maeA and maeB gene expression. maeA expression was elevated by malate, and maeB expression was inhibited by levels of both malate and pyruvate higher than 0.1 mM. Green fluorescent protein (GFP) reporter plasmids were also constructed by integration of the maeA/maeB promoter with the gfp gene. We showed that maeA driven GFP expression was positively and negatively correlated with extracellular malate and pyruvate induction. In contrast, no significant changes in maeB driven GFP expression were observed under both malate and pyruvate supplemented conditions.     

外源蛋白在大肠杆菌中的可溶性表达策略

长期以来,大肠杆菌一直是表达外源蛋白的首选表达系统. 但由于外源蛋白在表达过程中容易被宿主细胞蛋白酶降解或者形成包涵体,其应用受到了限制. 本文综述了在大肠杆菌中表达可溶外源蛋白的策略和进展,以期提高具有生物活性的外源基因的表达水平.     

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D_2S and D₃ belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D_2S and D₃ receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate K_D-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.     

Expression of a bispecific dsFv-dsFv' antibody fragment in Escherichia coli

A bispecific disulfide-stabilized Fv antibody fragment (dsFv–dsFv')consisting of two different disulfide-stabilized Fv antibodyfragments connected by flexible linker peptides was producedby secretion of three polypeptide chains into the periplasmof Escherichia coli. The dsFv–dsFv' molecules were enrichedby immobilized metal affinity chromatography and further purifiedby anion-exchange chromatography. The recombinant antibody constructsretained the two parental antigen binding specificities andwere able to cross-link the two different antigens. The describeddsFv–dsFv' design might be of particular value for therapeuticin vivo applications since improved stability is expected tobe combined with minimal immunogenicity.     

Creation of GPCR-based chemical sensors by directed evolution in yeast

G protein-coupled receptors (GPCRs) form a class of biological chemical sensors with an enormous diversity in ligand binding and sensitivity. To explore structural aspects of ligand recognition, we subjected the human UDP-glucose receptor (P2Y14) functionally expressed in the yeast Saccharomyces to directed evolution. We sought to generate new receptor subtypes with ligand-binding properties that would be useful in the development of practical biosensors. Mutagenesis of the entire UDP-glucose receptor gene yielded receptors with increased activity but similar ligand specificities, while random mutagenesis of residues in the immediate vicinity of the ligand-binding pocket yielded mutants with altered ligand specificity. By first sensitizing the P2Y14 receptor and then redirecting ligand specificity, we were able to create mutant receptors suitable for a simple biosensor. Our results demonstrate the feasibility of altering receptor ligand-binding properties via a directed evolution strategy, using standard yeast genetic techniques. The novel receptor mutants can be used to detect chemical ligands in complex mixtures and to discriminate among chemically or stereochemically related compounds. Specifically, we demonstrate how engineered receptors can be applied in a pairwise manner to differentiate among several chemical analytes that would be indistinguishable with a single receptor. These experiments demonstrate the feasibility of a combinatorial approach to detector design based on the principles of olfaction.