Exploring the Protein Stability Landscape: Bacillus subtilis Lipase A as a Model for Detergent Tolerance

A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/β‐hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.     

Directed evolution of Bacillus subtilis lipase A by use of enantiomeric phosphonate inhibitors: crystal structures and phage display selection

Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(-)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis-Menten parameters. The 3D structures of the Sc and Rc inhibitor-lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.     

基于脯氨酸理论提高枯草芽孢杆菌脂肪酶A的热稳定性

以枯草芽孢杆菌脂肪酶A(LipA)为研究对象,根据从RCSB数据库中获取的晶体结构,采用分子动力学模拟和分子生物学实验相结合的方法进行脂肪酶热稳定性位点突变的理性设计。首先,利用分子动力学模拟获得晶体结构中柔性较高的Loop区域;进而,结合"脯氨酸理论",将位于该区域附近的Gly残基突变为Pro,分析引入Pro突变对LipA热稳定性的影响,筛选得到Gly52和Gly158两个突变位点;最后,通过定点突变操作对突变株LipAG52P和LipAG158P进行热稳定性实验验证。结果显示,突变株LipAG52P、LipAG158P的比活力分别是野生型LipA的5.6倍和2.7倍,Tm值分别提高了15℃和7℃,催化效率分别提高了85%和22%。     

Manipulating the expression rate and enantioselectivity of an epoxide hydrolase by using directed evolution

We describe here a strategy to improve the expression efficiency and enantioselectivity of Aspergillus niger epoxide hydrolase (ANEH) by directed evolution. Based on a blue‐colony screening system using the LacZα (β‐galactosidase α peptide) complementation solubility reporter, several ANEH variants out of 15 000 transformants from a random‐mutagenesis library were identified that show improved recombinant expression in E. coli. Among them, Pro221Ser was subsequently used as a template for iterative saturation mutagenesis (ISM) at sites around the ANEH binding pocket. Following four rounds of ISM, a highly enantioselective mutant was identified that catalyzes the hydrolytic kinetic resolution of racemic glycidyl phenyl ether with a selectivity factor of E=160 in favor of the (S)‐diol compared to WT ANEH characterized by E=4.6. Expression of this mutant is 50 times higher than that of WT ANEH. It also serves as an excellent stereoselective catalyst in the hydrolytic kinetic resolution and desymmetrization of several other structurally diverse epoxides.     

Learning from directed evolution: Further lessons from theoretical investigations into cooperative mutations in lipase enantioselectivity

An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).     

Directed evolution of an esterase from Pseudomonas fluorescens yields a mutant with excellent enantioselectivity and activity for the kinetic resolution of a chiral building block

A triple mutant of an esterase from Pseudomonas fluorescens (PFE) that was created by directed evolution exhibited high enantioselectivity (E=89) in a kinetic resolution and yielded the building block (S)-but-3-yn-2-ol. Surprisingly, a mutation close to the active site caused the formation of inclusion bodies, but remote mutations were found to be responsible for the high selectivity. Back mutations gave a variant (double mutant PFE Ile76Val/Val175Ala) that showed excellent selectivity (E=96) and activity (20 min for 50% conversion, which corresponds to 1.25 U per mg of protein).     

Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.     

Multiplex-PCR-based recombination as a novel high-fidelity method for directed evolution

A new and convenient method for the in vitro recombination of single point mutations is presented. This method efficiently reduces the introduction of novel point mutations, which usually occur during recombination processes. A multiplex polymerase chain reaction (multiplex-PCR) generates gene fragments that contain preformed point mutations. These fragments are subsequently assembled into full-length genes by a recombination-PCR step. The process of multiplex-PCR-based recombination (MUPREC) does not require DNase I digestion for gene-fragmentation and is therefore easy to perform, even with small amounts of target DNA. The protocol yields high frequencies of recombination without creating a wild-type background. Furthermore, the low error rate results in high-quality variant libraries of true recombinants, thereby minimizing the screening efforts and saving time and money. The MUPREC method was used in the directed evolution of a Bacillus subtilis lipase that can catalyse the enantioselective hydrolysis of a model meso-compound. Thereby, the method was proved to be useful in producing a reliable second-generation library of true recombinants from which better performing variants were identified by using a high-throughput electrospray ionization mass spectrometry (ESI-MS) screening system.     

Constructing and analyzing the fitness landscape of an experimental evolutionary process

Iterative saturation mutagenesis (ISM) is a promising approach to more efficient directed evolution, especially for enhancing the enantioselectivity and/or thermostability of enzymes. This was demonstrated previously for an epoxide hydrolase (EH), after five sets of mutations led to a stepwise increase in enantioselectivity. This study utilizes these results to illuminate the nature of ISM, and identify the reasons for its operational efficacy. By applying a deconvolution strategy to the five sets of mutations and measuring the enantioselectivity factors (E) of the EH variants, DeltaDeltaG( not equal) values become accessible. With these values, the construction of the complete fitness-pathway landscape is possible. The free energy profiles of the 5!=120 evolutionary pathways leading from the wild-type to the best mutant show that 55 trajectories are energetically favored, one of which is the originally observed route. This particular pathway was analyzed in terms of epistatic effects operating between the sets of mutations at all evolutionary stages. The degree of synergism increases as the stepwise evolutionary process proceeds. When encountering a local minimum in a disfavored pathway, that is, in the case of a dead end, choosing another set of mutations at a previous stage puts the evolutionary process back on an energetically favored trajectory. The type of analysis presented here might be useful when evaluating other mutagenesis methods and strategies in directed evolution.     

Increased Enantioselectivity by Engineering Bottleneck Mutants in an Esterase from Pseudomonas fluorescens

Four hydrophobic and bulky amino acid residues (F126, F144, F159, and I225) were identified to form a bottleneck guarding the entrance to the active site of an esterase from Pseudomonas fluorescens (PFE I). Hence, a range of nonpolar amino acids were introduced into PFE I to broaden the substrate range and to increase enantioselectivity while preserving the hydrophobicity of the tunnel. First, single variants were created and then the most enantioselective ones were combined to find cooperative effects. This resulted in several mutants, which showed substantially enhanced enantioselectivity; for instance, in the kinetic resolution of 1‐phenyl‐1‐propyl acetate, with which the wild type only showed E=1.2, two mutants gave E>46. For 1‐phenyl‐1‐ethyl acetate enantioselectivity increased from ～50 to >100 for all mutants studied. Furthermore, higher conversions could be found at shorter reaction times; this indicates that the mutations not only enhanced selectivity, but that also the entrance into the active site was indeed facilitated by these mutations. The experimental results could be explained by computer modeling.     

Simultaneous Use of in Silico Design and a Correlated Mutation Network as a Tool To Efficiently Guide Enzyme Engineering

In order to improve the efficiency of directed evolution experiments, in silico multiple‐substrate clustering was combined with an analysis of the variability of natural enzymes within a protein superfamily. This was applied to a Pseudomonas fluorescens esterase (PFE I) targeting the enantioselective hydrolysis of 3‐phenylbutyric acid esters. Data reported in the literature for nine substrates were used for the clustering meta‐analysis of the docking conformations in wild‐type PFE I, and this highlighted a tryptophan residue (W28) as an interesting target. Exploration of the most frequently, naturally occurring amino acids at this position suggested that the reduced flexibility observed in the case of the W28F variant leads to enhancement of the enantioselectivity. This mutant was subsequently combined with mutations identified in a library based on analysis of a correlated mutation network. By interrogation of <80 variants a mutant with 15‐fold improved enantioselectivity was found.     

Achieving regio- and enantioselectivity of P450-catalyzed oxidative CH activation of small functionalized molecules by structure-guided directed evolution

Directed evolution of the monooxygenase P450-BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio- and enantioselectivity in the oxidative hydroxylation of cyclohexene-1-carboxylic acid methyl ester. Wild-type P450-BM3 is 84% regioselective for the allylic 3-position with 34% enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95% ee) or S selectivity (>95% ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93%. Control of the substrate-to-enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono-oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.     

枯草芽孢杆菌脂肪酶表达质粒的构建及其表达

以枯草芽孢杆菌168(B.subtilis 168)染色体为模板PCR扩增出P43启动子,与大肠杆菌-枯草杆菌穿梭质粒pUBC19相连得到表达载体pUBC-P43,然后将枯草芽孢杆菌脂肪酶基因lipA克隆到载体pUBC-P43启动子下游,得到重组质粒pUBCPL并转化B.subtilis TZ10.经中性红油脂平板、酶切和PCR方法鉴定得到重组菌TZ10/pUBCPL.宿主菌TZ10是B.subtilis DB104染色体缺失了lipA基因后获得.重组菌经初步发酵,以橄榄油为底物测定发酵上清液最高脂肪酶活力为49.1 U·L-1,而相应菌株DB104发酵最高酶活力仅为11.4 U·L-1.