Florence Bordes

Lipases: An Overview

Lipases are ubiquitous enzymes, widespread in nature. They were first isolated from bacteria in the early nineteenth century and the associated research continuously increased due to the particular characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes.

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Enhanced enantioselectivity: The resolution of 2‐bromo‐arylacetic acid esters by Lip2p lipase from Yarrowia lipolytica was improved through mutagenesis of the substrate binding site. Position 232 was identified as crucial for the discrimination. Saturation of this position led to the identification of variant V232S, which has a tremendously increased activity and E value as compared to the parental enzyme.

Isolation of a thermostable variant of Lip2 lipase from Yarrowia lipolytica by directed evolution and deeper insight into the denaturation mechanisms involved

Lip2 lipase from Yarrowia lipolytica is a very promising lipase with many potential applications (e.g. resolution of racemic mixtures, production of fine chemicals). Unfortunately this potential is impeded by a very low thermostability for temperatures higher than 40°C. Error-prone PCR and screening of the library in a high-performance yeast expression system (Y. lipolytica) enabled a thermostable variant to be identified. This variant presents only one mutation, the free cysteine 244 is changed into an alanine. At 60°C, the half-life time of the purified variant was 127-fold increased compared to the WT enzyme (from 1.5 min to 3 h). Saturation mutagenesis experiment at position 244 demonstrated that the presence of a cysteine at this position was responsible for the thermal denaturation. It was demonstrated that WT Lip2 and the thermostable variant are both inactivated through aggregation mechanisms, but that the kinetics and the nature of the aggregation were different. For the WT enzyme, rapid intermolecular disulphide bridge interchanges triggered by the free cysteine 244 mediates aggregation. For the variant C244A, aggregation still occurred but much slower than for the WT lipase and was mainly driven by hydrophobic forces.

Engineering and production of laccase from Trametes versicolor in the yeast Yarrowia lipolytica.

The lcc1 gene coding for the laccase from Trametes versicolor DSM11269 was cloned into the genome of Yarrowia lipolytica using either single or multiple integration sites. The levels of the recombinant laccase activity secreted in the culture media were 0.25 and 1 U ml(-1) for single and multiple integrations, respectively. The strain with a single integration was successfully used to express variant libraries which were screened on ABTS substrate. The strain encoding the double mutant L185P/Q214K (rM4A) showed a sixfold enhancement in secreted enzyme activity. The catalytic efficiency of the purified rM-4A laccase was respectively increased 2.4- and 2.8-fold towards ABTS and 2,6-dimethoxyphenol, compared to the rWT. Culture supernatants containing either rWT or rM-4A catalyzed the almost complete decolorization of an Amaranth solution (70 nMs(-1)). Taken together, our results open new perspectives for the use of Y. lipolytica as a molecular evolution platform to engineer laccases with improved properties.

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2-bromo phenyl acetic acid esters.

Inverting enzyme enantioselectivity by protein engineering is still a great challenge. Lip2p lipase from Yarrowia lipolytica, which demonstrates a low S‐enantioselectivity (E‐value = 5) during the hydrolytic kinetic resolution of 2‐bromo‐phenyl acetic acid octyl esters (an important class of chemical intermediates in the pharmaceutical industry), was converted, by a rational engineering approach, into a totally R‐selective enzyme (E‐value > 200). This tremendous change in selectivity is the result of only two amino acid changes. The starting point of our strategy was the prior identification of two key positions, 97 and 232, for enantiomer discrimination. Four single substitution variants were recently identified as exhibiting a low inversion of selectivity coupled to a low‐hydrolytic activity. On the basis of these results, six double substituted variants, combining relevant mutations at both 97 and 232 positions, were constructed by site‐directed mutagenesis. This work led to the isolation of one double substituted variant (D97A‐V232F), which displays a total preference for the R‐enantiomer. The highly reversed enantioselectivity of this variant is accompanied by a 4.5‐fold enhancement of its activity toward the preferred enantiomer. The molecular docking of the R‐ and S‐enantiomers in the wild‐type enzyme and the D97A‐V232F variant suggests that V232F mutation provides a more favorable stacking interaction for the phenyl group of the R‐enantiomer, that could explain both the enhanced activity and the reversal of enantioselectivity. These results demonstrate the potential of rationally engineered mutations to further enhance enzyme activity and to modulate selectivity. Biotechnol. Bioeng. 2010;106: 852–859.

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2‐bromo‐arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R‐enantiomer (E‐value = 15), while Lip1 and Lip3 showed an S‐enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S‐ or R‐enantiomer preference of C. rugosa lipases. This led to the construction by site‐directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2‐bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S‐enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)‐2‐bromo‐phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination. Biotechnol. Bioeng. 2011; 108:1749–1756.

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.

Yarrowia lipolytica lipase Lip2: An efficient enzyme for the production of concentrates of docosahexaenoic acid ethyl ester

The production of Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) was studied using lipase-catalysed hydrolysis of a mixture of ethyl esters from tuna oil. Lipases from Yarrowia lipolytica (YLL2), Thermomyces lanuginosus (TLL) and Candida rugosa (CRL1, CRL3 and CRL4) were tested. C. rugosa lipases discriminated esters on the basis of their chain length, with less affinity for γ-linolenate, 11-eicosenoate, arachidonate, EPA, DPA and DHA ethyl esters. However, YLL2 and TLL improved discrimination towards DHA, as enzyme selectivity was shown to be mainly based on the position of the double bond closest to the carboxylic group. From the point of view of kinetics, purity and yield, YLL2 was the most effective lipase for DHA purification. Using this enzyme in an open reactor process resulted in the highest concentrations of DHA ethyl ester (77%) and ω-3 esters (81%) with a recovery of 94% and 77% respectively.

The Yeast Yarrowia lipolytica as a Generic Tool for Molecular Evolution of Enzymes

It has been 20 years since strains of the yeast Yarrowia lipolytica were developed for the expression of recombinant proteins as alternative host to the commonly used yeasts, Pichia pastoris and Saccharomyces cerevisiae. Recently, a new strain, JMY1212, was engineered for protein evolution. With this new strain, a very high reproducibility in protein expression level was demonstrated, thus enabling its use for both rational and directed evolution strategies. Indeed, the coefficient of variation was shown to be of 10.7% for the whole process when all the steps are optimized, i.e. transformation of this strain with the gene of interest, cell growth, and protein production under oleic acid induction, and until activity screening assay. The object of this article is to summarize the fruit of these works and show the interest of Y. lipolytica strain JMY1212 for molecular evolution of enzymes, for both rational and directed evolution strategy. Lipase Lip2 from Y. lipolytica is taken here as an example to describe both strategies of molecular evolution. In these two methods, a first step of PCR creates either one targeted (rational design) or various random mutations (directed evolution), and is followed by the incorporation of the expression cassette in the genome of Y. lipolytica. An easy and direct comparison of variant properties is then allowed thanks to the extracellular and reproducible production of variants.

Functional characterization of a gene locus from an uncultured gut Bacteroides conferring xylo-oligosaccharides utilization to Escherichia coli.

In prominent gut Bacteroides strains, sophisticated strategies have been evolved to achieve the complete degradation of dietary polysaccharides such as xylan, which is one of the major components of the plant cell wall. Polysaccharide Utilization Loci (PULs) consist of gene clusters encoding different proteins with a vast arsenal of functions, including carbohydrate binding, transport and hydrolysis. Transport is often attributed to TonB‐dependent transporters, although major facilitator superfamily (MFS) transporters have also been identified in some PULs. However, until now, few of these transporters have been biochemically characterized. Here, we targeted a PUL‐like system from an uncultivated Bacteroides species that is highly prevalent in the human gut metagenome. It encodes three glycoside‐hydrolases specific for xylo‐oligosaccharides, a SusC/SusD tandem homolog and a MFS transporter. We combined PUL rational engineering, metabolic and transcriptional analysis in Escherichia coli to functionally characterize this genomic locus. We demonstrated that the SusC and the MFS transporters are specific for internalization of linear xylo‐oligosaccharides of polymerization degree up to 3 and 4 respectively. These results were strengthened by the study of growth dynamics and transcriptional analyses in response to XOS induction of the PUL in the native strain, Bacteroides vulgatus.