Porter JL

References (8)

Title : Improving on nature's shortcomings: evolving a lipase for increased lipolytic activity, expression and thermostability - Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
Author(s) : Alfaro-Chavez AL , Liu JW , Porter JL , Goldman A , Ollis DL
Ref : Protein Engineering Des Sel , 32 :13 , 2019
Abstract : An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37 degrees C, while the mutant with P291L of 42.2 degrees C and the mutant R7_47D with five mutations had a value of 52.9 degrees C, corresponding to an improvement of 16 degrees C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.
ESTHER : Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
PubMedSearch : Alfaro-Chavez_2019_Protein.Eng.Des.Sel_32_13
PubMedID: 31403166
Gene_locus related to this paper: drome-lip3

Title : Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search - Porter_2015_ACS.Chem.Biol_10_611
Author(s) : Porter JL , Boon PL , Murray TP , Huber T , Collyer CA , Ollis DL
Ref : ACS Chemical Biology , 10 :611 , 2015
Abstract : The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse alpha/beta hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
ESTHER : Porter_2015_ACS.Chem.Biol_10_611
PubMedSearch : Porter_2015_ACS.Chem.Biol_10_611
PubMedID: 25419863
Gene_locus related to this paper: psepu-clcd1

Title : Compensatory stabilizing role of surface mutations during the directed evolution of dienelactone hydrolase for enhanced activity - Porter_2015_Protein.J_34_82
Author(s) : Porter JL , Collyer CA , Ollis DL
Ref : Protein J , 34 :82 , 2015
Abstract : Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.
ESTHER : Porter_2015_Protein.J_34_82
PubMedSearch : Porter_2015_Protein.J_34_82
PubMedID: 25600287

Title : Crystallization of dienelactone hydrolase in two space groups: structural changes caused by crystal packing - Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
Author(s) : Porter JL , Carr PD , Collyer CA , Ollis DL
Ref : Acta Crystallographica F Struct Biol Commun , 70 :884 , 2014
Abstract : Dienelactone hydrolase (DLH) is a monomeric protein with a simple [alpha]/[beta]-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85 A resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.
ESTHER : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedSearch : Porter_2014_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_70_884
PubMedID: 25005082
Gene_locus related to this paper: psepu-clcd1

Title : Deciphering the genetic basis for polyketide variation among mycobacteria producing mycolactones - Pidot_2008_BMC.Genomics_9_462
Author(s) : Pidot SJ , Hong H , Seemann T , Porter JL , Yip MJ , Men A , Johnson M , Wilson P , Davies JK , Leadlay PF , Stinear TP
Ref : BMC Genomics , 9 :462 , 2008
Abstract : BACKGROUND: Mycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants (named A/B, C, D, E and F), produced by different species of very closely related mycobacteria including the human pathogen, Mycobacterium ulcerans. In M. ulcerans strain Agy99, mycolactone A/B is produced by three highly homologous type I polyketide megasynthases (PKS), whose genes (mlsA1: 51 kb, mlsA2: 7.2 kb and mlsB: 42 kb) are found on a 174 kb plasmid, known as pMUM001. RESULTS: We report here comparative genomic analysis of pMUM001, the complete DNA sequence of a 190 kb megaplasmid (pMUM002) from Mycobacterium liflandii 128FXT and partial sequence of two additional pMUM replicons, combined with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. These data reveal how PKS module and domain differences affecting MlsB correlate with the production of mycolactones E and F. For mycolactone E these differences from MlsB in M. ulcerans Agy99 include replacement of the AT domain of the loading module (acetate to propionate) and the absence of an entire extension module. For mycolactone F there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone F CONCLUSION: The mycolactone PKS locus on pMUM002 revealed the same large, three-gene structure and extraordinary pattern of near-identical PKS domain sequence repetition as observed in pMUM001 with greater than 98.5% nucleotide identity among domains of the same function. Intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls PKS genes is caused by frequent recombination-mediated domain replacement. This work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial PKS biochemistry.
ESTHER : Pidot_2008_BMC.Genomics_9_462
PubMedSearch : Pidot_2008_BMC.Genomics_9_462
PubMedID: 18840298

Title : Evolution of Mycobacterium ulcerans and other mycolactone-producing mycobacteria from a common Mycobacterium marinum progenitor - Yip_2007_J.Bacteriol_189_2021
Author(s) : Yip MJ , Porter JL , Fyfe JA , Lavender CJ , Portaels F , Rhodes M , Kator H , Colorni A , Jenkin GA , Stinear T
Ref : Journal of Bacteriology , 189 :2021 , 2007
Abstract : It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.
ESTHER : Yip_2007_J.Bacteriol_189_2021
PubMedSearch : Yip_2007_J.Bacteriol_189_2021
PubMedID: 17172337
Gene_locus related to this paper: mycul-a85a

Title : Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer - Stinear_2007_Genome.Res_17_192
Author(s) : Stinear TP , Seemann T , Pidot S , Frigui W , Reysset G , Garnier T , Meurice G , Simon D , Bouchier C , Ma L , Tichit M , Porter JL , Ryan J , Johnson PD , Davies JK , Jenkin GA , Small PL , Jones LM , Tekaia F , Laval F , Daffe M , Parkhill J , Cole ST
Ref : Genome Res , 17 :192 , 2007
Abstract : Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.
ESTHER : Stinear_2007_Genome.Res_17_192
PubMedSearch : Stinear_2007_Genome.Res_17_192
PubMedID: 17210928
Gene_locus related to this paper: mycmm-b2hds9 , mycmm-b2hg81 , mycmm-b2hgg2 , mycmm-b2hj55 , mycmm-b2hju3 , mycmm-b2hlr0 , mycmm-b2hlv2 , mycmm-b2hq96 , mycmm-b2hsm8 , mycmr-q5sdq4 , myctu-RV1683 , mycua-a0pkg7 , mycua-a0pki1 , mycua-a0pkn2 , mycua-a0pkn5 , mycua-a0pku2 , mycua-a0pl47 , mycua-a0plr3 , mycua-a0plu8 , mycua-a0ply4 , mycua-a0pm12 , mycua-a0pm14 , mycua-a0pme3 , mycua-a0pmj6 , mycua-a0pml9 , mycua-a0pmv0 , mycua-a0pmx9 , mycua-a0pn71 , mycua-a0png7 , mycua-a0png9 , mycua-a0pp56 , mycua-a0ppm6 , mycua-a0pqm2 , mycua-a0pqs2 , mycua-a0pr64 , mycua-a0pr98 , mycua-a0prq2 , mycua-a0prr7 , mycua-a0psb1 , mycua-a0psb4 , mycua-a0psi2 , mycua-a0psi3 , mycua-a0psu3 , mycua-a0pt08 , mycua-a0pt71 , mycua-a0pth6 , mycua-a0ptq0 , mycua-a0pu55 , mycua-a0pum4 , mycua-a0pv11 , mycua-a0pv36 , mycua-a0pv77 , mycua-a0pva4 , mycua-a0pwi8 , mycua-a0pwm4 , mycua-a0pwr6 , mycua-a0pwz5 , mycua-a0px52 , mycua-metx , mycua-a0pmc2 , mycua-a0pvg7 , mycua-a0pwz4 , mycmm-b2hqy3 , mycua-a0pmc3

Title : Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans - Stinear_2005_Microbiology_151_683
Author(s) : Stinear TP , Pryor MJ , Porter JL , Cole ST
Ref : Microbiology , 151 :683 , 2005
Abstract : The presence of a 174 kb plasmid called pMUM001 in Mycobacterium ulcerans, the first example of a mycobacterial plasmid encoding a virulence determinant, was recently reported. Over half of pMUM001 is devoted to six genes, three of which encode giant polyketide synthases (PKS) that produce mycolactone, an unusual cytotoxic lipid produced by M. ulcerans. In this present study the remaining 75 non-PKS-associated protein-coding sequences (CDS) are analysed and it is shown that pMUM001 is a low-copy-number element with a functional ori that supports replication in Mycobacterium marinum but not in the fast-growing mycobacteria Mycobacterium smegmatis and Mycobacterium fortuitum. Sequence analyses revealed a highly mosaic plasmid gene structure that is reminiscent of other large plasmids. Insertion sequences (IS) and fragments of IS, some previously unreported, are interspersed among functional gene clusters, such as those genes involved in plasmid replication, the synthesis of mycolactone, and a potential phosphorelay signal transduction system. Among the IS present on pMUM001 were multiple copies of the high-copy-number M. ulcerans elements IS2404 and IS2606. No plasmid transfer systems were identified, suggesting that trans-acting factors are required for mobilization. The results presented here provide important insights into this unusual virulence plasmid from an emerging but neglected human pathogen.
ESTHER : Stinear_2005_Microbiology_151_683
PubMedSearch : Stinear_2005_Microbiology_151_683
PubMedID: 15758215