International Journal of Molecular Sciences

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24 pages, 5676 KiB

Open AccessArticle

Deletion and Randomization of Structurally Variable Regions in B. subtilis Lipase A (BSLA) Alter Its Stability and Hydrolytic Performance Against Long Chain Fatty Acid Esters

by Ronny Martínez, Claudia Bernal, Rodrigo Álvarez, Christopher Concha, Fernando Araya, Ricardo Cabrera, Gaurao V. Dhoke and Mehdi D. Davari

Int. J. Mol. Sci. 2020, 21(6), 1990; https://doi.org/10.3390/ijms21061990 - 14 Mar 2020

Cited by 9 | Viewed by 3266

Abstract

The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated [...] Read more.

The continuous search for novel enzyme backbones and the engineering of already well studied enzymes for biotechnological applications has become an increasing challenge, especially by the increasing potential diversity space provided by directed enzyme evolution approaches and the demands of experimental data generated by rational design of enzymes. In this work, we propose a semi-rational mutational strategy focused on introducing diversity in structurally variable regions in enzymes. The identified sequences are subjected to a progressive deletion of two amino acids and the joining residues are subjected to saturation mutagenesis using NNK degenerate codons. This strategy offers a novel library diversity approach while simultaneously decreasing enzyme size in the variable regions. In this way, we intend to identify and reduce variable regions found in enzymes, probably resulting from neutral drift evolution, and simultaneously studying the functional effect of said regions. This strategy was applied to Bacillus. subtilis lipase A (BSLA), by selecting and deleting six variable enzyme regions (named regions 1 to 6) by the deletion of two amino acids and additionally randomizing the joining amino acid residues. After screening, no active variants were found in libraries 1% and 4%, 15% active variants were found in libraries 2% and 3%, and 25% for libraries 5 and 6 (n = 3000 per library, activity detected using tributyrin agar plates). Active variants were assessed for activity in microtiter plate assay (pNP-butyrate), thermal stability, substrate preference (pNP-butyrate, -palmitate), and compared to wildtype BSLA. From these analyses, variant P5F3 (F41L-ΔW42-ΔD43-K44P), from library 3 was identified, showing increased activity towards longer chain p-nitrophenyl fatty acid esters, when compared to BSLA. This study allowed to propose the targeted region 3 (positions 40–46) as a potential modulator for substrate specificity (fatty acid chain length) in BSLA, which can be further studied to increase its substrate spectrum and selectivity. Additionally, this variant showed a decreased thermal resistance but interestingly, higher isopropanol and Triton X-100 resistance. This deletion-randomization strategy could help to expand and explore sequence diversity, even in already well studied and characterized enzyme backbones such as BSLA. In addition, this strategy can contribute to investigate and identify important non-conserved regions in classic and novel enzymes, as well as generating novel biocatalysts with increased performance in specific processes, such as enzyme immobilization. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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14 pages, 1802 KiB

Open AccessArticle

Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

by Bangqiao Yin, Qinyan Hui, Muhammad Kashif, Ran Yu, Si Chen, Qian Ou, Bo Wu and Chengjian Jiang

Int. J. Mol. Sci. 2019, 20(24), 6224; https://doi.org/10.3390/ijms20246224 - 10 Dec 2019

Cited by 12 | Viewed by 3808

Abstract

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid [...] Read more.

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (k_cat/K_m of 561.72 mM⁻¹ s⁻¹) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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19 pages, 1667 KiB

Open AccessArticle

Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

by Miriam González-Villanueva, Hemanshi Galaiya, Paul Staniland, Jessica Staniland, Ian Savill, Tuck Seng Wong and Kang Lan Tee

Int. J. Mol. Sci. 2019, 20(22), 5737; https://doi.org/10.3390/ijms20225737 - 15 Nov 2019

Cited by 24 | Viewed by 7354

Abstract

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its [...] Read more.

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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24 pages, 2413 KiB

Open AccessArticle

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology

by Andrea C. Kneuttinger, Stefanie Zwisele, Kristina Straub, Astrid Bruckmann, Florian Busch, Thomas Kinateder, Barbara Gaim, Vicki H. Wysocki, Rainer Merkl and Reinhard Sterner

Int. J. Mol. Sci. 2019, 20(20), 5106; https://doi.org/10.3390/ijms20205106 - 15 Oct 2019

Cited by 8 | Viewed by 4346

Abstract

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a [...] Read more.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from Salmonella typhimurium (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network. To control TS allostery with light, we incorporated the unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) at seven strategic positions of TrpA and TrpB. Initial screening experiments showed that ONBY in position 58 of TrpA (aL58ONBY) inhibits TS activity most effectively. Upon UV irradiation, ONBY decages to tyrosine, largely restoring the capacity of TS. Biochemical characterization, extensive steady-state enzyme kinetics, and titration studies uncovered the impact of aL58ONBY on the activities of TrpA and TrpB and identified reaction conditions under which the influence of ONBY decaging on allostery reaches its full potential. By applying those optimal conditions, we succeeded to directly light-activate TS(aL58ONBY) by a factor of ~100. Our findings show that rational protein design with a photo-sensitive unnatural amino acid combined with extensive enzymology is a powerful tool to fine-tune allosteric light-activation of a central metabolic enzyme complex. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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21 pages, 7445 KiB

Open AccessArticle

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

by Yu Zhou and Spencer Whitney

Int. J. Mol. Sci. 2019, 20(20), 5019; https://doi.org/10.3390/ijms20205019 - 10 Oct 2019

Cited by 49 | Viewed by 8843

Abstract

Inaccuracies in biochemically characterizing the amount and CO₂-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and [...] Read more.

Inaccuracies in biochemically characterizing the amount and CO₂-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO₂-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (k_cat^C, up 27%), carboxylation efficiency (k_cat^C/K_m for CO₂, increased 17%), unchanged CO₂/O₂ specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L₈S₈) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility). Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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13 pages, 2100 KiB

Open AccessArticle

PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries

by Pawel Jajesniak, Kang Lan Tee and Tuck Seng Wong

Int. J. Mol. Sci. 2019, 20(16), 3908; https://doi.org/10.3390/ijms20163908 - 11 Aug 2019

Cited by 4 | Viewed by 5142

Abstract

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, [...] Read more.

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3′ overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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24 pages, 10460 KiB

Open AccessArticle

Directed Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles

by Gustavo de Almeida Santos, Gaurao V. Dhoke, Mehdi D. Davari, Anna Joëlle Ruff and Ulrich Schwaneberg

Int. J. Mol. Sci. 2019, 20(13), 3353; https://doi.org/10.3390/ijms20133353 - 8 Jul 2019

Cited by 14 | Viewed by 5784

Abstract

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the [...] Read more.

The O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ≈140-fold (WT: 8.3 ± 1.3 min⁻¹; R255L: 1168 ± 163 min⁻¹), total turnover number (TTN) increased ≈21-fold (WT: 40 ± 3; R255L: 860 ± 15), and coupling efficiency, ≈2.9-fold (WT: 8.8 ± 0.1%; R255L: 25.7 ± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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Review

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25 pages, 1305 KiB

Open AccessReview

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

by Francisca Contreras, Subrata Pramanik, Aleksandra M. Rozhkova, Ivan N. Zorov, Olga Korotkova, Arkady P. Sinitsyn, Ulrich Schwaneberg and Mehdi D. Davari

Int. J. Mol. Sci. 2020, 21(5), 1589; https://doi.org/10.3390/ijms21051589 - 26 Feb 2020

Cited by 75 | Viewed by 8248

Abstract

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production [...] Read more.

Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. Full article

(This article belongs to the Special Issue A Commemorative Issue to Celebrate the Nobel Success of Prof. Frances H. Arnold: Directed Evolution)

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