Dataset Information

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

ABSTRACT: Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting from the double variant W159H/S238F of Ideonella sakaiensis 201-F6 PETase, the W159H/F238A-ΔIsPET variant, possessing a higher hydrolytic activity on PET, was identified. This variant was stabilized by introducing two additional known substitutions (S121E and D186H) generating the TS-ΔIsPET variant. By using 0.1 mg mL-1 of TS-ΔIsPET, ~10.6 mM of degradation products were produced in 2 days from 9 mg mL-1 PET microparticles (~26% depolymerization yield). Indeed, TS-ΔIsPET allowed a massive degradation of PET nanoparticles (>80% depolymerization yield) in 1.5 h using only 20 μg of enzyme mL-1. The rationale underlying the effect on the catalytic parameters due to the F238A substitution was studied by enzymatic investigation and molecular dynamics/docking analysis. The present workflow is a well-suited protocol for the evolution of PHEs to help generate an efficient enzymatic toolbox for polyester degradation.

SUBMITTER: Pirillo V

PROVIDER: S-EPMC8745736 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

Pirillo Valentina V Orlando Marco M Tessaro Davide D Pollegioni Loredano L Molla Gianluca G

International journal of molecular sciences 20211227 1

Enzymatic degradation is a promising green approach to bioremediation and recycling of the polymer poly(ethylene terephthalate) (PET). In the past few years, several PET-hydrolysing enzymes (PHEs) have been discovered, and new variants have been evolved by protein engineering. Here, we report on a straightforward workflow employing semi-rational protein engineering combined to a high-throughput screening of variant libraries for their activity on PET nanoparticles. Using this approach, starting ...[more]

PMID: 35008691

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Project description:Background: Molecular breeding is an essential tool for accelerating genetic gain in crop improvement towards meeting the need to feed an ever-growing world population. Establishing low-cost, flexible genotyping platforms in small, public and regional laboratories can stimulate the application of molecular breeding in developing countries. These laboratories can serve plant breeding projects requiring low- to medium-density markers for marker-assisted selection (MAS) and quality control (QC) activities. Methods: We performed two QC and MAS experiments consisting of 637 maize lines, using an optimised genotyping workflow involving an in-house competitive allele-specific PCR (KASP) genotyping system with an optimised sample collection, preparation, and DNA extraction and quantitation process. A smaller volume of leaf-disc size plant samples was collected directly in 96-well plates for DNA extraction, using a slightly modified CTAB-based DArT DNA extraction protocol. DNA quality and quantity analyses were performed using a microplate reader, and the KASP genotyping and data analysis was performed in our laboratory. Results: Applying the optimized genotyping workflow expedited the QC and MAS experiments from over five weeks (when outsourcing) to two weeks and eliminated the shipping cost. Using a set of 28 KASP single nucleotide polymorphisms (SNPs) validated for maize, the QC experiment revealed the genetic identity of four maize varieties taken from five seed sources. Another set of 10 KASP SNPs was sufficient in verifying the parentage of 390 F 1 lines. The KASP-based MAS was successfully applied to a maize pro-vitamin A (PVA) breeding program and for introgressing the aflatoxin resistance gene into elite tropical maize lines. Conclusion: This improved workflow has helped accelerate maize improvement activities of IITA's Maize Improvement Program and facilitated DNA fingerprinting for tracking improved crop varieties. National Agricultural Research Systems (NARS) in developing countries can adopt this workflow to fast-track molecular marker-based genotyping for crop improvement.

Project description:The traditional strategy to improve the efficiency of an entire coupled enzyme system relies on separate direction of the evolution of enzymes involved in their respective enzymatic reactions. This strategy can lead to enhanced single-enzyme catalytic efficiency but may also lead to loss of coordination among enzymes. This study aimed to overcome such shortcomings by executing a directed evolution strategy on multiple enzymes in one combined group that catalyzes the asymmetric biosynthesis of l-phosphinothricin. The genes of a glutamate dehydrogenase from Pseudomonas moorei (PmGluDH) and a glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), along with other gene parts (promoters, ribosomal binding sites (RBSs), and terminators) were simultaneously evolved. The catalytic efficiency of PmGluDH was boosted by introducing the beneficial mutation A164G (from 1.29 s-1mM-1 to 183.52 s-1mM-1), and the EsGDH expression level was improved by optimizing the linker length between the RBS and the start codon of gdh. The total turnover numbers of the bioreaction increased from 115 (GluDH WTNADPH) to 5846 (A164GNADPH coupled with low expression of EsGDH), and to 33950 (A164GNADPH coupled with high expression of EsGDH). The coupling efficiency was increased from ∼30% (GluDH_WT with low expression of GDH) to 83.3% (GluDH_A164G with high expression of GDH). In the batch production of l-phosphinothricin utilizing whole-cell catalysis, the strongest biocatalytic reaction exhibited a high space-time yield (6410 g·L-1·d-1) with strict stereoselectivity (>99% enantiomeric excess).Importance: The traditional strategy to improve multienzyme-catalyzed reaction efficiency may lead to enhanced single-enzyme catalytic efficiency but may also result in loss of coordination among enzymes. We describe a directed evolution strategy of an entire coupled enzyme system to simultaneously enhance enzyme coordination and catalytic efficiency. The simultaneous evolution strategy was applied to a multienzyme-catalyzed reaction for the asymmetric synthesis of l-phosphinothricin, which not only enhanced the catalytic efficiency of GluDH but also improved the coordination between GluDH and GDH. Since this strategy is enzyme-independent, it may be applicable to other coupled enzyme systems for chiral chemical synthesis.

Dataset Information

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

Publications

An Efficient Protein Evolution Workflow for the Improvement of Bacterial PET Hydrolyzing Enzymes.

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