High-Throughput Screening for Thermostable Polyester Hydrolases

Branson, Yannick; Badenhorst, Christoffel P. S.; Pfaff, Lara; Buchmann, Carolin; Wei, Ren; Bornscheuer, Uwe T.

doi:10.1007/978-1-0716-2795-2_11

Cited by 2 publications

(1 citation statement)

References 33 publications

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“…Hits that grow in both media are false positives (containing the pabA gene) because they can grow in a PABA-free medium without acedoben, whereas the hits that grow only in the medium containing PABA are correct positive hits. Alternatively, positive hits can easily be transferred to another assay, like a halo formation assay (Branson et al 2022 ) or a microtiter plate-based assay (Henke and Bornscheuer 2003 ), increasing the throughput of these methods by far.…”

Section: Discussionmentioning

confidence: 99%

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity

Branson,

Schnell,

Zurr

et al. 2024

Appl Microbiol Biotechnol

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In the last decades, biocatalysis has offered new perspectives for the synthesis of (chiral) amines, which are essential building blocks for pharmaceuticals, fine and bulk chemicals. In this regard, amidases have been employed due to their broad substrate scope and their independence from expensive cofactors. To expand the repertoire of amidases, tools for their rapid identification and characterization are greatly demanded. In this work an ultra-high throughput growth selection assay based on the production of the folate precursor p-aminobenzoic acid (PABA) is introduced to identify amidase activity. PABA-derived amides structurally mimic the broad class of commonly used chromogenic substrates derived from p-nitroaniline. This suggests that the assay should be broadly applicable for the identification of amidases. Unlike conventional growth selection assays that rely on substrates as nitrogen or carbon source, our approach requires PABA in sub-nanomolar concentrations, making it exceptionally sensitive and ideal for engineering campaigns that aim at enhancing amidase activities from minimally active starting points, for example. The presented assay offers flexibility in the adjustment of sensitivity to suit project-specific needs using different expression systems and fine-tuning with the antimetabolite sulfathiazole. Application of this PABA-based assay facilitates the screening of millions of enzyme variants on a single agar plate within two days, without the need for laborious sample preparation or expensive instruments, with transformation efficiency being the only limiting factor. Key points • Ultra-high throughput assay (tens of millions on one agar plate) for amidase screening • High sensitivity by coupling selection to folate instead of carbon or nitrogen source • Highly adjustable in terms of sensitivity and expression of the engineering target

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Section: Discussionmentioning

confidence: 99%

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity

Branson,

Schnell,

Zurr

et al. 2024

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

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A High-Throughput Screening Platform for Engineering Poly(ethylene Terephthalate) Hydrolases

Groseclose,

Kober,

Clark

et al. 2024

ACS Catal.

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The ability of enzymes to hydrolyze the ubiquitous polyester, poly(ethylene terephthalate) (PET), has enabled the potential for bioindustrial recycling of this waste plastic. To date, many of these PET hydrolases have been engineered for improved catalytic activity and stability, but current screening methods have limitations in screening large libraries, including under high-temperature conditions. Here, we developed a platform that can simultaneously interrogate PET hydrolase libraries of 10 4 −10 5 variants (per round) for protein solubility, thermostability, and activity via paired, plate-based split green fluorescent protein and model substrate screens. We then applied this platform to improve the performance of a benchmark PET hydrolase, leaf-branch compost cutinase, by directed evolution. Our engineered enzyme exhibited higher catalytic activity relative to the benchmark, LCC-ICCG, on amorphous PET film coupon substrates (∼9.4% crystallinity) in pH-controlled bioreactors at both 65 °C (8.5% higher conversion at 48 h and 38% higher maximum rate, at 2.9% substrate loading) and 68 °C (11.2% higher conversion at 48 h and 43% higher maximum rate, at 16.5% substrate loading), up to 48 h, highlighting the potential of this screening platform to accelerate enzyme development for PET recycling.

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High-Throughput Screening for Thermostable Polyester Hydrolases

Cited by 2 publications

References 33 publications

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity

A High-Throughput Screening Platform for Engineering Poly(ethylene Terephthalate) Hydrolases

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