Creation of sequence-regulating polyhydroxyalkanoate (PHA) synthases with improved thermostability using a full consensus design for the biosynthesis of 2-hydroxyalkanoate-based PHA block copolymers

IF 6.3 2区化学 Q1 POLYMER SCIENCE

Polymer Degradation and Stability Pub Date : 2025-02-27 DOI:10.1016/j.polymdegradstab.2025.111295

Shoko Furukawa , Naoya Nakagawa , Shin-ichi Hachisuka , Shogo Nakano , Hiroya Tomita , Hiroshi Kikukawa , Ken'ichiro Matsumoto

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引用次数: 0

Abstract

Engineered polyhydroxyalkanoate (PHA) synthase PhaC_AR is a chimeric enzyme composed of PhaCs from Aeromonas caviae and Ralstonia eutropha (formally Cupriavidus necator). The enzyme has a broad substrate scope and unique sequence-regulating capacity: spontaneous synthesis of various block copolymers. This study aimed to acquire new sequence-regulating PHA synthases with a broader substrate scope and improved thermal stability. To meet this goal, artificial PHA synthases were designed using the full consensus design (FCD) algorithm. Based on amino acid sequences of PHA synthases with homology to PhaC from R. eutropha in the database, four artificial PHA synthases, FcPhaC1, FcPhaC2, FcPhaC4, and FcPhaC5, were created and expressed in Escherichia coli together with the monomer-supplying enzyme. Cells were cultivated with the supplementation of monomer precursors in the medium. As a result, these four FcPhaCs synthesized poly(3-hydroxybutyrate) [P(3HB)], although polymer production was slightly lower than that obtained using PhaC_AR, indicating that the algorithm successfully designed functional enzymes. FcPhaC1, FcPhaC4, and FcPhaC5 remained active after isothermal treatment at 60 °C for 30 min, whereas PhaC_AR and FcPhaC2 were inactivated. Therefore, the artificial PhaCs possessed improved thermal stability compared to PhaC_AR. The four FcPhaCs synthesized poly(glycolate-co-3HB) [P(GL-co-3HB)] with a block sequence as well as PhaC_AR. FcPhaC4 exhibited the highest GL fraction in P(GL-co-3HB) among the PhaCs tested. FcPhaC4 synthesized P(3HB)-b-P(2-hydroxybutyrate) and P(3HB)-b-polylactate, indicating its broad substrate scope. These results demonstrated the effectiveness of the FCD approach for creating sequence-regulating PHA synthases applicable to a wide range of polymer syntheses.

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来源期刊

Polymer Degradation and Stability 化学-高分子科学

CiteScore

10.10

自引率

10.20%

发文量

325

审稿时长

23 days

期刊介绍： Polymer Degradation and Stability deals with the degradation reactions and their control which are a major preoccupation of practitioners of the many and diverse aspects of modern polymer technology. Deteriorative reactions occur during processing, when polymers are subjected to heat, oxygen and mechanical stress, and during the useful life of the materials when oxygen and sunlight are the most important degradative agencies. In more specialised applications, degradation may be induced by high energy radiation, ozone, atmospheric pollutants, mechanical stress, biological action, hydrolysis and many other influences. The mechanisms of these reactions and stabilisation processes must be understood if the technology and application of polymers are to continue to advance. The reporting of investigations of this kind is therefore a major function of this journal. However there are also new developments in polymer technology in which degradation processes find positive applications. For example, photodegradable plastics are now available, the recycling of polymeric products will become increasingly important, degradation and combustion studies are involved in the definition of the fire hazards which are associated with polymeric materials and the microelectronics industry is vitally dependent upon polymer degradation in the manufacture of its circuitry. Polymer properties may also be improved by processes like curing and grafting, the chemistry of which can be closely related to that which causes physical deterioration in other circumstances.