Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

Wong, Jin Xiang; Ogura, Kampachiro; Chen, Shuxiong; Rehm, Bernd H. A.

doi:10.3389/fbioe.2020.00156

Cited by 39 publications

(35 citation statements)

References 175 publications

(194 reference statements)

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“…Among GAPs, phasins constitute the most abundant group of proteins covering the granule (8,9) and have been widely used as anchoring tags to functionalize PHA supports for a variety of applications, such as affinity bioseparation (10), protein purification (11)(12)(13), enzyme immobilization (14)(15)(16), protein delivery to natural environments (17), diagnostics (18,19), cell targeting (20)(21)(22)(23)(24), and antimicrobials (25).…”

mentioning

confidence: 99%

Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

Mato

Blanco

Maestro

et al. 2020

Appl Environ Microbiol

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Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro. The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP–β-galactosidase and MinP-CueO from Escherichia coli. IMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.

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mentioning

confidence: 99%

Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

Mato

Blanco

Maestro

et al. 2020

Appl Environ Microbiol

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“…Interestingly, these PHAs beads can also be functionalised in vivo with engineered fusion proteins, which decorate the materials surfaces [64]. Such functionalised PHAs beads have enabled novel protein purification strategies [65], as well as the development of PHAs-based vaccines [66].…”

Section: Resultsmentioning

confidence: 99%

AL-PHA beads: bioplastic-based protease biosensors for global health applications

Kelwick

Webb

Wang

et al. 2020

Preprint

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Proteases are multi-functional proteolytic enzymes that have complex roles in human health and disease. Therefore, the development of protease biosensors can be beneficial to global health applications. To this end, we developed Advanced proteoLytic detector PolyHydroxyAlkanoates (AL-PHA) beads, a library of over 20 low-cost, biodegradable, bioplastic-based protease biosensors. Broadly, these biosensors utilise PhaC-reporter fusion proteins that are bound to microbially manufactured polyhydroxyalkanoate beads. In the presence of a specific protease, superfolder green fluorescent reporter proteins are cleaved from the AL-PHA beads - resulting in a loss of bead fluorescence. The Tobacco Etch Virus (TEV) AL-PHA biosensor detected the proteolytic activity of at least 1.85 pM of AcTEV. AL-PHA beads were also engineered to detect cercarial elastase from Schistosoma mansoni-derived cercarial transformation fluid (SmCTF) samples, as well as cancer-associated metalloproteinases in extracellular vesicle and cell-conditioned media samples. We envision that AL-PHA beads could be further developed for use in resource-limited settings.

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“…Other plastic polymers such as poly-ethylene, poly-tetra-fluoro-ethylene, polycaprolactone can be used for cross-linking phages to prevent the formation of bacterial biofilms in the clinic for catheters or implants (92,109). Surgical threads that are composed of various polymers, including nylon, PE, and cellulose, have also been coated with phages (79,80,110,111). A successful attempt to develop phage-based washable and non-toxic wound dressings bacteriophages made use of Pseudomonas bacteriophages covalently immobilised on the surface of polycaprolactone (PCL) nanofibres and were shown to be effective even after 25 cycles of washing (92).…”

Section: Immobilisation With Fibresmentioning

confidence: 99%

Encapsulation and Delivery of Therapeutic Phages

Loh

Gondil

Manohar

et al. 2021

Appl Environ Microbiol

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Delivery of therapeutic compounds to the site of action is crucial. While many chemical substances such as beta-lactam antibiotics can reach therapeutic levels in most parts throughout the human body after administration, substances of higher molecular weight such as therapeutic proteins may not be able to reach the site of action (e.g. an infection), and are therefore ineffective. In the case of therapeutic phages, i.e. viruses that infect microbes that can be used to treat bacterial infections, this problem is exacerbated; not only are phages unable to penetrate tissues, but phage particles can be cleared by the immune system and phage proteins are rapidly degraded by enzymes or inactivated by the low pH in the stomach. Yet, the use of therapeutic phages is a highly promising strategy, in particular for infections caused by bacteria that exhibit multi-drug resistance. Clinicians increasingly encounter situations where no treatment options remain available for such infections, where antibiotic compounds are ineffective. While the number of drug-resistant pathogens continues to rise due to the overuse and misuse of antibiotics, no new compounds are becoming available as many pharmaceutical companies discontinue their search for chemical antimicrobials. In recent years, phage therapy has undergone massive innovation for the treatment of infections caused by pathogens resistant to conventional antibiotics. While most therapeutic applications of phages are well described in the literature, other aspects of phage therapy are less well documented. In this review, we focus on the issues that are critical for phage therapy to become a reliable standard therapy and describe methods for efficient and targeted delivery of phages, including their encapsulation.

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Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

Cited by 39 publications

References 175 publications

Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

AL-PHA beads: bioplastic-based protease biosensors for global health applications

Encapsulation and Delivery of Therapeutic Phages

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