Controlling phenolic radical coupling using engineered protein scaffolds for the synthesis of biodegradable plastics from sustainable phenolic monomers.

PI: Claudia Schmidt-Dannert

Introduction and Motivation Thermosetting phenolic resins made from reacting a phenol with an aldehyde have a long history as plastics (e.g., Bakelite). These resins exhibit high temperature and chemical resistance as well as mechanical strength and are used in numerous applications. Reaction of phenolic compounds (e.g., phenol, cresols, xylenols) with aldehydes (e.g. formaldehyde, furfural) results in a three dimensionally cross-linked polymer. Cross-linking can be controlled to some extent by varying phenol and aldehyde ratios and types. However, better control of the process in terms of being able to control the incorporation of certain substituted phenols, degree of cross-linking/branching and/or different monomer linkages would be desirable to obtain resins with more thermoplastic like properties.

To achieve a more controlled polymerization process that can incorporate different phenols, monomer linkages AND uses monomers that are not petroleumbased, we propose to develop a biocatalytic process that emulates the synthesis of plant derived polyphenols.

Fig. 1: Models of lignin polymers. Different linkages and monomers found in lignin are shown.

Fig. 2: Dirigent Protein (DP) mediated regioand enantioselective radical coupling vs. nonspecific, racemic coupling. Shown is the DP protein from Forsythia involved in the formation of the lignan (+) Pinoresinol.

Phenolic compounds make up a major class of plant metabolites and include the polyphenol lignin which is the second most abundant polymer on earth. Large quantities of phenols are released every year as waste products from unwanted lignin in the paper pulp industry and more recently, the conversion of biomass into biofuels. Plant derived phenols therefore represent an inexpensive resource for the synthesis of polymers. As polyphenols are degraded by microorganisms, the resulting polymers will have the additional benefit of being biodegradable. Polymers synthesized from these plant derived precursors have recently been shown to yield biodegradable high-performance plastics.

Lignin and lignans (phenol dimers) are synthesized in the plants by oxidative radical coupling of phenolic monomers (monolignols) derived from the aromatic amino acids phenylalanine or tyrosine (Fig. 1). Although the mechanism of lignin formation is not known in detail, it is known that polymerization is initiated via phenol radical formation by a laccase or peroxidase enzyme. The plant then controls oxidative radical coupling. Composition and structure of lignin polymers vary among different plants. How exactly this oxidative polymerization occurs in lignin formation is not known, but it has been proposed that so called dirigent proteins (DP) bind an orient phenoxy radicals prior to bond formation thereby controlling regio- and enantioselectively of bond formation.2 The function of DPs has been described in detail for the synthesis of bioactive lignan plant natural products (Fig. 2). Here, a small and non-catalytic dirigent protein acts as a scaffold that orients two phenoxy radicals (although lignans composed of more than two coupled phenol building blocks are also known). Large multigene families encoding DPs are found in many plant species. So far only a few have been functionally characterized. Their prevalence in plants underlines their importance in synthesis and suggests that DPs with variety of substrate-, regio- and enantioselectivities can be found in plants (or engineered once we have a better structural understanding of their function) for future biocatalytic applications in oxidative radical coupling.

In this application we will use DPs to explore controlled oxidative radical coupling for polyphenol synthesis. Specifically, we will develop a biocatalytic process which uses a laccase enzyme to generate a phenol radical together with DP and engineered DP scaffolds to achieve controlled radical coupling. Initially, we will test and develop a process that will be carried out in a small reaction vessel in vitro as a proof-of-principle application. Eventually we plan to transfer polymer synthesis into recombinant E. coli (using the same chosen proteins) to develop a cost-effective fermentation process where the phenolic monomers are either synthesized de novo by E. coli or added as inexpensive precursors o the medium.