Assay for monomer incorporation by polyester synthase to enable bioplastics engineering

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Origin: This problem statement originates from a workshop held by Homeworld Collective on Oct 10, 2023, where speakers presented ideas for how protein engineering could be applied for climate and sustainability. It is based on ideas presented by Nils Averesch, Research Engineer and Group Leader in Civil and Environmental Engineering at Stanford University and NASA's Center for the Utilization of Biological Engineering in Space.

Contributors: Nils Averesch, Paul Reginato, Ariana Caiati

Lead contact: Nils Averesch ([email protected])

General contact: Paul Reginato ([email protected])

Motivating Factor

Polymer production for manufacturing of plastic materials accounts for ~6% of global fossil fuel use [1] and over 100 Mt CO2e/yr of GHG emissions in the US [2]. Emissions could be avoided by synthesizing polymers in a one-step biological process from sustainable feedstocks. However, currently-produced bioplastics, like polyhydroxybutyrate (PHB) and polylactate (PLA), lack the material properties (tensile strength and stiffness, thermal stability, gas-barrier) to replace most fossil-derived plastics. A wider application spectrum of bioplastics would be a substantial step toward mitigating the emissions of synthetic polymer production by replacing fossil-derived plastics with biopolyesters.

Specific Constraint

Via whole-cell catalysis, microbes can directly utilize single-carbon (C1) compounds, derived from CO2 and sustainable energy, for the formation of various monomers and their polymerization into novel biopolyesters [3]. These polyesters are biodegradable as well as recyclable through deconstruction [4]. Further, it is possible to expand the utility of biopolyester in place of non-recyclable polymers by tuning their material properties via incorporation of alternative monomers [5]. Aromatic monomers are of particular interest, especially those with structural analogy to arylates (such as liquid-crystal polymers), which could enable the production of aromatic polyhydroxyalkanoates with previously unreached properties [6].

In practice, however, tunability of biopolyester material properties is limited by the monomer incorporation efficiency of polyhydroxyalkanoate (PHA) synthase. Protein engineering efforts are underway to increase the incorporation efficiency of diverse monomers by PHA synthase, which would unlock biocatalysis of biopolyesters with tunable material properties. However, those efforts are hampered by the lack of a scalable assay for monomer incorporation rate. Currently, analytics of biopolyesters are performed using 1H- and/or 13C-NMR spectroscopy or gas chromatography  to determine the molecular structure of the purified polymer. This is not only difficult to automate and to apply to rapid screening protocols, but perhaps also more detail than is needed for more qualitative screening purposes.

Actionable Goals

An assay should be developed to measure the incorporation rate of a range of aromatic monomers of significance, to be used in the development of polyester synthase enzymes that can synthesize a spectrum of polymers. Such monomers include terephthalic and furan-2,5-dicarboxylic acid, for PET and PEF synthesis, respectively; and aromatic hydroxy acids such as coumaric, phloretic, hydroxyphenylacetic, para-hydroxybenzoic, salicylic, and vanillic acid, for synthesis of polyhydroxyalkanoates with structural analogy to arylates [7][8][9][10].

An assay could be based on detection of free monomer, for example using an approach similar to ref [11], in which case depletion of the monomer pool would be a proxy for polymer synthesis. Alternatively, an assay could also be based on detection of polymerized monomer, which would provide a more sensitive and thus more ideal readout but may prove more challenging to develop. The required assay sensitivity is a few percent change in monomer content of the polymer or intracellular monomer concentration in the  µM to mM range, for detecting monomer incorporation or depletion, respectively. The required assay throughput is a minimum of thousands of variants, but ideally enough to accommodate FACs sorting.

Additional information

Related problem statements

• Polyester synthase for novel bioplastics with tunable properties

Open Questions

The Specific Constraint could be clarified by:

• Analysis describing the economic feasibility of replacing fossil-derived plastics with bioplastics manufacturing via whole-cell biocatalysis from various C1 feedstocks, and the key cost barriers to doing so.

  • ref [12] describes current challenges of readying sustainable bioplastics production for the consumer market

  • ref [3] tabulates production cost for various synthetic as well as biological plastics are compiled in Table 1, without emphasis on the feedstock

  • ref [13] performs a techno-economic assessment of PHB manufacture from methane, but PBH has limited applicability and methane is the least sustainable C1 feedstock

• Analysis of which biopolyesters, made from which C1 feedstocks, are likely to have the greatest sustainability impact via fossil-derived plastic replacement

Assumptions

• Manufacturing of bioplastics using whole-cell biocatalysis using C1 feedstocks can be done economically to compete with fossil-derived plastics

• The required volumes of sustainable C1 feedstock will become available to support a plastics economy. At current plastic production rates, ~ 1 Gt of C1 feedstock would be required. If recycling were to increase from the current ~10% to ~90%, that number could be reduced by an order of magnitude, which makes the required scale of infrastructure for production much more feasible.