Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Abstract

cProof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and V HH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced "target protein." Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.A ffinity chromatography in various forms has been a cornerstone of protein purification for decades. In its simplest form, the inherent interaction between a target enzyme and its substrate is used, for example, purifying amylase by adsorbing it on insoluble starch. However, this technique is limited by the availability of a naturally occurring interaction and the ability of the substrate to be immobilized on an insoluble matrix (1). A more common and versatile technique is to engineer a tag into the target protein and utilize a protein or matrix with affinity toward that tag (2). Though widely used, this approach is often unacceptable for structural studies and in many commercial production systems due to the chance that the affinity tag may affect the structure, function, or other characteristics of the target protein. Furthermore, the presence of tags is commonly not tolerated in the pharmaceutical industry (3). One approach to combat this is to remove the tag after purification by engineering specific protease cleavage sites or self-cleaving sites such as inteins between the target and the tag, but this introduces further processing and purification steps (to remove the protease and/or cleaved tag) and can often result in a "scar" (residual am...

Bioengineered Polyhydroxyalkanoates as Immobilized Enzyme Scaffolds for Industrial Applications

“…The PHA-anchoring characteristics of these PAPs via both covalent interactions (PhaC) and physical adsorption (PhaF and PhaP) to the surface of PHA particles have been exploited to fabricate task-specific designer PHA particles using recombinant DNA technology (Hay et al, 2015;Hooks and Rehm, 2015;Jahns and Rehm, 2015;Bello-Gil et al, 2018a,b). PAPs can be translationally fused to target proteins, including industrially relevant enzymes, to enable the recombinant production of functionalized PHA particles in vivo (Figures 2C,D).…”

“…*Kinetic parameters are not mentioned in the reference. Hay et al, 2015;Parlane et al, 2016a). Assembly of immobilized multiprotein complexes enables multi-enzymatic cascade systems with superior catalytic performance as recently reviewed (Hwang and Lee, 2019).…”

“…For example, a few studies reported that fusing different proteins to PhaC influences the PHA production yield over biomass, particle size distribution, surface charges, and purity of the target protein (Rubio-Reyes et al, 2016;Gonzalez-Miro et al, 2018a,b;Wong and Rehm, 2018). Decorating PHA particles with proteins using PhaC synthase as an anchoring domain can also cause varying distribution and density of respective proteins on the PHA particles (Hay et al, 2015;Wong and Rehm, 2018). Hooks et al (2013) also pointed out that displaying N-acetylneuraminic acid aldolase (NanA) from E. coli on PHA particles through N-and C-terminal fusion of PhaC resulted in varying catalytic performance .…”

Applications of Microbial Biopolymers in Display Technology

“…Recently, Hay and coworkers published a more extensive study that aimed at broadening the applicability of PHA bead affinity resins by identifying and testing several easily customizable affinity binding domains which they translationally fused to PhaC (Hay et al 2015b). This study demonstrated that V HH domains from camelid antibodies, designed ankyrin repeat proteins (DARPins) and OB-folds (OBodies), could be densely displayed on PHA beads resulting in high affinity binding resins for purification of various target proteins.…”

“…These binding domains were used to establish extensive libraries of variants enabling screening for binders specific for the target compound of interest (Binz et al 2004;Harmsen and De Haard 2007;Stumpp et al 2008;Steemson et al 2014). PHA-based affinity resins showed a purification performance at least equal to current commercial offerings (Hay et al 2015b). A recent surface topology study of the R. eutropha PhaC attached to PHA inclusions suggested new engineering strategies towards the development of PHA-based affinity resins with increased binding capacity via improved display (Hooks and Rehm 2015).…”