Physiochemical characterization of polyester synthase enzymes and production of polyhydroxyalkanoate

Abstract

Polyhydroxyalkanoate (PHA) synthases (PhaC) are the pivotal biocatalysts governing the biosynthesis of biodegradable polyesters. Current research on their structure and mechanisms remains limited, and the chemical mechanisms of all PHA synthases are still under debate. Despite some speculation, further physicochemical studies are needed to understand their structure and function.
The primary objective of this study was to investigate three enzymes from the class 1 PhaC family (PhaCCs, PhaCAc, and PhaCCn), including their structure, native molecular mass, molecular dynamics, and thermal stability. Furthermore, PhaCCn forms the phaCAB operon with other enzymes to produce PHB. In the model structure, the flexible N-terminus was removed, thus achieving soluble expression in E. coli. The melting points of all proteins were determined by differential scanning calorimetry (DSF), and the native molecular mass were determined by OMNISEC. Mass spectrometry analysis showed that all proteins underwent partial degradation, particularly in the N-terminal region. The C-terminus of one protein was crystallized with a crystal structure resolution of 1.84 Å, and structural refinement is currently underway. Finally, by using the phaCAB cluster inserted in E. coli, PHA was successfully produced, with dominant presence of PHB.
Comparative investigation of four candidates—PhaCCs-H, PhaCCs, PhaCAc, and PhaCCn—after affinity chromatography and recombinant production demonstrated that the N-terminal tag is critical for protecting sensitive regions from proteolytic degradation during purification. Subsequent structure prediction, molecular dynamics (MD) simulations, paired with nanoDSF, OMNISEC and Mass Spectrometry analyses uncovered distinct conformational and physicochemical properties divergences among the purified enzymes. In solution, the PhaCCs-H construct with a Histidine tag in the N-terminal demonstrated 98% monomeric homogeneity and great thermodynamic robustness. In contrast, PhaCAc adopted a highly stable dimeric conformation, whereas the intrinsic N-terminal flexibility of PhaCCn conferred severe thermodynamic fragility. The PhaCCs results of crystallization were directly determined by these biophysical dynamics: PhaCCs yielded high-quality macroscopic crystals, PhaCAc produced promising preliminary hits, while the structural plasticity of PhaCCn barred stable lattice nucleation. Parallel to this in vitro characterization, robust in vivo PHA synthesis was achieved in Escherichia coli using a native phaCAB operon, with Fourier-transform infrared (FTIR) spectroscopy, 1H-NMR and DSC analysis confirming the extracted product is polyhydroxybutyrate (PHB) homopolymer. Ultimately, this work connects high-resolution biophysiochemical profiling with metabolic application, clarifying how PhaC's biophysical integrity controls both its operational stability and the viability of its structural elucidation.

Popular Abstract

If we could directly utilize bacteria to produce 100% biodegradable and environmentally friendly plastics in vivo, humanity might be able to completely eliminate environmental pollution caused by petroleum-based plastics. Nature has long equipped bacteria with this superpower, the core of which is a miniature, "plastic-synthesizing enzyme" called PhaC. However, due to its extremely unstable structure and susceptibility to conformational deformation, its true form and operating mechanism have long been the subject of much debate in academia. This study focused on three different enzyme variants (PhaCCs, PhaCAc, and PhaCCn), first discovering a common weakness among them—a highly vulnerable, flexible "N-terminal tail." By molecularly protecting or locally modifying this tail, the research team successfully stabilized these fragile enzymes and revealed their distinctly different physical characteristics. Among them, the protected PhaCCs exhibited remarkable uniformity and thermal stability, successfully arranging themselves spontaneously into high-quality crystals in a perfect monomeric morphology. This allowed us to solve only the fifth high-resolution 3D atomic structure globally at an ultra-high resolution of 1.84 Å. PhaCAc, on the other hand, preferred to combine in pairs, forming highly stable dimer structures. PhaCCn, however, unfortunately could not settle down and form regular crystals due to its highly disordered and violently oscillating internal structure. In the real world outside the test tube, the team also successfully implanted a complete plastic synthesis gene cluster (phaCAB) into E. coli, directly transforming these bacteria into highly efficient micro-factories that successfully brewed bioplastics with a dominant presence of PHB within living cells. This research successfully connects the microscopic physical dynamics of proteins with macroscopic metabolic manufacturing applications, providing an indispensable and precise blueprint for the future rational design of more stable and efficient green plastic "cell factories.

• author: Wu, Nan
• supervisor: Javier Linares-Pastén
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• course: KBTM01 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Polyhydroxyalkanoate (PHA) synthase (PhaC), Class 1 PhaC family, phaCAB operon (or cluster), Polyhydroxybutyrate (PHB), Molecular dynamics, N-terminal flexibility, X-ray crystallography, Biophysical characterization, biotechnology
• language: English
• id: 9231537
• date added to LUP: 2026-06-08 15:27:06
• date last changed: 2026-06-08 15:27:06