Determination of substrate addition order strategy for bespoke Polyhydroxyalkanoates production: A molecular modeling based approach

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible materials with a wide range of biotechnological applications. An integrated study of the thermodynamic and kinetic behavior of the polymerization initiation process was developed for fine-tuning bespoke PHA production. Simplified molecular scale models were established to describe the initiation of PHA formation which cannot be captured with current physical tracking techniques in cells. A thermodynamic study and ab initio calculations demonstrate that the 4HB monomer exhibits higher reactivity in terms of PHA formation compared to 3HB, 3HV, 3HHx and 5HV. In order to produce terpolymer P(3HB-co-3HV-co-4HB) with a high fraction of 4HB, different scale fed-batch fermentation experiments using glucose, γ-butyrolactone, and propionic acid as carbon sources/precursors in Cupriavidus necator proved feeding in order of monomer reactivity led to double biomass and more than 50% of 4HB accumulation in terpolymer. The molecular model and substrate addition order strategy demonstrated in this work provide an efficient and practical fermentation method for controlling biopolymer production.