Engineering xylose metabolism for diverse polyhydroxyalkanoates synthesis in Halomonas TD

Engineering the biosynthesis of fully biodegradable polyhydroxyalkanoates (PHAs) from non-food and renewable feedstocks like lignocellulose is becoming an attractive strategy for sustainable biomanufacturing. However, the efficiency and diversity of PHA synthesis from lignocellulosic hydrolysate (LH), mainly containing glucose and xylose, still remains challenge. Here, Halomonas TD, a cost-effective PHA-producing chassis, was developed to utilize glucose and xylose (or LH) for effective production of poly-3-hydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] by engineering the phosphoketolase pathway-dependent xylose metabolism in the genome, yielding 50.1 g L-1 PHB and 42.6 g L-1 P(3HB-co- 11.4 mol% 4HB) under fed-batch condition. Subsequently, the introduction of Weimberg pathway was found be able to synthesize terpolymer consisting of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV), namely [P(3HB-co-4HB-co-3HV)], from glucose and xylose only due to the isoenzyme activity of keto-acid decarboxylase encoded by kivD, which converts xylose-derived intermediate 2,5-dioxopentanoate and -ketoglutarate into butanedial (4HB synthesis) and 2-ketobutyrate (3HV synthesis), respectively. Finally, a tailored-made xylose-induced system was constructed to achieve exquisite xylose transmembrane transportation control for improved synthesis of terpolymer P(3HB-co-4.5 mol% 4HB-co- 3.0 mol% 3HV), reaching to 6.3 g L-1 under shake-flask condition, together with the co-expression of fine-tuned phosphoketolase and Weimberg pathways. This study provides a feasible and sustainable alternative for lignocellulosic resources valorization powered by the engineered Halomonas TD capable of efficient xylose utilization and diverse PHAs synthesis.