Liu, P.; Xie, X.-Y.; Deng, Y.-H.; Li, Z.-F.; Wang, C.; Yang, H.; Li, Y.-X.; Zhao, L.-L.; Situ, W.; Shen, H.-W.; Yu, L.-S.; Lv, J.-Y.; Xiao, Y.-C.; Lin, Y.-N.; Ye, J.-W.

Halophilic chassis has emerged as a promising biomanufacturing platform for industrial polyhydroxyalkanoate (PHA) production. However, challenges still remain in improving the production capacity, scalability and robustness, thereby lowering cost to meet market demands. Here, a high-performing halophilic strain Halomonas LY03 was isolated with over 38% glucose-to-PHA conversion rate and broad non-grain substrate utilization capability. Multidimensional tools, including algorithm-guided high-expression neutral integration site (HENIS) screening toolkit designated 'SiteSeek', stop codon (TAA)-dependent enhancement of gene expression and recombinase-mediated large-fragment (> 9 kb) genomic integration, were then developed to enable precise, efficient and interference-free genomic integrative expression. Using these tools, various chromosomally engineered strains were rapidly constructed to achieve high-level production of poly-3-hydroxybutyrate (PHB, 151 g L-1) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB, 139 g L-1) under high cell-density fermentation (up to 186 g L-1 cell dry weight) in a 5-L bioreactor. Scalability was demonstrated at 2-m3 and 20-m3 industry-scale fermentations, yielding up to 134 g L-1 PHB and 127 g L-1 P34HB (6.1 mol% 4HB). Building on the proven robustness, a two-stage continuous fermentation (TCF) process was developed using a twin-bioreactor system at 5-L and 20-m3 scales, where stable and sustained PHA production lasted over 260 h and 160 h, respectively. Techno-economic analysis revealed a substantial cost-reduction space of 48% compared with conventional fed-batch process. This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-industry scales.