Identification and characterization of a potential strain for the production of polyhydroxyalkanoate from glycerol.

While poly (3-hydroxybutyrate) (PHB) holds promise as a bioplastic, its commercial utilization has been hampered by the high cost of raw materials. However, glycerol emerges as a viable feedstock for PHB production, offering a sustainable production approach and substantial cost reduction potential. Glycerol stands out as a promising feedstock for PHB production, offering a pathway toward sustainable manufacturing and considerable cost savings. The identification and characterization of strains capable of converting glycerol into PHB represent a pivotal strategy in advancing PHB production research. In this study, we isolated a strain, Ralstonia sp. RRA (RRA). The strain exhibits remarkable proficiency in synthesizing PHB from glycerol. With glycerol as the carbon source, RRA achieved a specific growth rate of 0.19 h-1, attaining a PHB content of approximately 50% within 30 h. Through third-generation genome and transcriptome sequencing, we elucidated the genome composition and identified a total of eight genes (glpR, glpD, glpS, glpT, glpP, glpQ, glpV, and glpK) involved in the glycerol metabolism pathway. Leveraging these findings, the strain RRA demonstrates significant promise in producing PHB from low-cost renewable carbon sources.

Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model.

Although cytochrome P450 enzymes are the most versatile biocatalysts in nature, there is insufficient comprehension of the molecular mechanism underlying their functional innovation process. Here, by combining ancestral sequence reconstruction, reverse mutation assay, and progressive forward accumulation, we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase (F6H) and proposed a "3-point fixation" model to elucidate the functional innovation mechanisms of P450s in nature. According to this design principle of catalytic pocket, we further developed a de novo diffusion model (P450Diffusion) to generate artificial P450s. Ultimately, among the 17 non-natural P450s we generated, 10 designs exhibited significant F6H activity and 6 exhibited a 1.3- to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1. This work not only explores the design principle of catalytic pockets of P450s, but also provides an insight into the artificial design of P450 enzymes with desired functions.