Engineering Naematelia aurantialba as a Robust Synthetic Biology Chassis: Stable Episomal Vector Construction and Metabolic Optimization for Enhanced Polysaccharide Synthesis.

Naematelia aurantialba is a promising basidiomycete chassis for bioproduction, yet its development is hindered by a lack of efficient genetic tools. This study established a robust genetic platform for N. aurantialba NX-20. First, protoplast regeneration was optimized to 46.16% using 20 mg/mL Lywallzyme and 0.6 M maltose. Genome-wide analysis revealed a strong G/C-ending codon preference (GC3s of 67.15%) driven primarily by natural selection, providing a basis for codon optimization. To address the common issues of genetic chimerism and fluorescence heterogeneity in transformants, a 475-bp minimal point centromere (CEN) core element was identified within a genomic GC3 valley. Integration of this CEN element into an expression vector enabled stable, autonomous episomal replication. The utility of this episomal system was validated through the heterologous expression of Vitreoscilla hemoglobin (VHb). Biochemical analysis confirmed VHb activity, which significantly enhanced cellular respiration and energy supply. In stirred-tank bioreactor fermentation, the VHb-expressing engineered strain achieved a Naematelia aurantialba polysaccharide (NAPS) yield of 25.01 g/L, representing a 44.8% increase over the wild-type strain. This versatile and stable genetic platform not only fills a critical gap in N. aurantialba research but also offers a scalable paradigm for the metabolic engineering of non-model basidiomycetes to produce high-value bioproducts.