通过工程改造，使卤虫（Halomonas）在将葡萄糖转化为聚羟基丁酸酯（PHB）时的效率显著提升。

Abstract

A PHB-induced protein degradation system was developed for Halomonas . • Application of this system to GltA degradation improved yield by 15% in shake flask studies. • CYL16 reached the highest >41% PHB yield in 7 L glucose-based fed-batch bioreactors. Halomonas bluephagenesis is a robust halophilic chassis capable of naturally accumulating polyhydroxybutyrate (PHB), a sustainable alternative to petroleum-based plastics. However, industrial production of PHB is currently constrained by low glucose to PHB conversion efficiency, leading to high production cost. Thus, a PHB-induced protein degradation system was established based on the ClpXP-SspB-SsrA machinery. The SsrA tag was systematically mutated to screen for variants capable of binding to the ClpXP protease only in the presence of SspB adaptor. When placing SspB expression under the control of the PHB-responsive PhaP1 promoter, protein degradation triggered by intracellular PHB accumulation was achieved. This system was applied to downregulate citrate synthase (GltA) during PHB accumulation, allowing more cell growth in the first stage followed by more PHB synthesis in the second one. PHB synthesis was further reinforced by overexpressing the precursor synthesis phaAB cn operon to capture more acetyl-CoA for PHB precursor 3-hydroxybutyryl-CoA (3HB-CoA) synthesis. The resulting H. bluephagenesis CYL16 was grown to 144.25 g/L cell dry weight (CDW) containing 84% PHB in a 7 L bioreactor, achieving a 43% (g/g; range: 41%–45%, n  = 2) glucose to PHB conversion efficiency, compared with the 31% (g/g) previously reported for H. bluephagenesis TD01Δ phaP1 . Thus, the inducible protein degradation system provides an efficient and antibiotic-free approach for post-translational regulation in metabolic engineering. Download: Download high-res image (102KB) Download: Download full-size image