With the development of the aquatic products processing industry, 6–8 million tons of shrimp and crab shell waste are produced globally annually, but, due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained are given as follows: a temperature of 30 °C, fermentation time of 42 h, substrate solid–liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39% from 350.67 × 10⁸ CFU/mL to 576.44 × 10⁸ CFU/mL. Combined with engineering modification, two engineered strains, KT_+IV and KT_+lasBT, expressing exogenous proteases, were obtained, and the maximum growth was increased from 316.44 × 10⁸ CFU/mL to 1268.44 × 10⁸ CFU/mL and 616.89 × 10⁸ CFU/mL, which were 300.84% and 94.94% higher, respectively. In addition, the engineered strain KT_+NtrcT-D55E, which regulates nitrogen metabolism, was obtained, and the accumulation of intracellular polyhydroxy fatty acid esters (PHA) was increased from 20.00 mg/L to 78.58 mg/L, which was a significant increase of 292.93% relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.

With the development of the aquatic products processing industry, 6-8 million tons of shrimp and crab shell waste are produced globally annually, but due to the lack of high-value conversion technology, crab shells are often discarded in large quantities as a by-product of processing. Pseudomonas-based microbial cell factories are capable of biosynthesis of high-value products using a wide range of substrates; however, there is currently no reliable fermentation model for producing high-value chemicals using crab shell waste by Pseudomonas strains. In this study, we first explored the culture conditions of shell fermentation using KT2440 through single-factor and orthogonal experiments, and the optimized fermentation parameters obtained were: with the temperature of 30°C, fermentation time of 42 h, substrate solid-liquid ratio of 7%, and rotational speed of 200 rpm. After optimization, the maximum cell growth was increased by 64.39%, from 350.67 × 10 8 CFU/mL to 576.44 × 10 8 CFU/mL. Combined with engineering modification, two engineered strains KT+IV and KT+lasBT expressing exogenous proteases were obtained, with the maximum growth increasing by 300.84% and 94.94%, respectively. Additionally, an engineered strain KT+NtrcT-D55E regulating nitrogen metabolism was obtained, significantly increasing the accumulation of intracellular polyhydroxyalkanoates (PHA) by 292.93%, relative to the control group. This study provides a theoretical basis and technical support for the high-value utilization of shrimp and crab shell resources and the development of environmentally friendly bioproducts.