Protein engineering, cofactor engineering, and surface display engineering to achieve whole‐cell catalytic production of chondroitin sulfate A

Abstract

Abstract Chondroitin sulfate A (CSA) is a valuable glycosaminoglycan that has great market demand. However, current synthetic methods are limited by requiring the expensive sulfate group donor 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) and inefficient enzyme carbohydrate sulfotransferase 11 (CHST11). Herein, we report the design and integration of the PAPS synthesis and sulfotransferase pathways to realize whole‐cell catalytic production of CSA. Using mechanism‐based protein engineering, we improved the thermostability and catalytic efficiency of CHST11; its T m and half‐life increased by 6.9°C and 3.5 h, respectively, and its specific activity increased 2.1‐fold. Via cofactor engineering, we designed a dual‐cycle strategy of regenerating ATP and PAPS to increase the supply of PAPS. Through surface display engineering, we realized the outer membrane expression of CHST11 and constructed a whole‐cell catalytic system of CSA production with an 89.5% conversion rate. This whole‐cell catalytic process provides a promising method for the industrial production of CSA.