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

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.

Reprogramming Metabolic Flux in Escherichia Coli to Enhance Chondroitin Production

“…Further, the purified chondroitin was sulfated by 4‐O sulfotransferase (C4ST), and we found that chondroitin sulfate A (CS‐A) was formed (Figure 1e ; Figures S2,S3 , Supporting Information). [ 14 ] These results indicated that the de novo biosynthesis pathway of chondroitin was constructed in E. coli successfully, suggesting that E. coli F01 can be used as a microbial reactor for chondroitin biosynthesis (Figure 1d ). However, the balance between the carbonization pool and the ammoniation pool still needs to be optimized to enhance the efficiency of the polymerization pool for chondroitin production.…”

“…To further confirm the biosynthesis of chondroitin, the purified enzymes (KlATPS, PcAPSK, and HsCHST11) were incubated with 60 m m ATP, 100 m m Na 2 SO 4 , and 10 g L −1 chondroitin to generate CSA. [ 14 ] The lysis and product identification methods of CSA were conducted similarly to the method used for measuring chondroitin.…”

Rational engineering of homospermidine synthase for enhanced catalytic efficiency toward spermidine synthesis

“…In recent years, there has been a steady stream of research reports on the application of whole-cell catalysis [ 20 , 21 ]. Among these, Escherichia coli has emerged as the most favored host cell for whole-cell catalysis due to its simple structure and extensive research history [ 22 , 23 ].…”