12.9 g/L！江南大学吴静/刘立明等创下硫酸软骨素C生物制造最高滴度记录

Core Viewpoint - Chondroitin sulfate C (CSC) is recognized as the "golden joint" with a significant total addressable market (TAM) in osteoarthritis treatment and health supplements. Recent advancements in bio-synthesis technology have enabled efficient and low-cost production of CSC, overcoming previous challenges in traditional extraction and chemical synthesis methods [2][3]. Group 1: Enzyme Selection and Optimization - The research team identified six candidate enzymes from a database, with RnCHST3 from Norway rats showing high regioselectivity for the 6-hydroxyl group of chondroitin. A truncated variant, ΔN106RnCHST3, was developed to improve solubility, increasing the conversion rate from 4.6% to 11.5%, although it exhibited a high Km value of 4.02×10³ mM and a half-life of only 8.1 hours at 37°C [5]. Group 2: Mechanistic Insights - The catalytic mechanism of RnCHST3 was elucidated using AlphaFold2 and molecular dynamics simulations, revealing that the E59 residue acts as a general base, while R38 and K169 stabilize the transition state, following a SN2-like mechanism. The dynamic nature of the Loop region above the substrate binding domain was found to hinder stable substrate binding, explaining the high Km value [6]. Group 3: Rational Design for Enhanced Activity - Through iterative saturation mutagenesis based on mechanistic insights, the enzyme's activity was improved. The double mutant M2 (F210R/Y310F) achieved a sulfation rate of 30.8%, while the combined mutant M4 (M2 + A209L + A303S) reached 40.1%. The final variant M5 (M4 + I173L) achieved a sulfation rate of 46.2%, significantly enhancing kcat/Km [9]. Group 4: Thermostability Improvements - To address the thermal instability of the engineered enzyme, a PROSS design approach was employed, identifying 18 stability sites. The variant M6 achieved a conversion rate of 59.8%, and further modifications to create M7 extended the half-life at 37°C to 12.3 hours (an increase of 51.9% compared to the wild type) with a sulfation rate of 74.5% [11][15]. Group 5: Whole-Cell Catalytic System Development - A six-enzyme cascade whole-cell catalytic system was constructed, integrating modules for PAPS biosynthesis, ATP regeneration, chondroitin sulfation, and PAPS regeneration. The core enzyme M7 was displayed on the cell surface, overcoming cofactor permeability barriers. The optimized system achieved a sulfation rate of 86.4% and a CSC titer of 12.9 g·L⁻¹, marking the highest level reported to date [16][19].