Engineering Komagataella phaffii cell factories for the production of chondroitin sulfate A with high sulfation degree

Chondroitin sulfate (CS), which is primarily extracted from animal tissues, has been widely used for maintaining joint and cartilage health. Despite a few innovations in CS biosynthesis, low titer or sulfation degree values remain bottlenecks for its potential applications. We developed robust Komagataella phaffii cell factories for producing highly sulfated chondroitin sulfate A (CSA). First, 82.3 mg/L CSA with a sulfation degree of 5.5 % was produced through the reconstruction of the chondroitin synthesis pathway, active expression of chondroitin 4- O -sulfotransferase (C4ST), and addition of adequate sulfates. Further overexpression of the sulfate transporter SUL1 elevated the sulfation level to 18.6 %. After revealing the quantitative relationship between chondroitin synthesis and its sulfation, the expression of C4ST improved with multicopy integration, and the sulfation degree of CSA increased to 57.7 %. Thereafter, a 3′-phosphoadenosine-5′-phosphosulfate regeneration system was introduced and optimized, which further elevated the sulfation degree to 90.3 %. Finally, a growth-coupled feeding approach was adopted to control the concentration of heavy metal ions, and the engineered strain CS10 produced 1.15 g/L (10.92 mg/g DCW) CSA with a sulfation degree of 96 % in fed-batch cultures. Our study provides an attractive alternative for the biomanufacturing of highly sulfated CSA. Furthermore, the engineered K. phaffii cell factories could be used for producing sulfated compounds of interest, such as heparin, and anticoagulant peptides.