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Chondroitin sulfate (CS) stands as a pivotal compound in dietary supplements for osteoarthritis treatment, propelling significant interest in the biotechnological pursuit of environmentally friendly and safe CS production. Enzymatic synthesis of CS for instance CSA has been considered as one of the most promising methods. However, the bottleneck consistently encountered is the active expression of chondroitin 4- O -sulfotransferase (C4ST) during CSA biosynthesis. This study meticulously delved into optimizing C4ST expression through systematic enhancements in transcription, translation, and secretion mechanisms via modifications in the 5′ untranslated region, the N-terminal encoding sequence, and the Komagataella phaffii chassis. Ultimately, the active C4ST expression escalated to 2713.1 U/L, representing a striking 43.7-fold increase. By applying the culture broth supernatant of C4ST and integrating the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) biosynthesis module, we constructed a one-pot enzymatic system for CSA biosynthesis, achieving a remarkable sulfonation degree of up to 97.0 %. The substantial enhancement in C4ST expression and the development of an engineered one-pot enzymatic synthesis system promises to expedite large-scale CSA biosynthesis with customizable sulfonation degrees. [Display omitted] [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS) is widely applied in the medical, clinical, and nutraceutical fields. However, all commercialized CS is extracted from animal tissues, which is associated with many problems, such as heavy reliance on antibiotics, animal excrement waste and long breeding time. Here, we developed a promising new green route for the de novo biosynthesis of non-animal CS from methanol, a one-carbon compound, using the engineered Pichia pastoris cell factory. Firstly, three exogenous genes, namely kfoC and kfoA from Escherichia coli K4 and tuaD from Bacillus subtilis, were introduced into P. pastoris to construct the chondroitin synthesis pathway, which yielded 5.5 mg L−1 chondroitin. After optimizing the expression of committed enzymes by codon optimization, the production of chondroitin improved by approximately 35-fold to 189.8 mg L−1. Thereafter, 6 kinds of Kozak sequences and 10 different endogenous promoters were compared and applied to achieve the expression of active chondroitin-4-O-sulfotransferase (41.3 U L−1). After integrating the chondroitin biosynthesis and sulfonation modules, the engineered strain, Pp006, successfully produced 182.0 mg L−1 CSA [GlcA-GalNAc (4S)] with a sulfation degree of 1.1%. We further improved the sulfation degree to 2.8% by strengthening the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) supply with the overexpression of adenosine-5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase. In the fed-batch cultivation, the final titer of CSA reached 2.1 g L−1 and the sulfation degree increased to 4.0%. Moreover, the results suggest that the P. pastoris cell factory could be used as a green route to produce other sulfated glycosaminoglycans. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are naturally occurring acidic polysaccharides with wide applications in pharmaceuticals, cosmetics, and health foods. The diverse biological activities and physiological functions of GAGs are closely associated with their molecular weights and sulfation patterns. Except for the non-sulfated hyaluronan which can be synthesized naturally by group A Streptococcus , all the other GAGs such as heparin and chondroitin sulfate are mainly acquired from animal tissues. Microbial cell factories provide a more effective platform for the production of structurally homogeneous GAGs. Enhancing the production efficiency of polysaccharides, accurately regulating the GAGs molecular weight, and effectively controlling the sulfation degree of GAGs represent the major challenges of developing GAGs microbial cell factories. Several enzymatic, metabolic engineering, and synthetic biology strategies have been developed to tackle these obstacles and push forward the industrialization of biotechnologically produced GAGs. This review summarizes the recent advances in the construction of GAGs synthesis cell factories, regulation of GAG molecular weight, and modification of GAGs chains. Furthermore, the challenges and prospects for future research in this field are also discussed. [Display omitted] • Various microbial cell factories have been constructed to efficiently synthesize unsulfured GAGs. • Many strategies have been developed for controlling the molecular weight of GAGs. • Artificial sulfation systems have been designed and constructed for preparing GAGs and its oligosaccharides. • The biosynthesis of GAGs with microbial cell factories has many advantages comparing the traditional extraction methods. [ABSTRACT FROM AUTHOR]

• The solubility of csABC I was improved with fusion of the MBP tag. • Mutation of I309 V significantly increased the catalytic activity. • OmpA signal peptide achieved efficient secretion of csABC I. • Deletion of a lipoprotein encoding gene lpp increased csABC I secretion. • Chondroitin sulfate oligosaccharides were produced with engineered csABC I. Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp , all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ± 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides. [ABSTRACT FROM AUTHOR]

• An efficient sulfation-modification system for CSA synthesis was constructed. • The soluble expression of C4ST was increased by 30 times. • The stability of AST IV was significantly improved with addition of glycerol. • CSA with high sulfation degree was produced with protein scaffold strategies. • 15 g CSA was produced within 24 h in 1 L system. Chondroitin sulfate (CS) extracted from animal tissues has been widely used as nutraceutical and pharmaceutical products for osteoarthritis treatment. Here we developed an efficient sulfation-modification system for large scale preparation of CSA in vitro. First, the expression level of C4ST was improved by 30 times with fusion of the chaperone SUMO. Then, glycerol as a protein stabilizer was found to improve rat AST IV stability during the regeneration of cofactor PAPS. Then peptide linkers or protein scaffolds were employed to assemble AST IV and C4ST into artificial complexes to bring the enzymes and PAPS spatially closer and enhance the catalytic efficiency of chondroitin sulfation. Eventually, the system was scaled up to 1 L system and 15 g chondroitin was converted to CSA in 24 h, with a 98 % conversion. The present study made a step further towards the industrial production of CSA with different sulfation degrees. [ABSTRACT FROM AUTHOR]