Sulfonated compounds are widely utilized in feed additives, daily commodities, industrial manufacturing, and healthcare applications. Their production relies on the sulfonate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS). This study identified a novel adenine-sulfotransferase with sulfotransferase activity toward adenosine monophosphate for adenosine phosphosulfate formation. The identified enzyme was rationally engineered, yielding the mutant Bta APSST H8M/L117D , which exhibited a 0.93-fold increase in sulfotransfer efficiency. The mutant Bta APSST H8M/L117D was subsequently combined with additional enzymes to reconstruct what we term the RPA pathway, enabling the synthesis of PAPS at titers of 7.6 g/l and 5.03 g/l in Escherichia coli and Bacillus subtilis , respectively, using adenine and ribose as substrates. The RPA pathway was further integrated into the chondroitin producing strain E. coli GZ17, to construct E. coli CSA-02, which produced 1.89 g/l chondroitin sulfate A (CSA) with a sulfation rate of 76%. These results offer a promising way to enhance the biosynthesis of sulfonated compounds in microbial cell factories.
Peichao Zhang,Jing Gao,Haiyang Zhang,Yongzhen Wang,Zhen Liu,Sang Yup Lee,Xiangzhao Mao
Zosteric acid (ZA) is a Zostera species-derived, sulfated phenolic acid compound with antifouling activity and has gained much attention due to its nontoxic and biodegradable characteristics. However, the yield of Zostera species available for ZA extraction is limited by natural factors, such as season, latitude, light, and temperature. Here we report the development of metabolically engineered Escherichia coli strains capable of producing ZA from glucose and glycerol. First, intracellular availability of the sulfur donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) was enhanced by knocking out the cysH gene responsible for PAPS consumption and overexpressing the genes required for PAPS biosynthesis . Co-overexpression of the genes encoding tyrosine ammonia-lyase, sulfotransferase 1A1, ATP sulfurylase, and adenosine 5′-phosphosulfate kinase constructed ZA producing strain with enhanced PAPS supply. Second, the feedback-resistant forms of aroG and tyrA genes (encoding 3-deoxy- d -arabinoheptulosonate 7-phosphate synthase and chorismate mutase , respectively) were overexpressed to relieve the feedback regulation of L- tyrosine biosynthesis. Third, the pykA gene involved in phosphoenolpyruvate-consuming reaction, the regulator gene tyrR , the competing pathway gene pheA , and the ptsHIcrr genes essential for the PEP:carbohydrate phosphotransferase system were deleted. Moreover, all genes involved in the shikimate pathway and the talA , tktA , and tktB genes in the pentose phosphate pathway were examined for ZA production. The PTS-independent glucose uptake system, the expression vector system, and the carbon source were also optimized. As a result, the best-performing strain successfully produced 1.52 g L −1 ZA and 1.30 g L −1 p -hydroxycinnamic acid from glucose and glycerol in a 700 mL fed-batch bioreactor .
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