Biosynthesis of non-animal chondroitin sulfate from methanol using genetically engineered Pichia pastoris
Biosynthesis of non-animal chondroitin sulfate from methanol using genetically engineered Pichia pastoris
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DOI：
10.1039/d1gc00260k
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发表时间：
2021-05-06
期刊：
影响因子：
9.8
通讯作者：
Kang, Zhen
中科院分区：
文献类型：
--
作者：
Jin, Xuerong;Zhang, Weijiao;Kang, Zhen
硫酸软骨素广泛应用于医学、临床和营养保健领域。然而，所有商业化的CS都是从动物组织中提取的，这与许多问题相关，例如严重依赖抗生素，动物粪便废物和养殖时间长。在这里，我们开发了一个有前途的新的绿色路线从头生物合成的非动物CS从甲醇，一个碳化合物，使用工程毕赤酵母细胞工厂。首先，将来自大肠杆菌K4的kfoC和kfoA以及来自枯草芽孢杆菌的tuaD三个外源基因导入毕赤酵母，构建了软骨素合成途径，获得了5.5 mg L-1的软骨素产量。在通过密码子优化来优化承诺酶的表达后，软骨素的生产提高了约35倍，达到189.8 mg L-1。随后，比较了6种Kozak序列和10种不同的内源性启动子，并应用于实现活性软骨素-4-O-磺基转移酶（41.3 U L-1）的表达。工程菌Pp 006在整合了软骨素生物合成和磺化两个模块后，成功地合成了182.0 mg L-1的CSA [GlcA-GalNAc（4S）]，硫酸化度为1.1%。我们进一步提高了硫酸化程度为2.8%，通过加强3 '-磷酸腺苷-5'-磷酸硫酸（PAPS）的供应与腺苷-5 '-三磷酸硫酸化酶和腺苷-5'-磷酸硫酸激酶的过表达。在流加培养中，CSA的最终效价达到2.1 g L-1，硫酸化度提高到4.0%。此外，结果表明，毕赤酵母细胞工厂可用作生产其他硫酸化糖胺聚糖的绿色途径。
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.