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Abstract：
BACKGROUND: Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production.
RESULTS: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.
CONCLUSIONS: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.
RESULTS: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.
CONCLUSIONS: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.
摘要：
背景：Oritavancin是针对革兰氏阳性菌的新一代半合成糖肽抗生素，它是第一种也是唯一一种单剂量治疗方案治疗ABSSSI的抗生素。天然存在的糖肽A82846B是奥利万星的直接前体。然而,其应用受到低产率和同源杂质的阻碍。本研究建立了多步骤组合策略，合理构建A82846B优质高效生物合成体系，并对其发酵工艺进行系统优化，突破微生物发酵生产瓶颈。
结果：首先，基于基因组测序和分析，我们删除了推定的竞争途径，并构建了一个更好的A82846B生产菌株，具有更干净的代谢背景，将A82846B产量从92mg/L提高到174mg/L。随后，在CRISPR-Cas12a系统的基础上引入了PhiC31整合酶系统。然后，通过构建的PhiC31系统过表达途径特异性调节因子StrR，将A82846B的发酵水平提高到226mg/L。此外，过表达糖基合成基因evaE将产量提高到332mg/L，这是由于中间体向目标产物的转化很大。最后,在发酵优化条件下,A82846B在15L发酵罐中的放大产量达到725mg/L,这是报道的A82846B的最高产量，没有产生同源杂质。
结论：在包括阻断竞争性途径在内的方法中，插入位点特异性重组系统，超压调节器，过表达糖基合成基因并优化发酵工艺，开发了A82846B高水平生产的多步组合策略，构建高产菌株AO-6。本文采用的组合策略可广泛应用于提高其他微生物次生代谢产物的发酵水平,为构建高效的高值天然产物微生物细胞工厂提供参考。
结果：首先，基于基因组测序和分析，我们删除了推定的竞争途径，并构建了一个更好的A82846B生产菌株，具有更干净的代谢背景，将A82846B产量从92mg/L提高到174mg/L。随后，在CRISPR-Cas12a系统的基础上引入了PhiC31整合酶系统。然后，通过构建的PhiC31系统过表达途径特异性调节因子StrR，将A82846B的发酵水平提高到226mg/L。此外，过表达糖基合成基因evaE将产量提高到332mg/L，这是由于中间体向目标产物的转化很大。最后,在发酵优化条件下,A82846B在15L发酵罐中的放大产量达到725mg/L,这是报道的A82846B的最高产量，没有产生同源杂质。
结论：在包括阻断竞争性途径在内的方法中，插入位点特异性重组系统，超压调节器，过表达糖基合成基因并优化发酵工艺，开发了A82846B高水平生产的多步组合策略，构建高产菌株AO-6。本文采用的组合策略可广泛应用于提高其他微生物次生代谢产物的发酵水平,为构建高效的高值天然产物微生物细胞工厂提供参考。
