CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus
CRISPR/dCas9-based metabolic pathway engineering for the systematic optimization of exopolysaccharide biosynthesis in Streptococcus thermophilus
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基于CRISPR/dCas9的代谢途径工程用于系统优化嗜热链球菌胞外多糖生物合成
DOI：
10.3168/jds.2021-21409
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Lianzhong Ai
中科院分区：
文献类型：
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作者：
Linghui Kong;Zhiqiang Xiong;Xin Song;Yongjun Xia;Lianzhong Ai
摘要嗜热链球菌广泛应用于乳品工业，并显示出作为高价值代谢物生物合成的底盘细胞的巨大潜力。然而，S.嗜热菌缺乏有效的遗传修饰工具来调节基因表达以减轻代谢负担并使所需化合物的产量最大化。在这里，我们开发了一个成簇的规则间隔的短回文重复序列干扰（CRISPRi）系统，用于S。嗜热菌我们的CRISPRi系统通常实现了单个或多个基因表达的66%至98%敲低。我们将CRISPRi用于新胞外多糖（EPS）的生物合成作为范例模型。在尿苷二磷酸葡萄糖糖代谢模块的galK的抑制和在EPS合成模块的epsA和epsE的过表达导致EPS滴度（277 mg/L）与对照菌株相比增加约2倍。这项研究证明了CRISPRi作为S.嗜热菌
ABSTRACT Streptococcus thermophilus is used extensively in the dairy industry and has shown great promise as a chassis cell for the biosynthesis of high-value metabolites. However, metabolic engineering in S. thermophilus lacks effective genetic modification tools to modulate gene expression to relieve metabolic burden and maximize the production of desired compounds. Here, we developed a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system for efficient gene transcriptional modulation in S. thermophilus. Our CRISPRi system typically achieved 66 to 98% knockdown of single or multiple gene expression. We used CRISPRi for the biosynthesis of a new exopolysaccharide (EPS) as a paradigm model. Repression of galK at module of uridine diphosphate glucose sugar metabolism and overexpression of epsA and epsE at EPS synthesis module resulted in an approximately 2-fold increase in EPS titer (277 mg/L) when compared with a control strain. This study demonstrated the effectiveness of CRISPRi as a powerful metabolic engineering tool and synthetic biology strategy for S. thermophilus.