Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in Lysobacter enzymogenes OH11.
Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in Lysobacter enzymogenes OH11.
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CRISPR/DCAS9的抗MRSA抗生素WAP-8294A的产生改善，并在溶血杆菌酶OH11中结合了重构自我保护基因。
DOI：
10.1021/acssynbio.7b00293
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发表时间：
2018-01-19
影响因子：
4.7
通讯作者：
Du L
中科院分区：
文献类型：
--
作者：
Yu L;Su W;Fey PD;Liu F;Du L
环状脂沉肽WAP-8294A是抗耐甲氧西林金黄色葡萄球菌（MRSA）的有效抗生素。该家族的一名成员WAP-8294A2（洛替比星）因其高活性和独特的化学成分而处于临床试验中。然而，溶杆菌生产WAP-8294A化合物的产率非常低，而且只有在非常严格的条件下才能生产。提高WAP-8294A的产率对这些抗mrsa化合物的研究和应用至关重要。在这里，我们报告了增加WAP-8294A产量的策略。我们首先使用CRISPR/dCas9系统，通过将RNA聚合酶的omega亚基与靶向操纵子启动子区域的dCas9融合，增加了WAP基因簇中5个共转录基因（orf1-5）的表达。这导致菌株dCas9-ω3中基因的转录增加了5-48倍。然后，我们重构了四个假定的自我保护基因（orf6、orf7、orf9和orf10），在强溶菌启动子PHSAF的控制下，将它们重组成一个操纵子。将重组的操纵子引入菌株dCas9-ω3中，其自我保护基因的转录量增加了20-60倍。WAP-8294A主要化合物WAP-8294A1、WAP-8294A2和WAP-8294A4的产率在工程菌株中分别提高了6倍、4倍和9倍。数据还表明，WAP-8294A化合物产量的增加主要是由于胞外分布的增加。WAP-8294A2在54株临床相关革兰氏阳性病原菌中表现出对金黄色葡萄球菌的强效（MIC为0.2 ~ 0.8 μg/mL）和特异性活性。
The cyclic lipodepsipeptides WAP-8294A are antibiotics with potent activity against methicillin-resistant Staphylococcus aureus (MRSA). One member of this family, WAP-8294A2 (Lotilibcin), was in clinical trials due to its high activity and distinct chemistry. However, WAP-8294A compounds are produced in a very low yield by Lysobacter and only under very stringent conditions. Improving WAP-8294A yield has become very critical for research and application of these anti-MRSA compounds. Here, we report a strategy to increase WAP-8294A production. We first used the CRISPR/dCas9 system to increase the expression of five cotranscribed genes (orf1–5) in the WAP gene cluster, by fusing the omega subunit of RNA polymerase with dCas9 that targets the operon’s promoter region. This led to the transcription of the genes increased by 5–48 folds in strain dCas9-ω3. We then refactored four putative self-protection genes (orf6, orf 7, orf 9 and orf10) by reorganizing them into an operon under the control of a strong Lysobacter promoter, PHSAF. The refactored operon was introduced into strain dCas9-ω3, and the transcription of the self-protection genes increased by 20–60 folds in the resultant engineered strains. The yield of the three main WAP-8294A compounds, WAP-8294A1, WAP-8294A2, and WAP-8294A4, increased by 6, 4, and 9 folds, respectively, in the engineered strains. The data also showed that the yield increase of WAP-8294A compounds was mainly due to the increase of the extracellular distribution. WAP-8294A2 exhibited potent (MIC 0.2–0.8 μg/mL) and specific activity against S. aureus among a battery of clinically relevant Gram-positive pathogens (54 isolates).
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影响因子：
11.4
作者：
PARRALOPEZ, C;BAER, MT;GROISMAN, EA
通讯作者：
GROISMAN, EA
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
3.2
作者：
Thompson, SA;Shedd, OL;Blaser, MJ
通讯作者：
Blaser, MJ
影响因子：
3
作者：
Schuster, Mariana;Schweizer, Gabriel;Kahmann, Regine
通讯作者：
Kahmann, Regine
影响因子：
6.4
作者：
Fey PD;Endres JL;Yajjala VK;Widhelm TJ;Boissy RJ;Bose JL;Bayles KW
通讯作者：
Bayles KW