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, orf7, orf9 and orf10) by reorganizing them into an operon under the control of a strong Lysobacter promoter, P_HSAF. 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).

中文翻译：

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CRISPR / dCas9结合重构自我保护基因在溶菌酶基因OH11中提高抗MRSA抗生素WAP-8294A的产量。

环状脂肽肽WAP-8294A是对耐甲氧西林的金黄色葡萄球菌（MRSA）具有有效活性的抗生素。由于其高活性和独特的化学性质，该家族的一个成员WAP-8294A2（Lotilibcin）正在临床试验中。但是，仅在非常严格的条件下，Lysobacter会以非常低的收率生产WAP-8294A化合物。对于这些抗MRSA化合物的研究和应用，提高WAP-8294A的产量已变得至关重要。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系统来增加五个共转录基因（orf1–5），通过将RNA聚合酶的ω亚基与靶向操纵子启动子区域的dCas9融合来实现。这导致dCas9-ω3菌株中基因的转录增加了5–48倍。然后，我们重构了四个假定自我保护基因（ORF6，ORF7，ORF9和ORF10通过重新组织他们到一个操纵子强烈的控制下）溶杆菌启动子，P _HSAF。重构的操纵子被引入到dCas9-ω3菌株中，并且在得到的工程菌株中自我保护基因的转录增加了20-60倍。在工程菌株中，三种主要的WAP-8294A化合物WAP-8294A1，WAP-8294A2和WAP-8294A4的产率分别提高了6倍，4倍和9倍。数据还表明，WAP-8294A化合物的收率增加主要是由于细胞外分布的增加。在一系列临床相关的革兰氏阳性病原体（54个分离株）中，WAP-8294A2表现出有效的浓度（MIC 0.2–0.8μg/ mL）和针对金黄色葡萄球菌的比活性。