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Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in Lysobacter enzymogenes OH11

文献类型： 外文期刊

作者： Yu, Lingjun 1 ; Su, Wei 2 ; Fey, Paul D. 3 ; Liu, Fengquan 1 ; Du, Liangcheng 2 ;

作者机构： 1.Jiangsu Acad Agr Sci, Inst Plant Protect, Nanjing 210014, Jiangsu, Peoples R China

2.Univ Nebraska Lincoln, Dept Chem, Lincoln, NE 68588 USA

3.Univ Nebraska Med Ctr, Dept Pathol & Microbiol, Omaha, NE 68198 USA

关键词： Lysobacter;natural products;antibiotics;WAP-8294A;CRISPR/dCas9;gene refactoring

期刊名称：ACS SYNTHETIC BIOLOGY （ 影响因子：5.11； 五年影响因子：5.239 ）

ISSN： 2161-5063

年卷期： 2018 年 7 卷 1 期

页码：

收录情况： SCI

摘要： 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-omega 3. 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-w3, 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 mu g/mL) and specific activity against S. aureus among a battery of clinically relevant Gram-positive pathogens (54 isolates).

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