TY - JOUR
T1 - Yield Improvement of the Anti-MRSA Antibiotics WAP-8294A by CRISPR/dCas9 Combined with Refactoring Self-Protection Genes in Lysobacter enzymogenes OH11
AU - Yu, Lingjun
AU - Su, Wei
AU - Fey, Paul D.
AU - Liu, Fengquan
AU - Du, Liangcheng
N1 - Funding Information:
This work was supported in part by NSFC (31329005, 31701839), the NIH (R01AI097260), and a UNL Revision Award and a UNL Faculty Seed Grant.
Funding Information:
We thank Drs. Ronald Cerny and Kurt Wulser for technical assistance. We are grateful to Dr. Weeks and Dr. Jiang (Department of Biochemistry, University of Nebraska-Lincoln) for providing the plasmid containing sgRNA and cas9. This work was supported in part by NSFC (31329005, 31701839), the NIH (R01AI097260), and a UNL Revision Award and a UNL Faculty Seed Grant.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2018/1/19
Y1 - 2018/1/19
N2 - 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, 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).
AB - 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, 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).
KW - CRISPR/dCas9
KW - Lysobacter
KW - WAP-8294A
KW - antibiotics
KW - gene refactoring
KW - natural products
UR - http://www.scopus.com/inward/record.url?scp=85040867134&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85040867134&partnerID=8YFLogxK
U2 - 10.1021/acssynbio.7b00293
DO - 10.1021/acssynbio.7b00293
M3 - Article
C2 - 29125739
AN - SCOPUS:85040867134
SN - 2161-5063
VL - 7
SP - 258
EP - 266
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 1
ER -