New Tools for Targeted Cloning and Over Expression of Biosynthetic Gene...
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1. Blin, K., Wolf, T., Chevrette, M. G., Lu, X., Schwalen, C. J., Kautsar, S. A., et al. (2017). antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Research. 2. Zhang, M. M., Wang, Y., Ang, E. L., & Zhao, H. (2016). Engineering microbial hosts for production of bacterial natural products. Natural Product Reports, 33(8), 963–987. 3. Montaser, R. & Kelleher, N. L. Discovery of the Biosynthetic Machinery for the Stravidins, Biotin Antimetabolites. ACS Chem. Biol. (2019). doi:10.1021/acschembio.9b00890 ❶ 5-day workflow for BGC cloning Monday Tuesday Wednesday Thursday Friday • gDNA purification • Vector preparation • Cas9 restriction • DNA assembly • Transformation • Pick clones • Colony PCR • Miniprep • Restriction digest • Submit sequencing • Sequence validation Introduction The genome sequencing revolution and corresponding development of biosynthetic gene cluster (BGC) prediction and analysis tools such as antiSMASH (1) have resulted in a wealth of new biosynthetic potential for further examination. Once a BGC of interest is identified, isolating a physical DNA clone for expression, refactoring, and other analyses can be a slow, expensive process. Classical methods of cloning can take months to complete, and gene synthesis is expensive and can be stymied by GC-rich and/or repetitive sequences (2). The development of CRISPR-Cas9 has revolutionized many fields within biology and medicine since it allows for precise, site-specific restriction with few off-target cuts. We developed a technique using CRISPR-Cas9 to directly clone large BGCs from genomic DNA without using gels or agarose plugs, to aid in the characterization of new BGCs and accelerate the pace of drug discovery. In order to facilitate the chemical analyses of the biosynthetic potential of gene clusters from genomic or metagenomic sources, we developed a method for heterologous expression of BGCs using a novel BAC vector that contains two inducible promoters flanking the cloning site for heterologous expression in Streptomyces. Here, we conducted experiments to determine: Can CRISPR-Cas9 be used to rapidly cut and assemble BGCs directly into a new vector that enables inducible, heterologous expression? Conclusions • 70+ large pathways ranging in size from 12–125 kb (median = 58) were cloned directly from genomic DNA, including BGCs from GC-rich Streptomyces, Bacillus, and other diverse bacteria and fungi • Through cloning and heterologous expression, new stravidin analogues and biosynthetic machinery were uncovered in only 2 months (3) • Cloning efficiencies of 100% were achieved for up to 125 kb after optimizing the technology • BGCs are cloned directly into an integrative vector ready for heterologous expression testing • pDualP dual-inducible vector allows for controlled heterologous expression of model (ACT and RED), novel genomic, and metagenomic BGCs • 20 metagenomic BGCs ranging from 16–154 kb were subcloned to pDualP • These results indicate that any sequenced biosynthetic gene cluster can be cloned intact from complex genomes and heterologously expressed to produce secondary metabolites, thereby expanding our available resources for natural product discovery References ❷ qPCR validation of Cas9 cuts left side > 90% cut uncut > 90% cut uncut right side ❹ Successful cloning of many clusters varigenbio.com B. C. ❸ 21 kb BGC: 100% cloning efficiency A. ❻ Inducible expression of novel BGCs Metagenomic BGC Heterologous Expression in S. coelicolor M1154 CRISPR-Cas9 restriction DNA assembly to BAC vector high-MW gDNA Cas9 + gRNA Cas9 + gRNA target BGC >20 kb integration vector for Streptomyces spp. target BGC >20 kb Methods ori2/V apraR φC31 int oriT P1 P2 Patent Pending 117 PKS-positive Contigs Detected by anti-SMASH 2 1,2 New Tools for Targeted Cloning and Over Expression of Biosynthetic Gene Clusters Robb Stankey 1 , Don Johnson 1 , Katherine Wozniak 1 , Rana Montaser 2 , Neil L. Kelleher 2 , Alinne Pereira 3 , Megan Sandoval-Powers 3 , Joyanne MacDonald 1 , Phil Brumm 1 , Håvard Sletta 4 , Trond Ellingsen 4 , Alexander Wentzel 4 , Mark Liles 3 , and David Mead 1 1 Varigen Biosciences (Madison WI, USA), 2 Northwestern University (Evanston IL, USA), 3 Auburn University (Auburn AL, USA), 4 SINTEF (Trondheim, Norway) Discovery of new Biosynthetic Machinery for Stravidins (3) ❺ Inducible expression of model BGCs –ind +ind2 +ind1 +ind1 +ind2 pBAC-S ACT pDualP ACT pBAC-S RED pDualP RED –ind +ind2 +ind1 +ind1 +ind2 Sco ACT and RED Heterologous Expression in S. lividans Δact Δred stravidin S4 [352.22] stravidin S5 [366.24]
Transcript of New Tools for Targeted Cloning and Over Expression of Biosynthetic Gene...
1. Blin, K., Wolf, T., Chevrette, M. G., Lu, X., Schwalen, C. J., Kautsar, S. A., et al. (2017). antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Research.
2. Zhang, M. M., Wang, Y., Ang, E. L., & Zhao, H. (2016). Engineering microbial hosts for production of bacterial natural products. Natural Product Reports, 33(8), 963–987.
3. Montaser, R. & Kelleher, N. L. Discovery of the Biosynthetic Machinery for the Stravidins, Biotin Antimetabolites. ACS Chem. Biol. (2019). doi:10.1021/acschembio.9b00890
❶ 5-day workflow for BGC cloning
Monday Tuesday Wednesday Thursday Friday
• gDNA purification
• Vector preparation
• Cas9 restriction• DNA assembly• Transformation
• Pick clones• Colony PCR
• Miniprep• Restriction digest• Submit sequencing
• Sequence validation
IntroductionThe genome sequencing revolution and
corresponding development of biosynthetic gene cluster (BGC) prediction and analysis tools such as antiSMASH (1) have resulted in a wealth of new biosynthetic potential for further examination. Once a BGC of interest is identified, isolating a physical DNA clone for expression, refactoring, and other analyses can be a slow, expensive process. Classical methods of cloning can take months to complete, and gene synthesis is expensive and can be stymied by GC-rich and/or repetitive sequences (2).
The development of CRISPR-Cas9 has revolutionized many fields within biology and medicine since it allows for precise, site-specific restriction with few off-target cuts. We developed a technique using CRISPR-Cas9 to directly clone large BGCs from genomic DNA without using gels or agarose plugs, to aid in the characterization of new BGCs and accelerate the pace of drug discovery.
In order to facilitate the chemical analyses of the biosynthetic potential of gene clusters from genomic or metagenomic sources, we developed a method for heterologous expression of BGCs using a novel BAC vector that contains two inducible promoters flanking the cloning site for heterologous expression in Streptomyces.
Here, we conducted experiments to determine:
Can CRISPR-Cas9 be used to rapidly cut and assemble BGCs directly into a new vector that enables inducible, heterologous expression?
Conclusions• 70+ large pathways ranging in size from 12–125 kb
(median = 58) were cloned directly from genomic DNA, including BGCs from GC-rich Streptomyces, Bacillus, and other diverse bacteria and fungi
• Through cloning and heterologous expression, new stravidin analogues and biosynthetic machinery were uncovered in only 2 months (3)
• Cloning efficiencies of 100% were achieved for up to 125 kb after optimizing the technology
• BGCs are cloned directly into an integrative vector ready for heterologous expression testing
• pDualP dual-inducible vector allows for controlled heterologous expression of model (ACT and RED), novel genomic, and metagenomic BGCs
• 20 metagenomic BGCs ranging from 16–154 kb were subcloned to pDualP
• These results indicate that any sequenced biosynthetic gene cluster can be cloned intact from complex genomes and heterologously expressed to produce secondary metabolites, thereby expanding our available resources for natural product discovery
References
❷ qPCR validation of Cas9 cutsleft side
> 90% cut
uncut
> 90% cut
uncut
right side
❹ Successful cloning of many clusters
varigenbio.com
B.
C.
❸ 21 kb BGC: 100% cloning efficiencyA.
❻ Inducible expression of novel BGCs
Metagenomic BGC Heterologous Expression in S. coelicolor M1154
CRISPR-Cas9 restriction
DNA assembly to BAC vector
high-MW gDNACas9+ gRNA
Cas9+ gRNA
target BGC>20 kb
integration vector forStreptomyces spp.
target BGC >20 kb
Methods
ori2/V
apraR
φC31 int
oriT
P1 P2
Patent Pending
117 PKS-positive Contigs Detected by anti-SMASH 21,2
New Tools for Targeted Cloning and Over Expression of Biosynthetic Gene ClustersRobb Stankey1, Don Johnson1, Katherine Wozniak1, Rana Montaser2, Neil L. Kelleher2, Alinne Pereira3, Megan Sandoval-Powers3,
Joyanne MacDonald1, Phil Brumm1, Håvard Sletta4, Trond Ellingsen4, Alexander Wentzel4, Mark Liles3, and David Mead1
1Varigen Biosciences (Madison WI, USA), 2Northwestern University (Evanston IL, USA), 3Auburn University (Auburn AL, USA), 4SINTEF (Trondheim, Norway)
Discovery of new Biosynthetic Machinery for Stravidins (3)
❺ Inducible expression of model BGCs–ind +ind2 +ind1
+ind1+ind2
pBAC-SACT
pDualPACT
pBAC-SRED
pDualPRED
–ind +ind2 +ind1+ind1+ind2
Sco ACT and RED Heterologous Expression in S. lividans Δact Δred
stravidin S4[352.22]
stravidin S5[366.24]
