In Vitro Packaging Mediated One-Step Targeted Cloning of NaturalProduct PathwayWeixin Tao,*,† Li Chen,† Chunhua Zhao,‡ Jing Wu,§ Dazhong Yan,§ Zixin Deng,† and Yuhui Sun*,†

†Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and Wuhan University School ofPharmaceutical Sciences, Wuhan 430071, People’s Republic of China‡School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republicof China§School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, People’s Republic of China

*S Supporting Information

ABSTRACT: Direct cloning of natural product pathways forefficient refactoring and heterologous expression has become animportant strategy for microbial natural product research anddiscovery, especially for those kept silent or poorly expressed inthe original strains. Accordingly, the development of convenient andefficient cloning approaches is becoming increasingly necessary. Herewe presented an in vitro packaging mediated cloning approach thatcombines CRISPR/Cas9 system with in vitro λ packaging system, fortargeted cloning of natural product pathways. In such a scheme,pathways of Tu3010 (27.4 kb) and sisomicin (40.7 kb) wererespectively cloned, and stuR was further depicted to positivelyregulate Tu3010 production. In vitro packaging mediated approachnot only enables to activate cryptic pathways, but also facilitatesrefactoring or interrogating the pathways in conjunction with variousgene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may bewidely adopted for targeted cloning of the natural product pathways.

KEYWORDS: direct cloning, natural product, in vitro packaging, CRISPR/Cas9, gene editing

Microbial natural products possess great potential inchemistry and biochemistry, and thus have long been

regarded as an excellent source for discovery of pharmaceuticaldrugs. Discovery of microbial natural products has latelystepped into a genomic era along with the advancements insequencing technology, which leads to an unprecedentedexplosion of microbial genomes and raises a huge potential toproduce novel natural products from the extensive untappednatural product pathways.1 Nevertheless, many of the in silicoidentified biosynthetic gene clusters have not identifiedcorresponding products under laboratory culture conditions.2

To activate these cryptic pathways, editing of the gene clustersis of indispensable necessity. In vivo strategies are mostcommonly used; however, the relevant approaches greatly relyon genetic operability of the original strains, since these areachieved through homologous recombination and involvelaborious screening processes. In addition, the application ofthe currently most valued CRISPR/Cas system for microbialgenome editing is still relatively limited, although itdemonstrates high accuracy and efficiency in some modelstrains.3−7

Alternatively, direct cloning of the biosynthetic pathway forrational refactoring and heterologous expression has raisedgreat concern. In this case, a randomly fragmented genomic

library has still been widely adopted since it provides excellentstability and reliability, while the experimental process isrelatively convenient. However, the screening process isnevertheless laborious. To directly clone a natural productpathway in a more specific manner, restriction endonucleasecleavage has been applied for targeted release of thebiosynthetic gene cluster, which is subsequently captured byusing approaches like RecE/T mediated homologous recombi-nation,8,9 Gibson assembly,10,11 and TAR cloning.12−16 A mainissue they may confront is the difficulty to figure out theappropriate digesting sites located just right at ends of the genecluster. Aiming at this issue, some functional elements, likeattP/attB,17 loxP,18 and I-SceI/PI-PspI,19 have been applied topre-embed on the flanks of the gene cluster for subsequentspecific releasing. These strategies enable to clone large-sizedpathways but still closely rely on the genetic operability of theoriginal strains, and involve multiple genetic manipulations.CRISPR/Cas system allows specific releasing of the geneclusters with the guidance of artificially designed sgRNAs.Most recently, it has been applied to targeted clone natural

Received: June 13, 2019Published: September 5, 2019

Letter

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© 2019 American Chemical Society 1991 DOI: 10.1021/acssynbio.9b00248ACS Synth. Biol. 2019, 8, 1991−1997

product pathways by coupling with capture approaches likeGibson assembly,20,21 TAR cloning,22,23 or RecE/T recombi-nation.24 However, these all involve tedious vector con-structions for the respective pathways, and some may requirespecific handling of genomic DNA (gDNA) like in-gel cell lysisor skillful operation experience on yeast. In vitro PCRamplification of the gene cluster does not require a largeamount of gDNA and is time-efficient.25,26 Nevertheless, PCR-based approach remains limited due to its potentialmutagenicity regardless of the use of high-fidelity DNApolymerases, and still, it confronts difficulties to clone large-sized pathways.In order to conveniently target clone natural product

pathways for exploring the biosynthetic potential in micro-organisms, here we presented an in vitro packaging mediatedapproach, which mainly involves routine DNA handlings, butpresents a rapid and convenient process for pathway cloningand enables targeted cloning of the pathway of interest within aweek. Using this approach, pathways of Tu3010 (stu) fromStreptomyces thiolactonus NRRL 15439 and sisomicin (sis) fromgenetically intractable Micromonospora inyoensis DSM 46123have been cloned, respectively. By applying in vitro CRISPR/Cas9-mediated editing system (ICE),27 stuR, a LuxR familytranscriptional regulator gene, has been interrogated in itspositive regulatory role in Tu3010 biosynthesis. In a word, invitro packaging mediated approach provides an alternative wayfor facile and expedited cloning of natural product pathways.Besides, it also facilities following refactoring or interrogatingpathways in junction with various gene editing systems, foractivating the cryptic pathways or elucidating the biosyntheticroute of valuable natural products.

■ RESULTS AND DISCUSSIONIn Vitro Packaging Mediated Approach. To access and

deeply exploit the biosynthetic potentials concealed inuntapped natural product pathways, we sought to develop asimple, rapid but easy-to-use method to clone and expressthese pathways in surrogate host strains for production ofnovel natural products. Randomly fragmented genomiclibraries have long been preferred as they perform in routineand reliable manipulations. However, random fragmentation ofgDNA also brings laborious screening processes. So, in orderto improve the specificity, we devised to introduce theCRISPR/Cas9 system, which has been approved high accuracyand efficiency, to specifically cleave and release the naturalproduct pathways of interest.20,22 Accordingly, an in vitropackaging mediated approach was developed that applies theCRISPR/Cas9 system to release the pathway, which is

subsequently ligated with a linearized universal vector and invitro packaged into phage particles to infect the Escherichia coli(Scheme 1).In vitro packaging mediated approach presents a universal

synthetic biology strategy with routine experimental processes,and that enables targeted cloning of a natural product pathwaywithin a week. Besides, more details may render it morefavorable: (i) it has no strict requirements for gDNApreparation and applies to the phenol/chloroform extractedgDNAs despite normally being contaminated with plentifulproteins and RNAs; (ii) EcoRV-linearized pJTU255428 isuniversal for most pathways and does not require extra-reconstructions for every single pathway; (iii) dephosphor-ylation of gDNA ahead of CRISPR/Cas9 cleavage increasescloning efficiency by avoiding random ligation of the nontargetgDNAs fragments; (iv) beyond routine experimental manip-ulations, reliable and ready-to-use λ packaging extractsguarantee high packaging efficiency; (v) the in vitro packagingsystem further lessens interference of gDNA fragments out ofrange, and is particularly suitable for cloning midsized naturalproduct pathways in a range of 30−45 kb; and (vi) it presentsa rapid and valid performance that only requires screeningdozens of colonies.

Targeted Cloning of Natural Product Pathways. Asproofs of concept, natural product pathways of 30−45 kb werechosen as targets for testing. We have previously reported themain stu gene cluster for production of Tu3010, composinggenes encoding polyketide synthase (PKS), nonribosomalpeptide synthetase (NRPS), iterative PKS/NRPS for backbonebiosynthesis, oxidoreductase genes for ramification, and genesfor regulation and self-resistance, which has truncated to a coregene cluster by duplicate ICE manipulations based on agenomic library clone.29 Here, stu gene cluster (27.4 kb, 69%GC) was applied to test cloning efficiency when the fragmentis close to the lower packaging limit of λ phage.Extraction of microbial gDNAs with phenol/chloroform

method normally involves multiple agitations for deproteina-tion, of which the mechanical shearing process makes gDNAsbe overfragmented and thus they are not applicable for cloninglarge-sized natural product pathways. Normally, gDNAsgenerated in this case are less than 100 kb. Among which,some nontarget gDNAs of 30−45 kb with blunt-ends mayinevitably ligate with linearized vector and subsequently bepackaged into the phage particles. To avoid this, dephosphor-ylation of S. thiolactonus gDNAs has been applied ahead ofCas9 cleavage. Rigorous design of sgRNA-stu-L and sgRNA-stu-R was strictly required for specific release of the blunt-ended stu gene cluster, which the target fragment should retain

Scheme 1. Overview of in Vitro Packaging Mediated Approach for Targeted Cloning of Natural Product Pathwaysa

aIn vitro packaging mediated approach applies CRISPR/Cas9 system for specific release of natural product pathways, which are subsequentlyligated with a universal vector, and further packaged into phage particles by in vitro λ packaging system to infect E. coli strains. This approachfacilitates rapid and efficient targeted cloning of mid-sized pathways and is promising for exploration of biosynthetic potential in microorganisms bycoupling with various gene editing systems.

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the PAM locus at both ends after Cas9 cleavage, to generateblunt ends at stu gene cluster (Figure 1a).21,27 After cleavage,intact stu gene cluster exposed blunt ends with 5′ phosphategroup at both terminals and theoretically only CRISPR/Cas9released stu fragments can ligate with pJTU2554. And actually,dephosphorylation of gDNA has significantly reduced pack-aging yield, the colonies of dephosphorylation assay dramat-ically decreased to only one-third of the undephosphorylationassay, indicating a significant reduction of undesired ligation ofgDNA fragments by dephosphorylation preprocessing. Even-tually, 180 colonies were obtained in total when dephosphor-ylation has been assayed, of which 48 colonies were randomlyselected for PCR screening by tracing the existence of bothterminal genes of stu gene cluster, stuE and stuF2. Nine of themwere determined to house both genes and supposed to harboran intact pathway (Figure S1). It reveals a cloning efficiencyabout 18% in the case of Tu3010 pathway when the pathway isclose to the lower packaging limit. Of these, three wereselected for further restriction mapping and junctionsequencing, and that harboring intact Tu3010 pathway wastermed pWHU2879 (Figure 1b and S2a).Usually, randomly fragmented genomic library is hardly

applied to clone an intact natural product pathway with theupper packaging limit size, since it is improbable to producesuch an exact target fragment by random fragmentation.Nevertheless, in vitro packaging mediated approach allowsspecific release of the target pathway in such a situation. Inallusion to this instance, sis gene cluster (40.7 kb, 70% GC) forsisomicin production from genetically intractable M. inyoensiswas selected as a suitable model system.30,31 Accordingly, sisgene cluster was specifically released from genome by Cas9cleavage in guidance of sgRNAs (Figure S3a), and wassubsequently ligated with pJTU2554 and in vitro packagedusing λ packaging extracts. Eventually, 48 out of 756 colonies

were randomly selected and be subjected to screening for sisgene cluster. It turned out to find that 54% of the colonies weresupposed to harbor intact sisomicin pathway (Figure S4). Toconfirm it, three colonies were subjected to restrictionmapping along with junction sequencing to validate thecorrect colony, and that termed pWHU2880 (Figure S3b,c).The practical cases of the successful cloning of Tu3010 and

sisomicin pathways presented a cloning efficiency in a range of18−54%. Cloning of Tu3010 pathway presented a lowerefficiency, and this is probably because the size of the resultedpWHU2879 (36.4 kb) is slightly beyond the range of 37.4−50.4 kb (78−105% of wild type λ phage genome), the confinesof a stable λ phage. Nevertheless, it could be substantiallyimproved when cloned pathways are in the range of packagingsizes, just as sisomicin pathway. In addition, recovery of thetarget gene clusters after Cas9 cleavage or capture by morespecific approaches may further increase the cloning efficiency,but these also lead to cumbersome operations. In vitropackaging mediated approach imposed the cloned pathwaysin the range of packaging capacity of λ phage; however,applying alternative delivery methods like electroporation tointroduce the ligation mixture into E. coli may bypass suchrestriction, at some expense, and the cloning efficiency maysignificantly decrease. Also, natural product pathways exceed50 kb may not be obtained in one-step, but these may still bealternatively cloned in parts first and that followed byassembling with TAR cloning or Gibson assembly. In contrastto the alternative pathway capture approaches, like TARcloning, Gibson assembly, and RecE/T recombination, whichall involved vector reconstructions for every specific pathway asthese approaches all required introducing overlapping area atterminals of pathway into the capturing vector, in vitropackaging mediated approach applied blunt-end ligation andthus EcoRV-linearized vector was universal for all pathways.

Figure 1. Targeted cloning of stu gene cluster by in vitro packaging mediated approach. (a) Schematic representation of sgRNAs design. A “G”started 20 nt protospacer along with PAM (5′-GX19NGG-3′, the PAM sequences are highlighted in blue) was screened at both outer edges of thegene cluster, and that designs required to retain the PAM at the end of the pathway to generate blunt ends after Cas9 cleavage. (b) Construct ofpWHU2879 harboring stu gene cluster and confirmation of the construct by junction sequencing.

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Moreover, above practical cases for Tu3010 and sisomicinpathways cloning have clearly illustrated the efficiency, celerity,accuracy and convenience of the approach, which may renderit to be widely adopted for targeted cloning the microbialnatural product pathways.In Vitro Editing for Heterologous Expression of

Pathways. Biosynthesis of thiotetronate antibiotics hasgarnered particular attention and achieved meaningful progressin recent years.29,32−34 However, the regulation of thesestructurally unique antibiotics remained inconclusive. Inaddition to genes coding for PKS, NRPS, P450, andcrotonyl-CoA reductase for thiotetronate backbone synthesisand ramification at C-5 position, stu gene cluster housed threegenes coding for β-ketoacyl−acyl carrier protein synthase(KAS) and stuR for LuxR family transcriptional regulator.Target-triplicated resistance genes that clustered with pathwaywere supposed to ensure a high level of KAS activity andendow self-resistance against Tu3010.29 To investigate thefunction of stuR, stu gen cluster was first heterologouslyexpressed in S. avermitilis MA-4680, and then LC-ESI-HRMSanalysis was performed to detect the production of Tu3010. Itwas found that in fermentation extracts of S. avermitilis

harboring pWHU2879, the production of Tu3010 is almost atsame level as that in S. thiolactonus (Figure 2c,d). stuR was thenin-frame deleted by ICE manipulation, which Cas9 cleaves thegene in guidance of sgRNA-stuR-L and sgRNA-stuR-R, toproduce a 2250 nt deletion in stuR and generated pWHU2881(Figure 2a,b). The resulting construct was introduced intoS. avermitilis for heterologous expression. LC-ESI-HRMSanalysis of fermentation extracts revealed that production ofTu3010 has been completely abolished (Figure 2c), suggestingstuR plays a positive role in Tu3010 biosynthesis, and it mayaccordingly be applied for pathway editing to improve theproduction of this unique thiotetronate antibiotic and developantibacterial analogue target to type II fatty acid synthasepathway of lipid biosynthesis.

■ CONCLUSION

Direct cloning of natural product pathways in a facile,expedited, and generic manner helps take an early lead inmicrobial natural product research for exploiting their hugebiosynthetic potentials. In vitro packaging mediated approachintegrates the high specificity of CRISPR/Cas9 system withhigh operability of in vitro λ packaging system, which features a

Figure 2. In vitro editing of stu gene cluster for heterologous expression. (a) Schematic representations of in-frame deletion of stuR for genefunction interrogation. The primers used for checking the expected deletion by PCR are indicated in blue half arrows. (b) PCR confirmation ofheterologous strain harboring native and recombinant stu gene cluster, respectively. (c) LC-ESI-HRMS analysis of Tu3010 production. (d) Theselective ion monitoring confirmation of Tu3010.

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precise, simple, rapid but easy-to-use pathway cloning manner.In the course of this pathway cloning approach, operationoptimizations have devised. These include the dephosphor-ylation preprocessing of gDNA, which not only significantlyreduced packaging yield but also led to a rapid and efficientscreening process, and the rigorous design of sgRNAs forgeneration of blunt-ended pathway was not only critical forprecise cloning but also simplified the cloning process byavoiding repairing sticky ends. By using this approach, one mayquickly access the natural product pathways from microbialgenomes, and subsequently available for pathway refactoring,to specifically awake cryptic pathways, interrogate biosyntheticroutes, and produce or structurally diversify the valuablenatural products; hence, it is strongly encouraged for cloningmidsized pathways and is conceivable, and it could be widelyadopted to the research community.

■ MATERIALS AND METHODSgDNA Isolation. gDNA isolation was operated according

to the salting out procedure with slight modification.35 In brief,the mycelium after centrifugation was first resuspended in 5mL SET buffer (75 mM NaCl, 25 mM EDTA, 20 mM Tris-HCl, pH 8.0) supplemented with 1 mg/mL lysozyme. Afterincubation at 37 °C for 1 h, 0.5 mg/mL proteinase K and 1%SDS were added and the reaction mixture was furtherincubated for 2 h at 55 °C until it became clear. 1.25 MNaCl was then supplemented and the cell lysate was mixedthoroughly with phenol:chloroform:isoamyl alcohol (25:24:1,pH8.0) for 15 min to remove the proteins. Repeatdeproteinations were performed until the middle layer hasno protein precipitated. gDNA was then precipitated byappending 0.6 volumes of isopropanol and redissolved in 10mM Tris-Cl (pH 8.0).Preparation of Cas9 Protein and sgRNAs. E. coli

Rosetta (DE3) harboring pWHU2739 was incubated in LB at37 °C.27 Cas9 induction was initiated by appending 0.2 mMisopropyl-β-D-1-thiogalactopyranoside when OD600 reached0.6−0.8. The culture was further incubated overnight at 18°C, the cells were then harvested and lysed in lysis buffer (20mM Tris-HCl, 500 mM NaCl, 1 mM tris(2-carboxyethyl)-phosphine pH 8.0) by sonication. Cas9 was purified by affinitychromatography using a NTA agarose (GE Life Sciences)column and eluted with elution buffer (250 mM imidazole, 20mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8.0). Afterdesalination using PD-10 desalting column (GE Life Sciences),Cas9 was concentrated to a final concentration of ∼4 mg/mLby Amicon Ultra centrifugal filters (Millipore).For sgRNA design, a “G” started 20 nt guide sequence along

with PAM sequence “NGG” (5′-GX19NGG-3′) was searchednear the outer edge of the natural product pathway, since the“G” beginning transcript is ideal for both Cas9 loading and T7RNA polymerase transcription. Additional “G” was opted toappend as starting nucleotide of guide sequence if there is no“G” available.21 Rigorous design of sgRNAs that retain the“NGG” sequence at end of the target pathway was required toensure both terminals of the released pathway harboring bluntend after Ca9 cleavage, which is critical to the following blunt-end ligation. The in vitro transcription template of sgRNA wasprepared by overlapping PCR using primers: sgRNA-Xcontaining the T7 promoter and guide sequence, andsgRNA/A containing the crRNA-tracrRNA chimera sequence.The primers used in this study were listed in Table S2. In vitrotranscription of the sgRNAs was performed by using

TranscriptAid T7 high-yield transcription kit (Thermo FisherScientific) according to the instructions of manufacturer. ThesgRNA was finally resuspended in RNase-free water and storedat −80 °C.

Protocols of in Vitro Packaging Mediated Approach.In vitro packaging mediated approach enables cloning apathway of interest in a facile, expedited, and generic manner.First, gDNA dephosphorylation was performed by incubatingthe gDNAs with thermosensitive alkaline phosphatase FastAP(Thermo Fisher Scientific) at 37 °C overnight. Dephosphory-lated gDNA was then deproteinated by phenol:chloroform:i-soamyl alcohol (25:24:1, pH 8.0) extraction to removephosphatase and then recovered by ethanol precipitation.Meanwhile, the sgRNAs was pretreated by incubating at 95 °Cfor 10 min and then was left to cool naturally. In vitro cleavageof gDNA was performed according to the ICE protocol.27 Inbrief, reaction mixture containing ∼20 μg sgRNAs, ∼6 μg Cas9protein, and ∼3 μg gDNA was incubated in reaction buffer(100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/mLBSA, pH 7.9) at 37 °C for 2 h. After cleavage, sgRNA wasremoved by appending 0.2 mg/mL RNase for a further 15 minincubation. The gDNA was then again deproteinated byphenol/chloroform extraction and recovered by ethanolprecipitation. Obtained gDNAs containing the pathwayfragments were incubated with EcoRV-linearized pJTU2554in a mixture containing 1 μL T4 DNA ligase (Thermo FisherScientific) and 15% PEG4000 at 16 °C overnight. Afterligation, the mixture was in vitro packaged using MaxPlax λPackaging Extracts (Epicentre) according to manufacturer’sinstruction and subsequently applied to infect E. coli EPI300.After incubating at 37 °C overnight, colonies that grew werecounted and selected for PCR screening of positive clonesusing two primer pairs respectively match to terminal genes ofthe pathway. Clones with both positive PCR products wereselected for further validation. Restriction mapping with threedifferent restriction endonucleases was performed, and correctrecombinant clones were then sequenced junction sites withplasmid-derived primers to determine the accurate sequence atboth joints.

In Vitro CRISPR/Cas9-Mediated Editing of stu,Heterologous Expression, and Detection. For construc-tion of stuR in-frame deletion, digestion of pWHU2879 withinstuR was performed by Cas9 nuclease equipping with sgRNA-stuR-L and sgRNA-stuR-R. After in vitro cleavage, linearizedcosmid DNA was self-cyclized to generate a recombinantpathway harboring stuR deletion. Recombinant construct wasintroduced into S. avermitilis MA-4680 for heterologousproduction of Tu3010.Isolation of Tu3010 was performed by extracting acidified

fermentation broth (pH 3.0) with two volumes of ethyl acetateand evaporating the organic layer to dryness under reducedpressure. The crude extract was dissolved in methanol for LC-ESI-HRMS analysis on LTQ XL Orbitrap (Thermo Scientific)coupled with HPLC system (Thermo Scientific) in a positiveion mode. Detection was performed as follows: equip a C18column (Phenomenex, 4.6 × 250 mm, 5 μm) and equilibrate itwith 20 mM ammonium acetate (A) and Acetonitrile (B)under a developed program (0−16 min, 95−20% A; 16−18min, 20−95% A; 18−20 min, 95% A) in a flow rate of 1 mL/min, and monitor UV absorption at 238 and 303 nm.

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■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acssyn-bio.9b00248.

The list of bacterial strains and plasmids; the list ofprimers; screening of recombinant vectors housing stugene cluster; confirmation of the recombinant constructsharboring stu gene cluster; targeted cloning of sis genecluster; screening of recombinant vectors housing sisgene cluster (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: yhsun@whu.edu.cn.*E-mail: taoweixin@whu.edu.cn.ORCIDWeixin Tao: 0000-0003-4122-9816Yuhui Sun: 0000-0002-9258-2639Author ContributionsW.T. and Y.S. designed the experiments. W.T., L.C., C.Z., andJ.W. performed the experiments. W.T, D.Y., Z.D., and Y.S.analyzed the data. W.T. and Y.S. wrote the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by National Key R&D Program ofChina (2018YFA0903203) and the National Natural ScienceFoundation of China (31800111).

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ACS Synthetic Biology Letter

DOI: 10.1021/acssynbio.9b00248ACS Synth. Biol. 2019, 8, 1991−1997

1997