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Letter

Direct Pathway Cloning (DiPaC) combined with Sequenceand Ligation-Independent Cloning (SLIC) for fast Biosynthetic Gene Cluster Refactoring and Heterologous Expression Paul D'Agostino, and Tobias A. M. Gulder ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00151 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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ACS Synthetic Biology

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Direct Pathway Cloning (DiPaC) combined with Sequence- and

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Ligation-Independent Cloning (SLIC) for fast Biosynthetic Gene

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Cluster Refactoring and Heterologous Expression

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Paul M. D’Agostino and Tobias A. M. Gulder*

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Biosystems Chemistry, Department of Chemistry and Center for Integrated Protein Science Munich

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(CIPSM), Technical University of Munich, Lichtenbergstraße 4, 85748 Garching bei München,

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Germany.

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Keywords: Synthetic biology, Direct Pathway Cloning (DiPaC), Sequence- and ligation-independent

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cloning (SLIC), Hapalosin, Heterologous Expression

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Abstract

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The need of new pharmacological lead structures, especially against drug-resistances, has led to a surge in natural

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product research and discovery. New biosynthetic gene cluster capturing methods to efficiently clone and

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heterologously express natural products have thus been developed. Direct Pathway Cloning (DiPaC) is an

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emerging synthetic biology strategy that utilises long-amplification PCR and HiFi DNA assembly for the capture

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and expression of natural product biosynthetic gene clusters. Here, we have further streamlined DiPaC by

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reducing cloning time and reagent costs by utilising T4 DNA polymerase (sequence- and ligation-independent

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cloning) for gene cluster capture. As a proof of principle, the majority of the cyanobacterial hapalosin gene

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cluster was cloned as a single piece (23 kb PCR product) using this approach, and predicted transcriptional

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terminators were removed by simultaneous pathway refactoring, leading to successful heterologous expression.

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The complementation of DiPaC with SLIC depicts a time and cost-efficient method for simple capture and

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expression of new natural product pathways.


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Microbial natural products are renowned for their bioactivity and structural complexity, making their discovery

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highly significant. For example, approximately 49% of anti-infective compounds and 61% of anti-cancer

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pharmaceutical agents currently in clinical use are natural products or their derivatives.1 The imminent rise of

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antibiotic resistance has further spurred the scientific community to identify more natural products and their

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corresponding biosynthetic gene clusters (BGCs). Aided by the rise of whole-genome sequencing,2 this revealed

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that many organisms encode more BGCs then there are yet discovered natural products.3 A range of bioinformatic

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tools, such as AntiSMASH4 and PRISM,5 have been developed for the in silico identification of BGCs within

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whole-genome sequences.6 Bioinformatic investigations of hundreds of (meta-)genomes across many genera have

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provided thousands of BGCs without a known corresponding natural product.7, 8

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In an attempt to activate BGCs, two broad methodologies which involve either untargeted altering of the physical

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bacterial culture environment (e.g. different medium9) or synthetic biology can be employed.10 Synthetic biology

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uses molecular engineering techniques to manipulate natural product pathways and can subsequently be coupled

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with heterologous expression.11 To experimentally validate the ever-increasing database of BGCs, a range of in

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vitro and in vivo methods for cloning and BGC capturing have been developed. Traditionally, the most common

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method has been the capture of large-insert genomic libraries via the packaging of partially digested genomic

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DNA into cosmid-, fosmid- or BAC-based (bacterial artificial chromosomes) libraries, which still has the

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advantage of not requiring genome sequence data. Additional in vivo methods require known sequence data and

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are based on Rec/ET recombineering,12 such as linear-linear-(LLHR)13 and linear-circular-homologous

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recombination (LCHR),14 exonuclease combined with RecET recombination (ExoCET),15 and Cas9-assisted

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targeting of chromosome segments (CATCH).16 The natural recombination capability of the yeast Saccharomyces

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cerevisiae has also been utilized for the capture of BGCs via transformation-associated recombination (TAR)

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cloning.17-19

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conditions and include circular polymerase extension cloning (CPEC),20 assembly of fragment ends after PCR

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(AFEAP),21 and sequence- and ligation-independent cloning (SLIC),22 amongst others.23 Whilst many of these

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methods have revolutionised natural products research, cloning and expression can still take an extended length

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of time due to the high amounts of genomic DNA and/or multiple steps required for successful in vivo vector

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construction and activation. Direct Pathway Cloning (DiPaC),23 used for cloning small- to mid-size BGCs, helps

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overcome many of these issues via the utilisation of long-amplicon PCR. This considerably decreases the time

Alternatively, in vitro methods commonly utilize specialized primer design and PCR cycling


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required for vector and insert construction whilst simultaneously allowing for cluster refactoring and free vector

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backbone choice during both the cloning and expression stage.

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Cyanobacteria are renowned for their ability to be impervious to genetic manipulation, yet they are prolific

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producers of bioactive natural products. One such natural product, hapalosin, is a small depsipeptide encoded by a

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hybrid non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) within the cyanobacteria

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Fischerella, Hapalosiphon and Westiella intricata.24, 25 Interest in hapalosin is due to its ability to reverse and

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overcome P-glycoprotein mediated multiple drug resistance making it an effective agent to improve the success

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of chemotherapy,24 thus making it an attractive target for total synthesis and activity based assays.26,

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hapalosin biosynthetic gene cluster (hap) spans almost 25.7kb in length and is encoded by the five genes hapA-

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hapE.25 However, there are inconsistencies regarding the true ORF of hapD, with two different translation start

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sites annotated amongst various hapalosin producing cyanobacteria. Biosynthetically, hapalosin utilises a rare

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adenylation-ketoreductase (A-KR) didomain responsible for incorporation of the non-amino acid 2-oxoisovaleric

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acid (Figure S1). Issues with genetic manipulation of cyanobacteria, slow growth times and inconsistent ORF

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annotation of hapD made the hap cluster a prime candidate for DiPaC and pathway refactoring. Through this

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study, we aimed to utilise the hap gene cluster as a proof of principle to further streamline DiPaC by replacing the

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HiFi DNA assembly with SLIC while still retaining high cloning efficiency. Further, we planned to show the

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applicability of DiPaC to refactor pathways to shed light on the true ORF of hapD.

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The

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Results and Discussion

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Cloning strategy and DiPaC of the hapalosin biosynthetic gene cluster

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The hap BGC was identified in the sequenced genome of Fischerella sp. PCC 9431 (NCBI accession:

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NZ_ALVX00000000)28 by utilising H. welwitschii HapA (JGI accession: 2529292566)25 as a query sequence.

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Both BGCs retained identical gene synteny and 100% nucleotide sequence similarity. The first step in the cloning

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strategy was to identify putative terminators within the hap gene cluster utilising the ARNold tool.29 A putative

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rho-independent transcriptional terminator was identified within the intergenic sequence directly downstream of

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hapA (Figure 1). Therefore, the cloning strategy included the removal of the 470bp intergenic region between

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hapA and hapB for the excision of the putative terminator. This involved cloning hapA as a single gene

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downstream of the PtetO promoter and within range of the promoter shine-dalgarno sequence, to generate the

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intermediate plasmid pET28b-ptetO::hapA. To further streamline time and cost efficiency of DiPaC, we

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envisioned to replace HiFi DNA assembly by SLIC for integration of the entire hapBCDE DNA fragment into the

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pET28b-ptet::hapA intermediate vector. SLIC has commonly been utilised for the ligation-independent cloning of

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single genes in enzyme overexpression experiments. To obtain homologous overhang sequences available for

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annealing of the insert of choice, SLIC utilises the 3ʹ→ 5ʹ exonuclease activity of T4 polymerase.22 By

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incorporating terminal homologous sequence via primers, the DNA anneals and stitching of the plasmid occurs in

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vivo by E. coli post transformation.22 Importantly, positioning of the primer target sequence provides flexibility in

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how the expression vector is generated. In general, we found amplification was more pronounced if homology

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sequences were placed on primers amplifying the smaller linear fragment. Accordingly, the entire native 23kb

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NRPS/PKS consisting of hapBCDE was successfully amplified by PCR using insert-specific primers as a clean

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and single product (Figure S2). After purification and concentration, the hapBCDE PCR product was integrated

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into the PCR amplified pET28b-ptetO::hapA vector backbone – equipped with 26-30 bp homology sequences

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introduced during PCR amplification – via SLIC to generate the expression vector pET28b-ptetO::hap.

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Preliminary clones were initially screened by colony PCR, where all 11 selected clones appeared to harbour the

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correct insert (Figure S3A). Successful cloning of the pET28b-ptetO::hap expression vector was then confirmed

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by restriction digest (Figure S4) and terminal insert sequencing. SLIC mediated DiPaC was therefore found to be

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extremely efficient at cloning DNA fragments up to 23kb, requiring only a 2.5 min incubation time compared to ACS Paragon Plus Environment

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the 60 min incubation time often used for HiFi DNA assembly. Consequently, SLIC is at least as efficient as HiFi DNA assembly while vastly quicker and more economical. To investigate the true ORF of hapD, a second expression vector was then generated, where the hap pathway was further refactored to remove a 316 bp intergenic region between hapC and hapD (Figure S5). Primers amplifying the pET28b-ptetO::hapA backbone were designed to include 25-30 bp homology sequences for the successful capture of hapB-hapC via SLIC. The pET28b-ptetO::refhapAC vector was then linearized and hapD-hapE inserted via SLIC to create pET28b-ptetO::refhapAC-hapDE. Thus, the expression plasmids pET28b-ptetO::hap and pET28b-ptetO::refhapAC-hapDE are identical with the exception of a 316 bp intergenic region between hapC and hapD. Colony PCR confirmed the removal of the 316 bp hapC-hapD intergenic region with a total of eight out of ten positive clones (Figure S3B). The final pET28b-ptetO::refhapAC-hapDE was confirmed by restriction digest and sequencing of the hapC-hapD insertion site. The DiPaC strategy utilizes long-amplicon PCR products and in vitro DNA hybridization. Here, we successfully ligated a 23kb PCR product within an 8kb vector, the largest cloned linear DNA fragments via SLIC to date. We found several factors vital for successful amplification and DiPaC of the hap gene cluster. Pure high molecular weight and stable gDNA was vital for successful amplification, with the observable loss of amplification after 2 months of gDNA storage at -20°C. Further, the successful cloning was dependent on the total absence of UV light exposure to the large DNA fragments, where UV exposure resulted in a complete loss of cloning efficiency. By basing our homology arm primer design as previously described by Greunke et al.,23 we found these resulted in compatible homology arms that could be used for both SLIC and HiFi DNA assembly. For future DiPaC cloning, we recommend utilizing SLIC cloning for the first attempt followed by HiFi DNA assembly if SLIC is unsuccessful, as the most economical DiPaC strategy. Overall, the factors described above were essential for the efficient cloning of the hap cluster.


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Heterologous expression of the hapalosin gene cluster The fully constructed pET28b-ptetO::hap and pET28b-ptetO::refhapAC-hapDE vectors were transformed into E. coli BAP1. The E. coli BAP1 strain is a genetically engineered derivative of E. coli BL21 (DE3) with the integration of the phosphopantetheinyl transferase sfp into the expression host genome,30 thereby facilitating the activation of NRPS/PKS pathways for heterologous expression.23, 31 Transcription of the expression plasmid was placed under the control of the PtetO promoter, which has been successful in activating a range of cyanobacterial natural product pathways.23,

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Hapalosin was identified in expression cultures harbouring the pET28b-

ptetO::hap expression vector but could not be identified within pET28b-ptetO::refhapAC-hapDE extracts (Figure 2). LCMS and HR-LCMS identified hapalosin at a RT of 7.9 min and m/z 490.3160 [M+H]+ in both pellet and supernatant fractions which was consistent with extracts of Fischerella sp. 9431 as a positive control (Figure S6 and Figure S7). These results confirm that the putative hap cluster is truly responsible for hapalosin biosynthesis, as predicted by Micallef et al.25 Comparison of supernatant samples indicate E. coli BAP1 heterologous expression cultures produce approximately 45% of hapalosin compared to the Fischerella sp. PCC 9431 native producer (Figure S8). Considering the very long growth times of Fischerella sp. PCC 9431 (30-40 days) compared to E. coli (5 day expression), this is a significant improvement towards production of hapalosin. Overall, the PtetO promoter and E. coli BAP1 expression system is proving to be efficient for the investigation of small to mid-sized cyanobacterial natural product pathways. The ORF of hapD within the genomes of Fischerella sp. PCC 9431, Hapalosiphon sp. MRB220, Hapalosiphon welwitschii IC-52-3 and Westiella intricata HT-29-1 begins with a TTG start codon and encodes a 1526aa protein. Alternatively, the ORF of hapD within H. welwitschii UTEX B 1830 is annotated to begin 181 bp downstream using the start codon ATG, thus encoding a 1466aa protein (Figure S5). Importantly, the Fischerella sp. PCC 9431 and H. welwitschii UTEX B 1830 hap gene clusters have a 100% nucleotide sequence similarity. The ability of DiPaC to efficiently refactor biosynthetic gene clusters via the selected integration of genetic blocks allowed for a simple platform to investigate the true ORF of hapD. We performed heterologous expression experiments using both full length (TTG start codon) and reduced length hapD (ATG start codon). From these expression cultures, hapalosin was only identified within full length hapD constructs (Figure 2). These results indicate the 60aa N-terminal region of HapD is essential for biosynthesis and therefore, TTG is the true start ACS Paragon Plus Environment

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codon of hapD. Cyanobacterial ORFs are predicted using bioinformatics, thus, they are prone to false positive identifications. Improvements to annotation software are decreasing the rate of false positive identification, but bioinformatics need to be further supported by experimentally validated data, particularly with the underrepresented bias of TTG start codons.34, 35

Conclusion DiPaC is proven to be an efficient method for the fast cloning of small- to medium-sized biosynthetic gene clusters.22 Here, we have utilised SLIC in combination with long-amplicon PCR to further streamline DiPaC by improvement of cloning time and reducing cost whilst retaining a high level of cloning efficiency. We expect these improvements to make the capturing of silent/orphan gene clusters more accessible to the research community. The ability to refactor the hap gene cluster during the cloning step was vital for the speed of cloning and proved a successful platform to investigate the true ORF of hapD. By providing a basis for the successful cloning of ever increasing DNA sizes and reducing cloning time and cost, the efficiency of DiPaC has been vastly improved. Cyanobacterial natural product pathways are prime candidates for DiPaC and refactoring due to the challenges towards the genetic manipulation of native producers and the difficulty in activating cyanobacterial native promoters in E. coli expression hosts.32, 33

Materials and Methods Bacterial strains, plasmids and genomic DNA extraction. Bacterial strains and plasmids generated in this study are listed in Table 1. Fischerella sp. PCC 9431 was obtained from the Collections des Cyanobactéries, Institut Pasteur, Paris, France. Fischerella sp. PCC 9431 was cultivated in BG-11 medium ([pH 8], Sigma-Aldrich, Germany) at room temperature under 24 hr light without shaking. E. coli strains were grown in LB medium supplemented with 50 µg/mL kanamycin. ACS Paragon Plus Environment

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The extraction of high molecular weight genomic DNA free of impurities was vital for long-amplicon PCR utilized in this study. Genomic DNA was extracted from cyanobacterial cultures using an optimized method adapted from D’Agostino et al.36 and Greunke et al.23 Briefly, extraction was performed as follows: fresh or frozen cell pellets were washed with 0.9% NaCl once and resuspended in 5 mL lysis buffer (25 mM EDTA, 0.3 M sucrose, 25 mM Tris-HCl [pH 7.5]). Extracted gDNA was stored in 0.1x TE buffer (1 mM Tris-HCl, 0.1 mM EDTA [pH8.0]) and stored in aliquots at -20°C until use.

Bioinformatic analysis and PCR For the development of cloning strategies and primer design, the Geneious37 software package (Version 8.1.9) and the NEBuilder assembly web tool (New England Biolabs; http://nebuilder.neb.com) were used. Maps of plasmids were constructed using the SnapGene software. The sequenced genomes of Fischerella sp. PCC 943128 was downloaded from the NCBI database using the accession number ALVX01000000. The putative hap gene cluster was analysed bioinformatically for the presence of transcriptional terminators using ARNold (http://rna.igmors.upsud.fr/toolbox/arnold).29 PCR used to generate linear fragments for cloning were performed in 20 µL reaction batch and long-amplicon cycling reactions consisted of: 1x Q5 reaction buffer, 200 µM deoxynucleotide triphosphates, 500 nM of forward and reverse primer, DNA template and 0.02 U/µL Q5 High-Fidelity DNA polymerase (NEB). Template DNA amounts were 15 ng for Fischerella sp. PCC9431 gDNA or 10 ng for plasmid DNA. Thermal cycling was performed in a Bio-Rad T100 Thermal Cycler and began with an initial denaturation cycle of 98°C for 2 min, followed by 30 cycles of DNA denaturation at 98°C for 20 s, primer annealing for 15 s, DNA amplification at 72°C for 30 s/kb amplified, and a final extension at 72°C for 5 min. The annealing temperature for specific primers was calculated using the NEB Tm Calculator tool (http://tmcalculator.neb.com) while the optimum annealing temperature for primers harbouring homologous sequences were experimentally determined using gradient PCR with annealing temperature 50-70°C. Screening of positive transformants was performed by colony PCR using Taq DNA polymerase (NEB). Reaction mixtures contained 1x Taq buffer (10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, pH 8.3 at 25 °C), 4% DMSO, 100 µM deoxynucleotide triphosphates, 500 nM of forward and reverse primer, DNA template (bacterial suspension in water) and Taq DNA polymerase (0.025 U/µL, NEB). ACS Paragon Plus Environment

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Cycling conditions were performed as previously described with the exception of a 5 min 98°C initial denaturation step. DiPaC strategy and construction of expression vector Linear fragments for all DiPaC steps were generated using PCR with all vectors listed in Table 1 and primers listed in Table S1. PCR products of any vector backbone was firstly treated by DpnI (NEB) to remove template plasmid that would act as transformation background. All linear DNA fragments used for cloning were further purified prior to cloning using the NEB Monarch Gel Purification Kit (NEB). An essential factor for the successful cloning of long DNA fragments was to ensure that agarose gel extracted DNA was not exposed to any UV light. Further, gel purified DNA was eluted in 10 µl to maximise possible concentration.

Firstly, primers were used to generate linear DNA fragments of the pET28b-ptetO vector and hapA insert (Table S1). The pET28b-ptetO vector is a derivative of pET28b where the T7 promoter has been exchanged for the tetracycline-inducible PtetO promoter. The purified linear fragments were used to generate pET28b-ptetO::hapA using HiFi DNA assembly as described by Greunke et al.23 Next, pET28b-ptetO::hapA was utilised as the vector backbone to create two expression vectors harbouring the hap gene cluster. The pET28b-ptetO::hapA backbone for cloning was generated by PCR. The first construct was generated with the PCR product spanning hapBCDE (23kb) to generate pET28b-ptetO::hap. The second construct utilised two rounds of cloning by firstly incorporating hapB-hapC (13kb) to generate pET28b-ptetO::refhapAC. This intermediate vector The pET28bptetO::hapAC (21kb) was amplified and utilised for the incorporation of hapD-hapE (10kb) to generate the expression vector pET28b-ptetO::refhapAC-hapDE. The pET28b-ptetO::hap and pET28b-ptetO:: refhapAChapDE vectors are identical with the exception that the 316 bp intergenic region between hapC and hapD has been deleted in the latter construct.

For the construction of the pET28b-ptetO::hap, pET28b-ptetO::hapAC and pET28b-ptetO::refhapAC-hapDE, the SLIC method was utilized as a cheaper and quicker alternative to HiFi DNA assembly. Concentrations of linear DNA fragments for assembly were calculated as described by Greunke et al.23 SLIC was performed by utilising 1x buffer 2.1 (NEB) and 0.5µl of T4 polymerase (NEB) in a 10 µL total reaction volume. The SLIC reaction mixture was incubated for 2.5 min at room temperature, followed by a 10 min incubation on ice. A total of 5 µl of ACS Paragon Plus Environment

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reaction mixture was transformed by heat shock into chemically competent E. coli DH5α. Positive clones for all constructs were initially screened by colony PCR followed by restriction digest analysis and terminal-end sanger sequencing. Heterologous expression of the hapalosin gene cluster Heterologous expression conditions were based on previously described experiments for the pET28b-ptetO plasmid with minor changes.23, 33 Briefly, prior to each heterologous expression experiment, E. coli BAP1 was chemically transformed with pET28b-ptetO (empty), pET28b-ptetO::hap or pET28b-ptetO::refhapAC-hapDE. A single colony was used to generate a 10 mL pre-expression culture grown in LB medium supplemented with 50 µg/mL kanamycin and incubated overnight at 37°C with shaking at 200 rpm. Pre-expression cultures were used to inoculate expression cultures (1% v/v) in 200 mL of LB or TB medium supplemented with 50 µg/mL kanamycin. Expression cultures were incubated at 30°C with shaking (200 rpm) until an OD600 of 0.4 (LB) or 0.8 (TB) was reached. Cultures were then cooled on ice for 30 min, induced with 0.5 µg/mL tetracycline and incubated for 5 days at 20 °C with shaking at 200 rpm. Flasks were covered in foil to reduce light-induced decomposition of tetracycline. Extraction of expression cultures and LCMS analysis Cyanobacterial or E. coli biomass was separated from growth medium by centrifugation at 10,000 g for 10 min. The supernatant was extracted in 1 vol of ethyl acetate and repeated three times followed by desiccation in vacuo at 40°C using a rotary evaporator. To extract cell biomass, dichloromethane was added to cell pellets and incubated in a sonicator bath for 30 mins. Extracted cell debris was removed via centrifugation (10,000 g for 10 min) and the solvent desiccation in vacuo at 40°C using a rotary evaporator. Desiccated extracts were dissolved in HPLC-grade methanol and filtered through a Millex-GP, syringe driven 0.22 µm PES membrane filter (Millipore, USA) prior to injection into LCMS systems. LCMS experiments were conducted on an UltiMate 3000 LC System coupled to a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific). The chromatographic HPLC separation was carried out on a Hypersil Gold aQ C18 column (150 × 2.1 mm, 3 µm particle size). Buffers consisted of water (A) and acetonitrile (B) as the eluents, both supplemented with 0.1% formic acid. Chromatographic separation was performed at 0.7 mL/min using a gradient as follows: 5% B at 0 min to 95% B by 8 mins followed by washing the column at 100% B for 2 minutes ACS Paragon Plus Environment

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and re-equilibration of the column at 5% B for 2 minutes prior to the next injection. HR-ESI-MS spectra were recorded with a Thermo LTQ-FT Ultra coupled with a Dionex UltiMate 3000 HPLC system. Separation was achieved using a C18 column with a 10 min gradient from 10% to 98% solvent B (solvent A = water + 0.1% FA, solvent B = 90% ACN, 10 % water, 0.1% FA). The mass spectrometer was operated in a positive mode, collecting full scans from m/z = 100 to m/z = 2000. Interpretation of all recorded MS data was performed using the Thermo Xcalibur Qual Browser 2.2 SP1.48 software.


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Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: List of nucleotides used for cloning and sequencing (Table S1), proposed hapalosin biosynthetic pathway (Figure S1), Amplification of hapBCDE from Fischerella sp. PCC 9431 gDNA (Figure S2), Colony PCR of pET28ptetO::hap and pET28-ptetO::refhapAC-hapDE E. coli DH5α transformants (Figure S3), Restriction digest confirmation of pET28-ptetO::hap expression vector (Figure S4), Organisation of the Fischerella sp. 9431 hap gene cluster (Figure S5), LCMS of expression extracts (Figure S6), High-resolution LCMS of hapalosin (Figure S7). Relative production of hapalosin in E. coli and Fischerella sp. PCC 9431 (Figure S8).

Author Information. Corresponding Author *Tobias A. M. Gulder E-mail: [email protected] Telephone number: +49-(0)89-289-13833

ORCID P. M. D’Agostino: 0000-0002-8323-5416 T. A. M. Gulder: 0000-0001-6013-3161

Postal Address Technische Universität München Department of Chemistry and Center for Integrated Protein Science Munich (CIPSM) Biosystems Chemistry Lichtenbergstraße 4 85748 Garching

Author Contributions P. M. D. performed all experiments. P. M. D. and T. A. M. G. designed the research project and wrote the manuscript.


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Acknowledgments We thank Catharina Seel (Biomimetic Catalysis, Prof. Dr. Tanja Gulder, , TUM) and Barbara Hofbauer (Chair of Organic Chemistry II, Prof. Dr. Stephan A. Sieber, TUM) for HR-LCMS analysis of hapalosin. We would like to thank Prof. Bang-Guo Wei and Dr. Chang-Mei Si (Fudan University, China) for providing a synthetic hapalosin standard. We also thank Anna Glöckle for reading and editing the manuscript. Funding: P.M.D thanks the TUM Foundation Fellowship and the Marie Skłodowska-Curie Actions Individual Fellowship(Project ID: 745435) for funding. We thank the DFG for generous financial support of the work in our laboratory (Emmy Noether program and Center for Integrated Protein Science Munich CIPSM).

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Table 1: bacterial strains and plasmids used in this study Strains

Description

Reference or Source

E. coli DH5α E. coli BAP1 Fischerella sp. PCC 9431

Host strain for cloning Heterologous expression strain Native producer of hapalosin

NEB

Plasmids

Description

pET28b-ptetO (6,029bp)

Tetracycline inducible expression plasmid, ColE1, KanR

pET28b-ptetO::hapA (8,156bp)

Tetracycline inducible expression plasmid, ColE1, This study KanR harbouring the first gene of the hap cluster, hapA

pET28b-ptetO::hap (31,163bp)

Built using pET28b-ptetO::hapA as the vector and This study hapBCDE single piece nucleotide insert.

pET28b-ptetO::refhapAC (20,969bp)

Built using pET28b-ptetO::hapA as the vector and This study hapB-hapC single piece nucleotide insert.

pET28b-ptetO::refhapAC-hapDE (30,847bp)

Constructed using pET28b-ptetO::refhapAC as the This study vector and hapD-hapE single piece nucleotide insert. This plasmid was refactored to completely remove all intergenic regions of the hap cluster

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Institut Pasteur

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Figure 1: Cloning strategy for the production of pET28b-ptetO::hap and pET28bptetO::refhapAC-hapDE. Bioinformatic analysis of the hap cluster identified a putative transcriptional terminator (red triangle) which was removed with the selective amplification and cloning of hapA (green) to produce pET28b-ptetO::hapA (1). The entire hapBCDE (blue) PCR fragment was amplified and incorporated into pET28b-ptetO::hapA via SLIC to produce pET28b-ptetO::hap (2). To shed light on the true ORF of hapD, a further refactored expression vector was generated by amplifying the ATG start site of hapBC (orange) and incorporated into the pET28b-ptetO::hapA to produce pET28bptetO::refhapAC (3). To complete the cluster, hapDE (red) was then incorporated into pET28bptetO::refhapAC to produce pET28b-ptetO::refhapAC-hapDE (4). The blue triangle indicates the site of excision of the hapC-hapD intergenic region.


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Figure 2: Structure of hapalosin and LCMS of heterologous expression extracts. A) Structure of hapalosin with calculated and observed high-resolution ion masses (Figure S7). HR-LCMS detection of hapalosin is presented in the supporting information. B) Heterologous expression extracts identified hapalosin solely within pET28b-ptetO::hap expression cultures at a RT of 7.9. Hapalosin could not be detected in fully refactored expression plasmid cultures (pET28b-ptetO::refhapAC-hapDE) and empty plasmid controls. Peak intensity is extracted ion mass of m/z 490. LCMS comparison to extracts of Fischerella sp. 9431 can be found in Figure S6.


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