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Assessment of Anabaena sp. strain PCC 7120 as a heterologous expression host for cyanobacterial natural products: production of lyngbyatoxin A. Patrick Videau, Kaitlyn Wells, Arun J Singh, William H Gerwick, and Benjamin Philmus ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00038 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 17, 2016

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ACS Synthetic Biology

Assessment of Anabaena sp. strain PCC 7120 as a heterologous expression host for cyanobacterial natural products: production of lyngbyatoxin A.

Patrick Videau1, Kaitlyn N. Wells2, Arun J. Singh1, William H. Gerwick3, Benjamin Philmus1*

1

Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331 2 Undergraduate Honors College, Oregon State University, Corvallis, OR 97331 3 Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093 *Correspondence: B. Philmus, Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, 203 Pharmacy Bldg., Corvallis, OR 97331. Email: [email protected]

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Abstract Cyanobacteria are well known producers of natural products of highly varied structure and biological properties. However, the long doubling times, difficulty in establishing genetic methods for marine cyanobacteria, and low compound titers have hindered research into the biosynthesis of their secondary metabolites. While a few attempts to heterologously express cyanobacterial natural products have occurred, the results have been of varied success. Here we report the first steps in developing the model freshwater cyanobacterium Anabaena sp. strain PCC 7120 (Anabaena 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that Anabaena 7120 can heterologously synthesize lyngbyatoxin A in yields comparable to the native producer, Moorea producens, and detail the design and use of replicative plasmids for compound production. We also demonstrate that Anabaena 7120 recognizes promoters from various biosynthetic gene clusters from both free-living and obligate symbiotic marine cyanobacteria. Through simple genetic manipulations, the titer of lyngbyatoxin A can be improved up to 13-fold. The development of Anabaena 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways.

Freshwater and marine cyanobacterial strains are prolific producers of natural products that display a wide array of bioactivities.1-4 Freshwater strains garner press coverage due to the production of compounds with toxicity to humans and other animals, among which are the microcystins, cylindrospermopsin, saxitoxin, and anatoxin-a.5 Screening efforts focused on marine cyanobacteria have yielded biologically active compounds that could serve as drug leads, including patellamide D (P-glycoprotein pump inhibitor),6 the jamaicamides (neurotoxins),7 curacin A (anti-tubulin),8 coibamide A (cytotoxin),9 and lyngbyatoxin A (protein kinase C activator).10 From the numerous compounds isolated to date from cyanobacteria, a derivative of one has been developed into an FDA approved drug, the dolastatin 10 analog monomethyl auristatin E (MMAE), as part of the antibody drug conjugate brentuximab vedotin.11 Despite the ability to isolate compounds in sufficient quantities for structural and initial biological characterization, several factors have impaired drug discovery efforts involving cyanobacterial compounds: (1) compound yields from field collections are typically insufficient to support biological trials (animal or human clinical trials) and require an alternate supply route, usually involving synthetic organic chemistry; (2) the producing cyanobacterium can be difficult to identify and isolate as an axenic strain; (3) cyanobacteria, particularly marine strains, are generally slow-growing with doubling times in the range of days and cultures can readily be overgrown by faster growing bacteria and fungi; (4) continuous culture in the laboratory can also result in strains that cease to produce the desired compound.12 Additionally, the cyanobacterial production strains are not usually amenable to genetic manipulation, which precludes strain engineering and synthetic biology approaches. To address and circumvent the problems outlined above, a general heterologous expression host is needed specifically for cyanobacterial natural products. To date, no such host has been described in the literature.


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Heterologous expression of five cyanobacterial natural products in bacterial hosts has met with varied success. The ribosomal peptide natural products (RiPPs) patellamides A and C13, 14 and the microviridins15 were produced in Escherichia coli from the native promoters. Although yields for the patellamides were low (approximately 20 µg/L),13 yields of the microviridin analogs ranged from 20 – 7,280 µg/L. In the latter case, multiple analogs, in addition to the expected products, were observed as a result of incorrect proteolytic processing of the leader peptide.15 The expression of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) natural products has also met with limited success. Attempts at producing barbamide A in Streptomyces venezuelae were unsuccessful; however, the closely related product 4-demethylbarbamide A was produced in low yield.16 Similarly, lyngbyatoxin A (LTXA) production in S. coelicolor A3(2) was unsuccessful, likely due to premature transcriptional termination within ltxA, the first gene in the biosynthetic gene cluster (BGC).17 Termination of the ltxA transcript was probably caused by the presence of intrinsic or rho-dependent terminators or by the appearance of rare codons, a potential result of the large difference in G+C content between S. coelicolor (~70% G+C) and the lyngbyatoxin producer, Moorea producens (~45% G+C). In another experiment, E. coli was used to produce LTXA and its precursors in good yield. However, promoter exchange was required for successful expression as the native cyanobacterial promoter(s) were not recognized by the heterologous host.18 These and other issues continue to hinder the use of E. coli or Streptomyces sp. as general hosts for the production of cyanobacterial compounds. Lyngbyatoxin A is a protein kinase C activator originally isolated from a Hawaiian strain of M. producens (formerly Lyngbya majuscula).10 The 11.3 kb BGC, identified using a fosmid library, was proposed to consist of four genes (ltxA-D).19 The biosynthesis of LTXA begins with the formation of N-methyl-L-valyl-L-tryptophanol (NMVT) through the action of a di-domain NRPS (LtxA). NMVT is released through a four-electron reduction of N-methyl-valyl-tryptophan thioester by the terminal reductase domain.20 NMVT is then cyclized through the action of LtxB, a cytochrome P450, to form indolactam V (ILV).21 Finally, LTXA is formed by prenylation of ILV by LtxC in the presence of geranyl diphosphate.17, 19 The fourth gene, ltxD, has been postulated to encode a protein that converts LTXA into lyngbyatoxin B and C (LTXB and LTXC, respectively); however, no in vitro data have been reported for this latter reaction (Figure 1). Anabaena sp. strain PCC 7120 (herein Anabaena 7120) is a filamentous freshwater cyanobacterial strain that has been used as a model organism to investigate cellular differentiation,22 hydrogen production,23 and the reduction of dinitrogen.24 Anabaena 7120 has a modest doubling time (14-15 h),25 is not known to produce bioactive metabolites of its own from NRPS/PKS clusters (B. Philmus and T.K. Hemscheidt, unpublished data), and is genetically tractable with established protocols for conjugation and genetic manipulation.26 Here we describe and validate Anabaena 7120 as a heterologous expression host by expressing the lyngbyatoxin A (ltx) gene cluster from the native M. producens promoter(s), and detail conditions and genetic methods to increase compound titer. We also present evidence that Anabaena 7120 is a suitable general host for cyanobacterial natural products because it recognizes promoters from a



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diverse set of marine cyanobacterial BGCs. We also use Anabaena 7120 to show that LtxD is not involved in the formation of LTXB and LTXC. Results and Discussion Anabaena 7120 has been utilized in research worldwide since the 1970s and is relatively simple and inexpensive to cultivate using BG-11 medium.27 The non-toxic nature, ease of culture, and genetic tractability make Anabaena 7120 a ready host for heterologous expression. As Anabaena 7120 is a freshwater cyanobacterium, its use as a heterologous expression host for marine cyanobacterial natural products requires a proof of concept study. To demonstrate the utility of Anabaena 7120 in this regard, an approach involving both heterologous expression of the lyngbyatoxin (ltx) BGC and promoter fusion assays was utilized. Heterologous expression of ltxA-C in Anabaena 7120 results in lyngbyatoxin A production. Most cyanobacterial natural products are produced by NRPS, PKS, or NRPS-PKS hybrids.28 NRPS and PKS enzymes are multi-modular proteins that assemble a wide variety of natural products. Each module contains individual domains that select [adenylation (NRPS), acyltransferase domains (PKS)], elongate [condensation (NRPS), ketosynthase domains (PKS)], and modify (methylation, ketoreductase, dehydratase, enoyl reductase domains) the building block molecule (amino acids and α-keto acids for NRPS, malonyl-coenzyme A and derivatives for PKS).29, 30 During assembly, the growing natural product is tethered to the enzyme complex through a thioester linkage derived from a phosphopantetheinyl arm attached to the thiolation domain.29, 31 This posttranslational modification of the enzyme occurs at a conserved serine residue and is mediated by 4’-phosphopantetheinyl transferases (4’-PPTase). Genome sequencing revealed that Anabaena 7120 encodes a single open reading frame (ORF) annotated as a 4’-PPTase (all5359/hetI)32 and classified as a HetI-type 4’-PPTase, similar to the well studied Sfp 4’-PPTase from Bacillus subtilis.31 Previous research has indicated that hetI is essential for Anabaena 7120 viability.33 To explore its substrate specificity, recombinant HetI was produced, purified from E. coli, and shown to be capable of modifying a diverse set of thiolation domains including BarA from M. producens34 and ApnA from Planktothrix agardhii CYA 126/835 (B. Philmus and T.K. Hemscheidt, unpublished data). These results indicated that HetI has broad specificity and would perform the required post-translational modification of thiolation domains during heterologous BGC expression in Anabaena 7120. To validate Anabaena 7120 as a suitable heterologous host, we chose to express the ltx BGC for the following reasons: (1) it is relatively small (11.3 kb) allowing for easier cloning; (2) its biosynthesis involves a bi-modular NRPS that requires modification by a 4’-PPTase; (3) LTXA was the focus of previous attempts at heterologous expression in both S. coelicolor17 and E. coli.18 The ltx BGC is proposed to produce lyngbyatoxins A, B, and C; we chose to work with ltxA-C initially to produce only lyngbyatoxin A (LTXA), thus simplifying the analytical challenges that might be associated with producing three compounds. The ltxA-C genes were cloned into pPJAV361, a replicative plasmid based on pAM50436 that confers spectinomycin and


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streptomycin resistance and replicates in E. coli and Anabaena 7120. The resulting construct, pPJAV500, was introduced into Anabaena 7120 utilizing triparental conjugation. Cultures of Anabaena 7120 containing either the empty vector (pPJAV361) or ltxA-C (pPJAV500) were grown on BG-11 (nitrate) agar plates for 28 days. The cells were extracted as described in the Supplemental Experimental, and the extract was analyzed by HPLC, LC-HRMS, and LC-MS/MS. A peak at 21 min was present only in extracts from cultures harboring ltxA-C (pPJAV500) and displayed UV absorption maxima identical to those of LTXA (Figure 2).10 This peak possessed a protonated ion of 438.3115 Da, identical to that calculated for LTXA (calc. 438.3115, 0 ppm error, Supplementary Figure 1). The biosynthetically produced molecule co-eluted with a synthetic standard of LTXA (a kind gift from N. K. Garg) and possessed an identical MS/MS fragmentation pattern (Supplementary Figure 2).18, 37 Isolation of the produced compound from large-scale culturing efforts followed by 1H NMR analysis confirmed it to be LTXA (Supplementary Figure 3, Supplementary Table 1).37 As with other cyanobacterial natural products, LTXA was isolated only from the cellular biomass and was not exported into the growth medium.38-40 Taken together, these data indicate that LTXA can be heterologously produced by Anabaena 7120 using native promoters. Construction of a TAR capable vector (pPJAV550) and cloning of a large (> 40 kb) insert using λ/Red recombination. PCR amplification and ligase-mediated cloning of large inserts can be a complicated and often unproductive approach because cloning efficiency is inversely related to insert size; this is problematic for bacterial BGCs as they typically range from 10-70 kb, and clusters over 100 kb have been reported.41, 42 Genetic manipulation of larger BGCs requires recombination-based methods including lambda red (λ/Red) recombination in E. coli43 and transformation-associated recombination (TAR) in Saccharomyces cerevisiae.44 To address this, we created a vector for the TAR-mediated capture of cyanobacterial BGCs (pPJAV550). As TAR requires selection in S. cerevisiae, the original vector (pPJAV361) was modified to contain a TRP selection marker and an origin of replication for plasmid maintenance in yeast, creating pPJAV550 (Supplementary Figure 4). TAR was then used to clone ltxA-C into pPJAV550 generating pPJAV633, which was introduced into Anabaena 7120 via conjugation and used as described below. Production of LTXA from Anabaena 7120 harboring pPJAV500 or pPJAV633 was not statistically different, demonstrating that the yeast element does not interfere with LTXA production (Table 2, line 3; Supplementary Tables 3 and 4). To demonstrate the ability of pPJAV550 to harbor large DNA fragments, a ~42 kb fragment of M. producens DNA was introduced (pPJAV654) and was found to be stably maintained in both E. coli and Anabaena 7120 (Supplementary Figure 5). These vectors allow the cloning of BGCs through the use of traditional PCR and ligation, λ/Red, or TAR mediated cloning, which facilitates the manipulation of larger BGCs from cyanobacteria (up to 100 kb) without using multi-step cloning methodologies. Anabaena 7120 can produce lyngbyatoxin A at levels comparable to Moorea producens. Because the question of isolated yield is of prime concern in any heterologous expression system, we determined yields of LTXA produced by Anabaena 7120 harboring ltxA-C on pPJAV500 under a variety of conditions. Using LC-DAD MS/MS



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analysis for quantitation, two standard curves were established to quantitate LTXA from 0.44-8.7 ng (1 pmoles-20 pmoles) and (8.7-875 ng (20 pmoles-2 nmoles). For the lower range (0.44-8.7 ng), we utilized a multiple reaction-monitoring (MRM) program by isolating the protonated molecular ion of LTXA at m/z 438.2 and observing the primary product ion at m/z 410.2 (Supplementary Figure 2). For the higher range (8.7-875 ng), we integrated the area under the UV curve at 300 + 2 nm. Using these standard curves, LTXA yield could be quantified over the large dynamic range present in our samples (Table 1, Figure 3, and Supplementary Table 2). A single extraction with ethyl acetate (see Supplemental Experimental) resulted in consistent extracted yields of LTXA with a standard deviation of less than 1% (Supplementary Table 4), while subsequent extractions with ethyl acetate increased the yield of isolated LTXA by less than 4% (data not shown). Addition of LTXA to extracts of Anabaena 7120 harboring the empty vector, both during the initial ethyl acetate extraction and to the redissolved extract just prior to quantification, were utilized to ensure that the quantification of LTXA was not underestimated due to signal suppression. We typically observed LTXA at a 95% or higher recovery of the spiked amounts (data not shown). These data demonstrate that our extraction and processing procedures are robust, accurate, and precise. Anabaena 7120 is usually grown in media containing 17.6 mM nitrate [BG-11 (nitrate)] or 6 mM ammonia [BG-11 (NH4)] as a nitrogen source and can differentiate specialized nitrogen-fixing heterocyst cells to grow diazotrophically in the absence of combined nitrogen. To optimize LTXA production on solid medium, Anabaena 7120 harboring pPJAV500 was grown on plates containing BG-11 medium supplemented with either nitrate or ammonia, and cultures were harvested every week for four weeks. When ltxA-C were expressed from the native promoter following three weeks of growth on BG11 (NH4), we observed maximal production at 174.9 ng of LTXA per mg of dry cell mass (Table 1, line 2). In contrast to the results from BG-11 (NH4) plates, growth on BG-11 (nitrate) plates yielded a lower maximal quantity of LTXA after four weeks of growth (39.8 ng LTXA per mg of dry cell mass, Supplementary Table 2, line 2). We obtained and extracted M. producens and note that the concentration of LTXA was 270.1 ± 10.9 ng LTXA per mg of dry cell mass, which is similar to levels observed from the original isolation (200 ng LTXA per mg of dry cell mass).10 These results indicate that Anabaena 7120 can recognize the promoter(s) within the ltxA-C gene cluster and efficiently transcribe and translate the biosynthetic genes. Moreover, the native 4’-PPTase HetI can modify LtxA to the holoenzyme, and the heterologous host is able to produce LTXA at levels comparable to the native producer when grown on BG-11 (NH4) plates. Cultures grown on BG-11 (NH4) produce a maximum amount of LTXA at three weeks; however, after four weeks a 2-fold reduction relative to the maximum LTXA yield was observed (Supplementary Table 2). This decrease in production may be attributable to the degradation of LTXA by Anabaena 7120 during extended growth on BG-11 (NH4) medium. We hypothesized that prolonged growth on BG-11 (NH4) medium results in nitrogen starvation, which could account for the degradation of LTXA as a nutrient scavenging mechanism. In support of this hypothesis, heterocyst formation was observed by light microscopy in cultures of Anabaena 7120 after three weeks of cultivation on BG-11 (NH4) and four weeks on BG-11 (nitrate). Heterocysts only differentiate in response to nitrogen starvation, which demonstrates the lack of combined


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nitrogen remaining in the media (Supplementary Figure 6). We also observed a large decrease in LTXA titer when strains were grown on solid BG-11 agar plates lacking a source of combined nitrogen (Table 1, Supplementary Table 5). Beaches in Honolulu, HI, on the south shore of Oahu where M. producens was originally collected, have been reported to have low amounts of total nitrogen ranging from 0.01 mg/L – 0.42 mg/L (0.71 µM – 30.0 µM) while a nearby stream has been reported to have similar concentrations of total nitrogen (0.01 mg/L – 0.56 mg/L; 0.71 µM – 40 µM).45 These nitrogen concentrations are far below those present in BG-11 media (17 mM). The slow growth rate of M. producens in low nitrogen conditions may allow accumulation of LTXA, while nitrogen limitation coupled with the faster growth of Anabaena 7120 in laboratory culture may result in LTXA degradation. To quantify the amount of LTXA produced in liquid media, we cultivated Anabaena 7120 with pPJAV500 in both BG-11 (nitrate) and BG-11 (NH4) media for two weeks and eight days, respectively (Table 1, line 2) as growth beyond these time points resulted in decreased culture viability. The amount of LTXA produced was lower from all liquid cultures than from cultivation on solid media (Table 2). To determine if decreased production in liquid media resulted from reduced gene expression, we created a plasmid-borne transcriptional fusion of the ltxA promoter region (containing 1 kb directly upstream of ltxA) to a chloramphenicol acetyltransferase (cat) gene (pPJAV562). Anabaena 7120 harboring pPJAV562 was grown in conditions identical to those used for LTXA production and assessed for chloramphenicol acetyltransferase (CAT) activity.46, 47 Determination of CAT activity revealed that levels of cat expression were not predictive of the amount of LTXA observed from a particular growth condition; liquid cultures displayed a higher level of CAT expression but decreased LTXA production compared to solid cultures, which yielded higher LTXA levels but lower CAT expression (Tables 1 and 2). Because promoter strength and transcript abundance/stability are separate levels of genetic regulation, levels of ltxA-C mRNA were assessed using RTPCR to investigate whether transcription of ltxA-C, mediated by PltxA, and/or mRNA stability was predictive of LTXA yields. Correlational analysis of the mRNA levels of any of the ltx genes was not predictive of the amount of LTXA observed as higher amounts of mRNA were found in liquid cultures, which produced low LTXA titers (r = 0.34, p = 0.74; Table 1, Figure 4). We note that mRNA levels most closely correlate to CAT activity suggesting that neither gene expression or mRNA stability are solely responsible for determining the yield of LTXA (r = 0.19, p = 0.85; Table 2, Figure 4). Based on these results, we posit that cultivation time plays a significant role in LTXA production. The reduced cultivation time in liquid media versus solid media (2 weeks vs. 4 weeks, BG-11 (nitrate), 1.1 weeks vs. 3 weeks, BG-11 (NH4)) and the reduced cultivation time in BG-11 (NH4) medium compared to BG-11 (nitrate) medium (1.1 weeks vs. 2 weeks) is likely the primary reason that lower yields of LTXA were achieved in liquid media (Table 1 and Supplementary Table 5). This indicates that conditions capable of extending the growth time in liquid media would enhance LTXA yield. Experiments are currently underway in our lab to fully understand the biological variables (e.g. light, medium components) controlling LTXA production. To this end, preliminary experiments in our lab have shown that the addition of fructose to Anabaena 7120 cultures extends the length of viable culture time in liquid media, which results in an increase in the yield of LTXA (data not shown).



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Nitrogen limitation effects lyngbyatoxin A production and/or accumulation. Because we observed significant changes in LTXA production on media containing different nitrogen sources (Table 1) and decreased LTXA yields during extended culture times on BG-11 (NH4) plates (Supplementary Table 2, line 1), we hypothesized that changes in nitrogen availability could alter LTXA production and accumulation. LTXA yields were compared between nitrogen rich, nitrogen limited, and diazotrophic culture conditions (Figure 3). Liquid cultures of Anabaena 7120 harboring pPJAV500 were inoculated into BG-11 (NH4) or BG-11 (nitrate) media and grown for four or seven days, respectively. At this time, the cultures were either (1) left alone in the original medium; (2) pelleted and resuspended in fresh medium; (3) pelleted and resuspended in BG-11 medium lacking combined nitrogen (diazotrophic). The cultures were then grown for an additional seven days [BG-11 (nitrate) cultures], or four days [BG-11 (NH4) and diazotrophic cultures], respectively. The cells were collected and LTXA was quantified (Figure 3). Replenishing nitrate repressed LTXA production while replenishing ammonia had lead to no effect on LTXA yield (Figure 3B, line 1, Supplementary Tables 6 and 8). Cells starved for nitrogen under diazotrophic growth conditions produced significantly less LTXA, in analogy to what was observed earlier during growth on agar plates lacking combined nitrogen (Table 1 and Figure 3, column 4). This decrease in LTXA yield is presumably through LTXA degradation triggered by nitrogen starvation as discussed above. Promoter exchange results in increased lyngbyatoxin A production. In an effort to increase the yield of LTXA, we exchanged the native promoter, PltxA, with the constitutive glnA (PglnA; pPJAV503),48 the copper-inducible petE (PpetE; pPJAV501),49 or nitrate-inducible nirA (PnirA; pPJAV502)50 Anabaena 7120 promoters. Following introduction of these constructs into Anabaena 7120, the resulting strains were cultivated under the conditions described above, extracts processed, and LTXA yield was quantified by LCMS/MS as above. As with the native promoter, LTXA production was far greater on solid medium than from liquid cultures presumably due to the increased growth times (Table 1, line 4-6). Though growth on solid BG-11 (NH4) medium resulted in an overall increase in LTXA production, the greatest increase in LTXA yield occurred by a maximum of 2.3-, 10.2-, and 13.2-fold from the PnirA, PpetE, and PglnA constructs on BG-11 (nitrate) plates, respectively, compared to the highest level of LTXA observed with the native promoter [BG-11 (NH4) plates]. Because the nirA promoter is repressed by ammonia,50 growth on BG-11 (NH4) media repressed the PnirA construct and resulted in low titers of LTXA as expected. Production was highest from the PglnA construct on solid BG-11 (nitrate) resulting in roughly 3.2 mg L-1 estimated from a plate volume of 40 mL (2307.2 ng LTXA per mg dried cell mass * 56.5 mg dried cell mass * 25 plates/L), which is lower than the 25.6 mg L-1 produced in E. coli using the Ptet promoter.18 Despite the lower total yield, expression of the ltx cluster in E. coli resulted in higher production of the NMVT and ILV precursor molecules than of LTXA. Complete conversion of the biosynthetic precursors to LTXA indicates that the Anabaena 7120 host is a more efficient producer of the desired final product, as neither NMVT nor ILV were observed in any of these experiments.


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To determine whether LTXA production correlated with expression from the different promoters employed, transcriptional fusions of the petE, nirA, and glnA promoters with cat were created, introduced into Anabaena 7120, and expression was assessed using the CAT assay described above. In nearly all conditions tested, CAT activity was highest for the glnA promoter fusion, followed by the petE and nirA promoters (Table 2, lines 2-5); this is the same trend of transcriptional levels found in previously published transcriptomic work on Anabaena 7120.51 The only exception was on BG-11 (nitrate) plates where PpetE had 1.7-fold higher CAT activity than PglnA. Additionally, CAT activity was typically elevated in cultures grown in liquid media compared to those grown on solid media. In almost all cases when expression from the petE, nirA, and glnA promoters was higher than from the native ltxA promoter, LTXA yield was also increased (Tables 1 and 2); the only exception occurring during cultivation on BG-11 (NH4) plates in which CAT activity was 2.5-fold higher and LTXA yield was 3.6-fold lower from PnirA than PltxA. Analysis of the relationship between promoter strength, as determined by CAT activity, and LTXA yield found statistically significant positive correlations among the four promoters tested within each media condition (r > 0.60, p < 0.05; Supplementary Table 8). When RT-PCR was used to assess ltxA-C expression from all four promoters tested as discussed for the native promoter above (Figure 4), statistically significant correlations between mRNA levels from PltxA and LTXA yields were observed within each media type (r > 0.64, p < 0.03; Supplementary Table 8); comparison between media types or growth conditions did not yield significant correlations. CAT activity and ltxA mRNA levels also displayed significant positive correlations in each condition tested (r > 0.89, p < 0.001) except during growth on BG-11 (nitrate) plates (r = 0.20, p = 0.54). Analysis of ltxB mRNA and CAT activity also displayed significant positive correlations within each media condition except from BG11 (nitrate) plates. It is likely that the similar correlations observed between ltxA and ltxB expression and CAT activity are the result of co-transcription of these two genes on an ~9 kb mRNA. Positive correlations between ltxB mRNA and LTXA production were generally observed but did not reach significance, which suggests that additional levels of regulation influence the abundance of ltxB mRNA and/or LTXA yield. These results indicate that, as discussed for PltxA (vide supra), transcription and/or mRNA stability is not solely responsible for regulating the production of LTXA but ltxA mRNA level is a strong predictor within each media type individually. And as noted earlier, we credit the reduced cultivation time in liquid media for the decreased LTXA yield compared to solid media. We also observed that ltxC expression was slightly downregulated during expression of the ltx gene cluster using heterologous promoters (Figure 4); negative correlations were observed between CAT activity and LTXA production compared to ltxC expression (Supplementary Table 8). Despite the decrease in gene expression, it is apparent that LtxC was produced in sufficient quantities to fully convert ILV to LTXA because no ILV was observed in any condition tested (data not shown). The difference in gene expression between ltxA/B and ltxC provides additional evidence for the presence of a promoter upstream of ltxC as proposed previously.17 Growth of the Anabaena 7120 strains was not generally hampered by LTXA production as evidenced by similar final dried cell masses originating from the individual



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culture conditions (Supplementary Tables 6 and 7) except during the expression of ltxA-C from the glnA promoter and in BG-11 (NH4) media. These final dried cell masses correlate with the final optical density achieved during a growth curve experiment, which also showed that Anabaena 7120 harboring the PglnA construct (pPJAV503) had a growth impairment (Supplementary Table 6 and 7, Supplementary Figure 7), yielding between 13.3-70.1% of the dried cell mass when compared to empty vector. We attribute this growth defect to the increased metabolic burden derived from expressing the cluster from a highly transcribed promoter. Despite the growth impairment, PglnA yielded the highest quantities of LTXA both in per mg dried cell mass and total amount. The final cell mass of all strains harboring the ltx genes was decreased in liquid BG-11 (NH4) media despite low LTXA production in this culture condition. We also note that the Anabaena 7120 harboring the PpetE construct (pPJAV501) was not viable on BG-11 (NH4) plates, we do not know the cause of this result. Work is currently underway to understand the complex relationship between promoter strength, gene expression, protein production, and compound production. Overall, using strong heterologous promoters resulted in increased gene expression levels that correlate with higher LTXA yields although media conditions and growth mode influenced LTXA production considerably. It is noteworthy that the native producer Moorea producens is a slow-growing benthic organism, a growth characteristic that may allow accumulation of the toxins. Previous studies involving the curacin A-producing strain M. producens 19L demonstrated that the yield per mg dry cell mass of curacin A was at a minimum during the culture’s fastest growth phase. After stationary phase was reached, the curacin A yield per mg dry cell mass increased until cell death occurred.52 It is possible that during the later weeks of growth on plates, Anabaena 7120 similarly reaches stationary phase, with growth slowing to allow LTXA accumulation. Anabaena 7120 recognizes a variety of promoters from marine cyanobacterial BGCs. To further validate Anabaena 7120 as a general heterologous host for cyanobacterial natural products, we explored its ability to recognize promoters derived from different cyanobacterial BGCs. To test the promiscuity of the Anabaena 7120 transcriptional machinery, we created transcriptional fusions of both green fluorescent protein (gfp) and cat reporter genes to the promoter regions upstream of ltxA, ltxD, barA, curA, jamA, and patA, which are found in the BGCs for lyngbyatoxin (ltx),19 barbamide A (barA),53 curacin A (curA),8 jamaicamide (jamA),7 and patellamide (patA),13 respectively. These clusters represent NRPS, PKS, NRPS/PKS hybrids and ribosomally produced (RiPP) natural products from free-living (M. producens) and symbiotic (Prochloron didemni) cyanobacteria. We used 1 kb upstream of ltxA, ltxD, barA, curA, jamA and 226 bp upstream of patA to create the reporter plasmids. Fusions of PltxA, PltxD, PcurA, and PpatA to gfp displayed fluorescence in all cells within Anabaena 7120 filaments during growth in the presence of combined nitrogen (Supplementary Figure 8). When the nitrogen source was removed from the growth medium, a condition that induces the formation of nitrogen-fixing heterocyst cells, fluorescence was evident in heterocysts as well (Supplementary Figure 9). Plasmids containing fusions with PbarA-gfp or PjamA-gfp were not stably maintained by Anabaena 7120, which we attribute to toxicity caused by overexpression of GFP. To demonstrate that PbarA and PjamA were recognized by


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Anabaena 7120, mosaic filaments were created.54, 55 A mosaic filament is a mutant strain in which a plasmid is introduced into the filament without antibiotic selection; this results in a small fraction of its cells harboring the plasmid. If the promoters are recognized by Anabaena 7120 under these conditions, individual cells or small clusters of cells within filaments fluoresce green. The introduction of both PbarA-gfp and PjamA-gfp into mosaic filaments resulted in clusters of green fluorescent cells (Supplementary Figure 10), demonstrating that the Anabaena 7120 transcriptional machinery recognizes all of the promoters chosen for in study. Following qualitative analysis that demonstrated each tested cyanobacterial promoter functioned in both cell types, the relative strength of each promoter was quantified using the in vitro CAT assay described above. Transcriptional fusions of the barA, curA, ltxA, ltxD, jamA, and patA promoters to cat were created, introduced into Anabaena 7120, and cultured in the same conditions utilized for LTXA production. Like the PjamA-gfp fusion, the PjamA-cat fusion was not maintained by Anabaena 7120 so CAT assays could not be conducted. The results from the five remaining promoter fusions demonstrated that the region upstream of ltxA was one of the weaker promoters, while the promoter regions upstream of curA and barA displayed far more cat expression: 364- and 470-fold higher on average, respectively (Table 2, lines 6-10). This suggests that other natural products could be obtained in greater yields during heterologous expression in Anabaena 7120. In general, transcription from all promoters was higher in liquid than on solid media, and was higher when the medium was supplemented with ammonia rather than nitrate; this is consistent with the trend of LTXA yield in the various media types using the native promoter. Comparison to the Anabaena 7120 petE and glnA promoters indicates that the barA and curA promoters are of comparable strength and may be useful for strong expression of desired genes during future heterologous expression studies. Thus, Anabaena 7120 is capable of recognizing promoters from a wide range of natural product BCGs (NRPS, PKS and RiPP) from both free-living and symbiotic cyanobacterial strains, suggesting it may be an ideal heterologous host. LtxD does not form lyngbyatoxins B and C. The roles of LtxA, -B and –C have been characterized by in vitro studies,17, 19, 21 but a role for LtxD has yet to be shown. During heterologous expression of ltxA-D in E. coli, NMVT, ILV, and LTXA were observed, but the oxidized derivatives LTXB and LTXC56 were not reported.18 We hypothesized that E. coli may not recognize a putative internal promoter present between ltxC and ltxD (vide infra), and therefore LtxD is not expressed in this heterotrophic bacterial system. Due to the fact that Anabaena 7120 is capable of recognizing promoters from M. producens including PltxD, we reasoned that our system would be ideal for examining the role of LtxD. To determine the role of LtxD, ltxA-D was heterologously expressed in Anabaena 7120. A replicative plasmid containing ltxA-D (pPJAV571), was created using λ/Red, introduced into Anabaena 7120, and grown under identical conditions to those used for LTXA production. LC-MS analysis of Anabaena 7120 extracts containing ltxA-D (pPJAV571), ltxA-C (pPJAV500), and an empty vector (pPJAV361) showed that LTXA was produced by both the ltxA-C- and ltxA-D-containing strains; however, no new peaks



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with an m/z consistent with LTXB/LTXC ([M+H]+ 454.3) were observed in the strain containing ltxA-D. We also noted that the amount of LTXA produced in the ltxA-Dcontaining cultures was higher than that from the ltxA-C-containing strains although not significantly different at p < 0.05 (Table 1, line 7, Supplementary Table 5). We posit that this change in yield may be caused by increased stability of the ltxA-D mRNA compared to the ltxA-C mRNA. The increase in LTXA yield further suggests that LTXB and LTXC were not formed, because their formation would result in a decreased amount of LTXA isolated from the ltxA-D-containing strain as compared to the ltxA-C-containing strain. The failure to produce LTXB and LTXC by the ltxA-D-containing strain could be explained by one of the following: (1) ltxD is not transcribed; (2) LtxD is not translated; (3) LtxD does not perform the reaction converting LTXA to LTXB/LTXC. To determine whether ltxD was transcribed, the 356 bp intergenic region between ltxC and ltxD was examined, and a putative promoter region was identified (Supplementary Figure 11). Transcriptional fusions to the cat gene were created with regions 1 kb upstream of the ltxA and ltxD genes to demonstrate gene expression. These plasmids, along with a negative control containing a promoterless cat gene, were introduced into Anabaena 7120 and cultured in the conditions used for LTXA production. Expression from the ltxA or ltxD promoters was then assayed using the CAT assay described above (Table 2, lines 13). We noted that CAT activity from expression by the ltxD promoter region was 35-fold greater on average than that from the ltxA promoter across all conditions. We then created a version of the plasmid-borne ltxA-D BGC in which ltxD contained a 3’-extension resulting in the production of LtxD-His6. Growth of Anabaena 7120 containing this construct under conditions that resulted in LTXA production displayed a large band, detected by Western blotting, which corresponded to a purified recombinant His6-LtxDHis6 standard produced in E. coli (Supplementary Figure 12). These results demonstrated that ltxD is efficiently transcribed and translated in Anabaena 7120 under conditions that produce LTXA. Because LtxD is present in Anabaena 7120 harboring ltxA-D, it is clear that LtxD did not catalyze the formation of LTXB or LTXC from LTXA. It is possible that LtxD requires a co-factor absent from both Anabaena 7120 and E. coli, because neither LTXB nor LTXC have been detected from heterologous expression of the lyngbyatoxin BGC.18 It is equally likely, however, that LtxD does not catalyze the proposed reaction. Using bioinformatics, we determined that LtxD contains an NADH Rossmann fold, similar to that found in FabG (a 3-ketoacyl acyl carrier protein reductase involved in fatty acid biosynthesis).57 BLAST analysis indicated that the closest homologs were NAD(P)Hdependent 3-oxo-acyl-ACP reductases from Streptomyces sp. with amino acid identities ranging from 55-60% for the top 10 hits (Supplementary Table 12). Because LTXB and LTXC were absent from Anabaena 7120 cultures (and E. coli cultures in previous work)18 despite expression of LtxD, we propose that LtxD does not catalyze this transformation. It is possible that LTXA undergoes an ene reaction with molecular oxygen to generate a hydroperoxide,58 which is subsequently reduced to LTXB or LTXC (Supplementary Figure 13). Interestingly, the teleocidin B producer S. blastmyceticus NRBC 12747 encodes a methyltransferase-cyclase tailoring protein, TleD, in a different chromosomal locus from the BGC encoding TleA, -B and –C (homologs of LtxA, -B, and –C, respectively). The similarity of the ltx and teleocidin B BGCs suggests that tleD


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was acquired separately from tleA-C and represents an interesting case of bacterial combinatorial biochemistry. Taken together, these data strongly suggest that, while LtxD is produced in the Anabaena 7120 host, it does not catalyze the formation of LTXB and C from LTXA and the function of LtxD, if any, in lyngbyatoxin biosynthesis is currently unknown. Moreover, these observations validate the use of Anabaena 7120 to investigate the function of unknown genes found in cyanobacterial BGCs. Conclusions. The work presented herein demonstrates that Anabaena 7120 is a suitable general heterologous host for the production of cyanobacterial natural products. We were able to produce LTXA from the BGC’s native promoters, utilize different nitrogen sources on solid and in liquid growth media to enhance compound production, and introduce heterologous promoters to greatly increase LTXA yield. Genetic tools were developed and implemented to introduce BGCs into replicative plasmids or for integration into the genome using three different cloning techniques (traditional ligation, λ/Red, and TAR). Furthermore, cyanobacterial promoters derived from five different BGCs were all recognized and efficiently expressed by the Anabaena 7120 transcriptional machinery. The Anabaena 7120 system overcomes most of the problems encountered in other host strains during heterologous expression of cyanobacterial BGCs, and these features will be necessary for expression of more complicated BGCs. Due to the large size of many BGCs, lambda-red recombination or TAR will be required for cloning. Similarly, the promiscuity of the Anabaena 7120 transcriptional machinery is ideal for efficient expression from the many promoters likely present in large BGCs. As the host is of cyanobacterial origin, it is likely that optimal expression levels of individual genes within large BCGs will be attained such that the desired final metabolite will be produced rather than a buildup of metabolic precursors. The implementation of this heterologous expression system will allow further probing and manipulation of cyanobacterial BGCs to investigate the biosynthetic roles of proteins with unknown functions and mechanisms, and will improve compound production, create stable compound sources for continued testing, and enable combinatorial biosynthesis. In addition, the photosynthetic nature of Anabaena 7120 allows for the heterologous expression of natural products in an ecologically sustainable and economical fashion. Experimental Section. Microbiological techniques. Specifics on the bacterial strains and growth conditions, genetic manipulations, plasmid and strain creations, microscopy, chloramphenicol acetyltransferase assays, protein purification, Western blot analysis, LTXA isolation and characterization are contained in the Supplemental Experimental section. Production, purification, and analysis of LTXA. Assays for lyngbyatoxin production were initiated by resuspending the desired Anabaena strains in BG-11 (nitrate) to an optical density at 750 nm (OD750) ~4.0 and spreading 200 µl onto the surface of BG-11 (nitrate)/ BG-11 (NH4) plates or inoculating 100 µl into 100 mL BG-11 (nitrate)/ BG-11 (NH4) liquid medium in 500 mL flasks. After the specified duration of incubation, plates were scraped with a sterilized flat spatula to remove the Anabaena filaments and the total



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biomass was then transferred into a 20 mL scintillation vial. Alternatively, liquid cultures were concentrated onto a GF/C glass microfiber filters (GE Healthcare, Pittsburgh, PA) via vacuum filtration. All Anabaena cultures were lyophilized, extracted overnight with ethyl acetate, and the extract dried to completeness in vacuo. Extracts were dissolved in methanol at 10 mg/mL, particulates removed by filtration through a 0.2 µm nylon syringe filter, and 10 µL injected for quantification by LC-MS/MS as described below. Quantification by LC-MS/MS. LC-MS/MS analyses were accomplished using a Shimadzu Prominence HPLC (consisting of a degasser, two LC-10AD HPLC pumps, an autosampler, a photodiode array and system controller, Columbia, MD) upstream of a 3200 QTrap mass spectrometer (AbSciex, Framingham, MA). Separation was achieved using a Luna C18(2) column (2.0 x 150 mm, 3 µm, Phenomenex, Torrance CA) with a flow rate of 0.2 mL/min with line A containing water + 0.1 % (vol/vol) formic acid, and line B containing acetonitrile + 0.1 % (vol/vol) formic acid, operated under the following program. The column was pre-equilibrated in 95% A/5% B and upon injection this composition was held for 1 min. The composition of mobile phase was then changed to 0% A/100% B over 29 min utilizing a linear gradient. This composition was held for 5 min followed by changing to 95% A/5% B over 3 min. The column was equilibrated in 95% A/5% B for 5 min prior to the next injection. Under these chromatographic conditions, LTXA eluted at 29 min. The mass spectrometer was operated with the following settings in positive polarity mode: MS (EMS mode, 50-1500 m/z), Curtain gas, 30.0; Collision gas, High; IonSpray voltage, 5500.0; Temperature, 400.0; Ion Source Gas 1, 30.0; Ion Source Gas 2, 30.0; Interface heater, ON; Declustering potential, 30.0; Entrance potential, 4.0; Collision energy, 10.0, number of scans to sum, 2; scan rate, 1000 Da/sec. MS/MS (MRM mode, Q1, 438.3; Q3, 410.2; time 40 msec) Curtain gas, 30.0; Collision gas, High; IonSpray voltage, 5500.0; Temperature, 400.0; Ion Source Gas 1, 30.0; Ion Source Gas 2, 30.0; Interface heater, ON; Declustering potential, 30.0; Entrance potential, 4.0; Collision energy, 45.0; Collision cell exit potential, 3.0;. The machine was operated with Analyst 1.5.1, Build 5218) and data analysis was performed with PeakView, ver. 2.1.0.11041 (AbSciex). Keywords Secondary metabolites, non-ribosomal peptide synthetase, promoter fusion Conflict of interest The authors declare no competing financial interests. Acknowledgements Fosmid fos-DE3-86 was provided by Lena Gerwick (University of California, San Diego) and Daniel Edwards (California State University, Chico). Sean M. Callahan (University of Hawaii) kindly supplied Anabaena sp. strain PCC 7120 strains and plasmids and critically read the manuscript. Thomas K. Hemscheidt (University of Hawaii) is thanked for allowing us to use unpublished data on HetI characterization. Stephen Atkinson (Oregon State University) is thanked for his aid in microscopy. Andrew M. Senner is acknowledged for initial attempts at lyngbyatoxin A purification. Synthetic lyngbyatoxin A was a kind gift from Neil K. Garg (University of California,


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Los Angeles). Vladimir Larionov (National Cancer Institute, NIH) provided S. cerevisiae strain VL6-48N. pET28-BarA was a kind gift of Christopher T. Walsh (Harvard Medical School). Samantha O’Hanlon-Videau is thanked for aid in statistical analysis. We would like to thank Philip Proteau (Oregon State University) and Lizbeth Videau (Duke University) for critical reading of the manuscript and helpful suggestions. Funding for this project was provided from the College of Pharmacy, Oregon State University and the Medical Research Foundation of Oregon (New Investigator Grant #1415).

+H

O-

3N

H N

O

L-Val

N H

LtxA

OH

N H

O-

3N

N H

N-Me-L-ValylL-Tryptophanol (NMVT)

O

L-Trp

OH

O

H N

+H

H N

N

LtxB

O

Indolactam V (ILV)

H N

N

OH

O N H H N

N

LtxC

Geranyl diphosphate

O

PP i

OH

OH

Lyngbyatoxin B (LTXB)

LtxD

N H

H N

N

OH

O

Lyngbyatoxin A (LTXA) HO

N H

Lyngbyatoxin C (LTXC)

Figure 1: Biochemical route for the formation of the lyngbyatoxins.



Intensity (254 nm, mAU)!

A!

H N

N

OH

O

!!!! !!

N H

Lyngbyatoxin A! [M+H]+ = 438.3115!

!!

Time (min)!

B!

C! Ion Count!

Intensity (mAU)!

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! Absorbance (nm)!

!! m/z (Da)!

2. Heterologous expression of ltxA-C from pPJAV500 in Anabaena 7120 yields lyngbyatoxin A. (A) HPLC chromatograms for the organic extracts from a strain containing ltxA-C (red) and an empty vector (blue); inset is an expansion of the putative lyngbyatoxin A peak. (B) UV spectrum of the peak at 21 min. (C) Mass spectrum of peak at 21 min (MS/MS collision energy 10V). Figure


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!

A! Cultures left in original media Grow

Grow

Extract cultures and quantify lyngbyatoxin A production

Media replaced with fresh media

B!

Anabaena 7120 containing ltxA-C Media replaced with BG-11 lacking combined nitrogen

Line

Growth condition

Combined Nitrogen

1 2

Nitrate Ammonia

15.9 ± 9 5 9.2 ± 6.2

Combined Nitrogen Replaced 4.6 ± 4.7 9.8 ± 2.3

Diazotrophic 2 ± 0.7 6.2 ± 1.7

Figure 3. Examining the effect of nitrogen limitation on lyngbyatoxin A production and accumulation. Schematic outline of experiment (A). Lyngbyatoxin A quantification results (B). The strain harboring pPJAV500 was grown in liquid cultures containing nitrate or ammonia as the sole nitrogen source for two weeks or eight days, respectively, in triplicate. Halfway through the experimental duration, three cultures were washed and fresh medium containing the same nitrogen source was added, three cultures were stepped down to medium lacking a combined nitrogen source (diazotrophic), and three cultures left growing unaltered. At the conclusion of the growth period, the cultures were individually filtered and lyophilized. The quantity of LTXA is presented as ng of compound per mg of dried cell mass ± the standard deviation.



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A

B

C

D

E

Figure 4. Expression of the ltxA-C genes controlled by the native or heterologous promoters during growth in the presence of ammonia (NH4; B, D) or nitrate (N+; C, E) in liquid (D, E) or on solid (B, C) medium. Cultures were grown in the same conditions used for LTXA quantification, total RNA was extracted, and RT-PCR was conduced with four different primer sets. Data was compared to expression of the 16S rRNA as a reference gene. Fold changes, shown as relative mRNA expression, were calculated based on comparison to expression of the ltxA-C genes from the native promoter. Expression of ltxA-C on either NH4 or N+ media in liquid or plate culture is assessed in panel A. In all other panels, ltxA-C expression controlled by the petE (checkered bars), nirA (horizontal lined bars), or glnA (vertical lined bars) promoters is compared to expression from the native promoter (unfilled bars).


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Table 1: Lyngbyatoxin A production from replicative plasmids containing the ltxA-C or ltxA-D genes expressed from the native or heterologous promoters in Anabaena 7120. The quantity of LTXA is presented as ng of compound per mg of dried cell mass ± the standard deviation. N/D, not determined. Line

Diazotrophy Platesc 0 8.4 ± 3.6 N/D

Ammonia Liquidc 0 9.0 ± 0.6 N/D

Nitrate Liquidd 0 17.1 ± 1.8 N/D

1787 ± 69.5 498 ± 170.5 404.8 ± 78.5 ± 6.6 149.1 6 PglnA-ltxA-C pPJAV503 379.9 ± 58.2 2307.2 ± 101.5 ± 20.5 163.9 7 PltxA-ltxA-D pPJAV571 238.2 ± 37.5 63.3 ± 11.4 5.5 ± 1.9 a , grown for 3 weeks; b, grown for 4 weeks; c, grown for 8 days; d, grown for 2 weeks

11.2 ± 2.6 1.2 ± 2.2

27.8 ± 8.9 33.6 ± 14.1

18.4 ± 1.2

197.2 ± 69.3 24.1 ± 2.2

1 2 3 4 5

Genes Introduced Empty vector PltxA-ltxA-C PltxA-ltxA-C with Yeast Element PpetE-ltxA-C PnirA-ltxA-C

Plasmid pPJAV361 pPJAV500 pPJAV633

Ammonia Platesa 0 174.9 ± 70.2 170.6 ± 14.2

pPJAV501 pPJAV502

Not Viable 48.4 ± 20.9

Nitrate Platesb 0 40.2 ± 11 43.6 ± 19.9


14.9 ± 2.6


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Table 2. Quantification of CAT activity from an in vitro assay. Anabaena 7120 harboring transcriptional fusions of various promoter regions to cat were grown under the same conditions used for LTXA production. CAT activity was assayed in triplicate and is presented as the activity in units, determined by comparison to a standard curve made with purified CAT, per mg of protein ± standard deviation. The plasmid containing PjamAcat was not viable in Anabaena 7120. Line

Plasmid

Promoter

1

pPJAV561

2 3 4 5 6 7 8 9 10

pPJAV560 pPJAV608 pPJAV609 pPJAV610 pPJAV562 pPJAV574 pPJAV576 pPJAV575 pPJAV578

Promoterless cat Pcat PpetE PnirA PglnA PltxA PltxD PcurA PbarA PpatA

Ammonia Plates

Nitrate Plates

Ammonia Liquid

Nitrate Liquid

0

0

0

0

1.30 ± 0.03 4.66 ± 0.02 0.15 ± 0.01 20.90 ± 0.58 0.06 ± 0.03 3.24 ± 0.05 24.10 ± 0.34 15.70 ± 0.26 0.13 ± 0.01

0.44 ± 0.01 16.70 ± 0.26 1.67 ± 0.14 9.82 ± 0.39 0.01 ± 0.01 0.21 ± 0.01 3.69 ± 0.04 6.03 ± 0.57 0.01 ± 0.01

16.97 ± 0.72 42.91 ± 1.89 0.11 ± 0.04 84.92 ± 1.46 0.26 ± 0.02 10.83 ± 0.11 91.80 ± 6.94 43.70 ± 4.42 0.55 ± 0.02

2.04 ± 0.02 9.72 ± 0.06 2.05 ± 0.03 10.90 ± 0.02 0.09 ± 0.01 2.59 ± 0.04 32.94 ± 4.82 79.91 ± 1.64 0.15 ± 0.11

TOC Graphic


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