Design of engineering experiments based on classical modular boundaries. The stambomycin PKS comprises 25 modules distributed among 9 polypeptides (Pks1 − 9)23 (Fig. 1a) (Note: throughout the text, the stambomycin genes have been numbered in accordance with ref. 23). To access abridged derivatives using the classical module boundaries, we reasoned that we could engineer novel intersubunit interfaces by suitable manipulation of docking domains. Encouragingly, the extreme C- and N-termini of all subunits (with the exception of the N-terminus of Pks1 and the C-terminus of Pks9) contain sequences with convincing homology to previously-identified DDs22,27 (the C-terminal DDs are referred to hereafter as CDDs and their partner N-terminal DDs as NDDs). By bioinformatics analysis, we were able to confidently assign the DDs acting at 6 of the 8 interfaces to the type ‘1a’ class22, and the remaining two sets of DDs as type ‘1b’27 (Supplementary Fig. 1). In both cases, docking occurs between an α-helical CDD and a coiled-coil formed by the NDD, with specificity achieved via strategically-placed charge:charge interactions at the complex interface (Supplementary Fig. 1)22,27.
Among the type 1a junctions, there were notably two which appeared compatible in terms of the translocated substrate: PKSs 3/4 + 7/8 and Pks 4/5 + 8/9 (Supplementary Fig. 2). Specifically, the functional groups at the critical α- and β- positions14,28 of the transferred chains are identical at these junctions, and correspondingly, the downstream KSs show similarities across several sequence motifs previously correlated with substrate specificity14,20,29 (Supplementary Fig. 2). Targeting such interfaces thus allowed us, at least in principle, to overcome the functional block to the engineered systems represented by poor recognition of the incoming substrate by the directly downstream KS domain21. Ultimately, we targeted a new interface between Pks subunits 4 and 9 for two principle reasons. Firstly, Pks4 is at the origin of the structural variation between the stambomycin family members, and thus we anticipated that maintaining the subunit within the hybrid system would give rise to a corresponding series of truncated analogues, providing important evidence for their identity. Secondly, it was genetically more practical to modify the second set of interfaces due to splitting of the PKS subunits between two loci (Fig. 1).
To establish the novel junction, we initially modified the CDD of Pks4 (CDD4) to match that of Pks8 (the natural partner of the NDD of Pks9 (NDD9)), either by site-directed mutagenesis of residues previously identified as key mediators of interaction specificity (construct CDD4 SDM; Supplementary Fig. 3 and Supplementary Table 1)22, or by exchange of the complete CDD docking α-helix of CDD4 for that of CDD8 (construct CDD4 helix swap; Supplementary Fig. 3 and Supplementary Table 1)30. Modifying the CDD4 specificity ‘code’ to match that of CDD8 required mutation of 3 residues, while for the CDD4 helix swap, the terminal 16 amino acids of CDD4 were exchanged for the corresponding 15 residues of CDD8 (Supplementary Fig. 3 and Supplementary Table 1). The genetic alterations were carried out in two distinct PKS contexts: (i) in the presence of the intervening subunits 5 − 8, which allowed for the possibility of competitive interactions between modified Pks4 and both Pks5 and Pks9; and ii) removing the intervening subunits 5 − 8, thus eliminating competition for binding of Pks4 by Pks5, and of Pks9 by Pks8 (Supplementary Fig. 3). We further generated a mutant in which Pks subunits 5 − 8 were deleted but no modification was made to CDD4, in order to judge the intrinsic capacity of Pks4 and Pks9 to interact. Furthermore, genetic engineering was carried out in parallel by both PCR-targeting31 and CRISPR-Cas932, in order to directly compare the efficacy of these two approaches, as well as evaluate the effect of the short scar sequence remaining in the chromosome following PCR-targeting.
Engineering the stambomycin PKS based on the classical module definition. The CDD4 SDM and CDD4 helix swap sequences were introduced in parallel into the S. ambofaciens genome using PCR targeting and CRISPR-Cas9 (full experimental details are provided in the Supplementary Methods). As discussed previously, the modifications were made both in the presence of the intervening subunits Pks5 − 8 and in their absence (Supplementary Fig. 3). As previous work has shown that production from the stambomycin biosynthetic gene cluster requires activation by constitutive overexpression of a pathway-specific LAL (Large ATP-binding regulators of the LuxR family) regulator23, we additionally introduced the LAL overexpression plasmid (pOE484) into each of the mutants, using the empty parental plasmid (pIB13933) as a control. In total, this strategy resulted in 20 targeted strains harboring interface mutants (where K7N refers to PCR targeting and CPN to CRISPR-Cas9 engineering): K7N1/pIB139, K7N1/OE484, K7N2/pIB139, K7N2/OE484, K7N3/pIB139, K7N3/OE484, K7N4/pIB139, K7N4/OE484, K7N5/pIB139, K7N5/OE484, K7N6/pIB139, K7N6/OE484, CPN1/pIB139, CPN1/OE484, CPN2/pIB139, CPN2/OE484, CPN4/pIB139, CPN4/OE484, CPN5/pIB139, CPN5/OE484 (Table 1, Supplementary Tables 2 − 4; despite extensive efforts the CPN3 mutant strain was not obtained). The principal difference between the K and CPN series of constructs is the presence of a 33 bp ‘scar’ sequence between the modified pks4 and pks9 genes (Supplementary Fig. 4). Construct K7N6 was assembled specifically to test the effect of this region, without any further modification to CDD4 and the intervening pks5 − pks8 genes.
With the exception of K7N3, CPN4 and CPN5, extracts of the engineered mutant strains harboring pOE484 were analyzed by high performance liquid chromatography heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) on a Dionex UItiMate 3000 HPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, and compared to extracts of the control strain containing pIB13933 as well as the wild type S. ambofaciens containing pOE484, using SIEVE 2.0 screening software. K7N3, CPN4 and CPN5 were analyzed subsequently, and the data inspected manually. Yield quantification was carried out with reference to a calibration curve generated with purified stambomycins A/B 1 (the limit of detection was found to be between 10 and 1 µg L− 1, and so any yields < 10 µg L− 1 must be considered an estimate). Novel metabolites not present in the control strains, and for which we obtained reliable exact masses, are listed in Table 1 and Supplementary Fig. 5.
The first notable result is that the K7N6/OE484 mutant yielded a similar metabolic profile to S. ambofaciens wt (22 ± 3 mg L− 1, 73% relative yield (Supplementary Table 5)), showing that the scar sequence negatively impacted stambomycin production, but not dramatically (Fig. 3). By contrast, no stambomycins were observed, as anticipated, in all constructs in which Pks5 − Pks8 had been removed (K7N1 − 3; CPN1,2) (Fig. 3). Stambomycins 1 were present, however, in strains K7N4 and CPN4 harboring CDD4 site-directed mutations and in the CDD4 helix swap strain CPN5, all of which still contained Pks5 − Pks8, albeit at reduced amounts relative to the wild type (18%, 23% and 14% of wt, respectively) (Fig. 3 and Supplementary Table 5). (Surprisingly, the metabolic profile of K7N5 reproducibly differed from that of CPN5, as no stambomycin-related metabolites were detected (Fig. 3)). These data suggested that while the mutations introduced into CDD4 reduced interaction with NDD5, they were not sufficient to disrupt natural chain transfer between Pks4 and Pks5, arguing that DD engineering to alter partner choice should be accompanied by removal of competing intersubunit interactions.
We did not find any evidence in the DD engineering experiments for any of the target 37-membered metabolites (Supplementary Figs. 3 and 5). However, all strains in which stambomycin production was abolished (Table 1) exhibited four new peaks in common (Fig. 3b and Supplementary Fig. 5) (other peaks corresponding to potentially novel compounds were observed, but none were shared between multiple strains). The determined exact masses and mass spectra (as exemplified by strain CPN2/OE484, Fig. 3b) correspond to truncated derivatives of stambomycins A/B and C/D respectively, following premature release from modules 13 and 12 of Pks4 (compounds 2 − 5, Fig. 3d and Supplementary Fig. 5; ca. 8-fold greater yield of the module 13 products (Supplementary Table 5)). Further support for the identity of these shunt compounds was obtained by grafting the chain-terminating (type I) thioesterase (TE) domain from the C-terminal end of Pks9 to the C-terminus of Pks4 in order to force chain release at this stage. Indeed, identical compounds were produced, but at 17-fold increased yield relative to CPN2/OE484, consistent with active off-loading of the chains (Fig. 3c, Supplementary Fig. 6 and Supplementary Table 5).
Based on their masses, both sets of shunt metabolites were hydroxylated on a single carbon, while none were found to bear the β-mycaminose of the mature stambomycins, consistent with the absence of the tetrahydropyran moiety to which it is normally tethered. To determine the location of the hydroxylation and therefore the hydroxylase responsible, we inactivated in mutant CPN2/OE484 the genes samR0478 and samR0479 encoding respectively, the stambomycin C-28 and C-50 cytochrome P450 hydroxylases34. While extracts of CPN2/OE484/Δ478 were unchanged relative to CPN2/OE484 (i.e. the hydroxyl group was still present), the CPN2/OE484/Δ479 mutant exhibited four new peaks with masses corresponding to the dehydroxylated shunt products (Fig. 3, Supplementary Fig. 7 (compounds 6 − 9) and Supplementary Table 5). Taken together, these data show that the unusual on-line modification catalyzed by SamR047934 which is necessary for macrocyclization, occurs prior to chain extension by Pks5. While SamR0478 has also been speculated to act during chain assembly34, hydroxylation evidently occurs downstream of Pks4, at least. The intriguing substrate structural and/or protein-protein recognition features controlling the timing of hydroxylation by these P450 enzymes remain to be elucidated.
Role of TE domains in release of the shunt metabolites. We attributed the observed shunt metabolites 2 − 5 to the lack of productive chain translocation between Pks4 and Pks9, causing intermediates to accumulate on ACPs 12 and 13. To evaluate whether these were released by spontaneous hydrolysis or enzymatically, we further investigated the role of Pks9 TEI34 in chain release, as well as that of SamR0485, a proof-reading type II TE35 located in the cluster. Both TEs were disabled by site-directed mutagenesis of the active site serines (Ser ◊ Ala) (Supplementary Fig. 6).
Interestingly, inactivation of both the type I and type II TEs reduced the yields of shunt products 2 − 5 relative to the parental strain CPN2/OE484 (by 66% and 27%, respectively; average of duplicate experiments) (Supplementary Fig. 6 and Supplementary Table 5). These data clearly show that premature release of the chains is catalyzed, at least in part, by both TEs in the cluster, although spontaneous liberation also occurs. While type II TEs typically interact with acyl-ACPs in trans to release blocked chains35, the effect of the Pks9 TEI is less readily explained. One possibility is that the new productive docking interaction between Pks4 and Pks9 allows Pks9 to adopt an alternative conformation from which the TE can off-load intermediates bound to ACPs 12 and 13 of Pks4 (Supplementary Fig. 6). Although this mechanism is reminiscent of that used by the pikromycin PKS to generate both 12- and 14-membered rings36, the pikromycin TEI is separated from its alternative ACP target by a single module, while Pks9 TEI is located five or four modules downstream from ACPs 12 and 13 in the engineered system, which would seem to necessitate substantial inter-subunit acrobatics.
Understanding the docking domain engineering via studies in vitro with recombinant domains. To better understand the results of the DD engineering, we studied in vitro the wild type DD pairs (CDD4/NDD5 and CDD8/NDD9), as well as binding between the modified versions of CDD4 and wild type NDD9. Design of suitable expression constructs in E. coli (Supplementary Tables 1 − 3) was based on bioinformatics analysis of the C-terminal ends of Pks4 and Pks8, and the N-termini of Pks5 and Pks9, and secondary structure analysis using PSIPRED37 (Supplementary Fig. 8). Overall, we expressed and purified the following proteins in recombinant form from E. coli: CDD4 wt, CDD4 SDM, CDD4 helix swap, NDD5, and CDD8 (Supplementary Fig. 8, Supplementary Table 4). As NDD9 proved insoluble when expressed in E. coli, two versions with alternative start sites were obtained as synthetic peptides (Met and Val; Supplementary Fig. 8, Supplementary Table 4). Analysis of the individual CDDs by circular dichroism (CD) confirmed their expected high α-helical content (CDD4 wt (100 µM): 58%; CDD8 wt (100 µM): 49%), and showed no evident effect of the introduced mutations on secondary structure (Supplementary Fig. 8). All of the constructs were further confirmed to be homodimeric by size exclusion chromatography-multi-angle light scattering (SEC-MALS) (Supplementary Fig. 8).
The two NDDs also exhibited α-helical character, though less pronounced than the CDDs (NDD5 (100 µM): 27%; NDD9 Met (100 µM): 21%; NDD9 Val (100 µM): 25%), and were monomeric by SEC-MALS (Supplementary Fig. 8). The latter result was surprising, as type 1a NDDs classically form a homodimeric coiled-coil domain (Fig. 1, Supplementary Fig. 1), but we recently identified functional, monomeric type 1 NDDs38. Indeed, we detected binding between the native pairs by isothermal titration calorimetry (ITC), with affinities in the range of those determined previously for matched pairs of DDs27,38−40 (CDD4 + NDD5, Kd = 14.5 ± 0.9 µM; CDD8 + NDD9 Met, Kd = 33 ± 2 µM; CDD8 + NDD9 Val, Kd = 22 ± 1 µM) (Supplementary Fig. 8). Thus, while stable homodimerization of the NDDs may depend on the presence of a downstream homodimeric KS domain, their monomeric character did not preclude interaction with their CDD partners. Based on the higher affinity of the interaction, we could identify the NDD9 Val as the physiologically relevant construct. The observed binding stoichiometry (1 homodimeric CDD:2 monomeric NDDs), is consistent with the known structure of a type 1a complex in which two monomers of each DD are present (Fig. 1, Supplementary Fig. 1)22. As expected, no non-specific interaction was detected between native CDD4 and NDD9, explaining the lack of productive interaction between unmodified subunits Pks4 and Pks9 when the intervening multienzymes are deleted (strain K7N3) (Fig. 3a).
Analysis by ITC of binding between CDD4 SDM or CDD4 helix swap and NDD5 revealed the complete absence of interaction (Supplementary Fig. 8), and therefore that the introduced modifications were sufficient to disrupt communication between the native pair. Thus, the continued production of stambomycins 1 by K7N4, CPN4 and K7N5 harboring Pks5 − Pks8 must be due to additional contacts between Pks4 and Pks5 beyond the docking domains, likely including the compatible ACP13/KS14 interface. On the other hand, no interaction was detected between CDD4 SDM and NDD9, showing that this limited number of mutations was inadequate to induce productive contacts. This result is fully in accord with the absence of the expected mini-stambomycin products from these strains (K7N1/CPN1, Fig. 3a). By contrast, the CDD4 helix swap exhibited essentially the same binding to NDD9 Val as CDD8 (Kd = 21.0 ± 0.3 µM), demonstrating that exchange of just this helix is sufficient to redirect docking specificity30. Thus, inefficient docking is not at the origin of the failure of the CDD4 helix swaps to yield chain-extended products in vivo (strains K7N2/CPN2, Fig. 3a). We could therefore conclude at this stage that the problem arose from the non-native interface generated between ACP13 and KS21, poor acceptance by KS21 of the incoming substrate during chain transfer and/or chain extension, and/or low activity towards the modified chain of domains/modules acting downstream.
Attempted optimization of the stambomycin DD mutants. We aimed next to improve the novel Pks4/Pks9 intersubunit interface in strain CPN2 (CDD4 helix swap + deletion of Pks5 − 8) by targeting helix αI of ACP13, as the first 10 residues of this helix have been implicated previously in governing the interaction with the downstream KS domain at hybrid junctions26. Notably, multiple sequence alignment of all ACPs in the stambomycin PKS located at intersubunit junctions, revealed a unique sequence for each ACP in the helix αI region, consistent with a recognition ‘code’ for the KS partner, and the idea that mismatching these contacts might hamper productive chain transfer (Supplementary Fig. 9). Indeed, as mentioned previously, even when docking is interrupted, contacts between ACP13 and KS14 are apparently sufficient to enable chain translocation between Pks4 and Pks5 (Fig. 3a). In addition, an analogous strategy of optimizing the ACPn/KSn+1 chain transfer interface was shown recently to substantially improve interaction between an ACP (JamC) derived from the jamaicamide B biosynthetic pathway, and the first chain extension module of the lipomycin PKS (LipPKS1)41.
In our case, the first six residues of ACP13 helix αI were modified using CRISPR-Cas9 (EADQRR ◊ PSERRQ), so that the full 10-residue recognition sequence matched that of ACP20, the natural partner of KS21 (Supplementary Fig. 9). Analysis of extracts of the resulting strain CPN2/OE484/ACP13 SDM by HPLC-MS revealed at best minute amounts (highest yield of 0.5 µg mL− 1) of target cyclic mini-stambomycins A/B (11), lacking the hydroxyl group introduced by SamR0478 (Fig. 4 and Supplementary Fig. 9). Thus, while this experiment finally yielded the first evidence for successful chain transfer between Pks4 and Pks9 followed by subsequent chain extension by Pks9 and TE-catalyzed release, the overall efficiency of the system remained poor. Interestingly, however, the yields of the four shunt metabolites 2 − 5 were as much as 48-fold higher from the ACP13 helix swap mutant than from CPN2/OE484, showing that improved interactions between ACP13 and KS20 facilitated release of the stalled intermediates from ACPs 12 and 13, presumably via remote action by the TEI domain.
Engineering mini-stambomycins by maintaining the native ACP 13 /KS 14 junction (alternative module definition). Cumulatively, the results obtained with the docking domain engineering identified KS21 as one potential bottleneck in the engineered PKS. Our parallel strategy based on the alternative module definition (Fig. 2) allowed us to directly test this idea. Specifically, we investigated the effects of preserving the native CDD4/NDD5 pair and either the majority of KS14, or a little more than half of the domain, resulting in a KS14/KS21 hybrid. For this, we used two different splice sites in KS14: i) at the end of the domain in a highly-conserved region (GTNAHV) exploited recently to efficiently swap downstream AT domains42; and, ii) at a site corresponding to a recombination hot spot identified during induced evolution of the rapamycin (RAPS) PKS43, yielding the KS14/KS21 chimera (Fig. 4 and Supplementary Fig. 10). Both of these modifications were introduced into S. ambofaciens using CRISPR-Cas9, while simultaneously removing Pks5 − Pks8, yielding respectively after co-transformation with pOE484 and the control plasmid pIB139, strains ATCC/OE484/hy59_S1, ATCC/pIB139/hy59_S1, ATCC/OE484/hy59_S2, and ATCC/pIB139/hy59_S2.
Analysis of culture extracts revealed the presence in both ATCC/OE484/hy59_S1 and ATCC/OE484/hy59_S2 relative to the controls, of a novel series of 37-membered metabolites (Fig. 4). The measured masses were consistent with the desired mini-stambomycins either as their free acids or in cyclic form (metabolites 10 − 12, Fig. 4). Signals corresponding to the A/B and C/D derivatives of all metabolites were detected, providing important evidence for their identities, as well as both the C-14 hydroxylated 12 and non-hydroxylated 11 forms of the cyclic mini-stambomycins (C-14 corresponds to C-28 in the parental compounds (Fig. 1)). It is not surprising that the corresponding E and F forms were not detected, as their yields even from the wild type are much lower than the A − D derivatives (Fig. 3a). The observation of non-hydroxylated 11 shows notably that internal hydroxylation by SamR0478 is not an absolute prerequisite for TE-catalysed macrolactonization, and argues that hydroxylation of the mini-stambomycins only takes place on the macrocyclic compound. Although compounds 11 and 12 incorporate the tetrahydropyran moiety of the parental stambomycins 1 which undergoes glycosylation, derivatives bearing β-mycaminose were not observed, presumably due to poor recognition of the overall modified macrocycle by glycosyl transferase SamR048123.
The yields of the target compounds were minor relative to wild type stambomycins (metabolites 10, 11 and 12 from ATCC/OE484/hy59_S1 were obtained at highest yields of 0.3, 3.2 and 1.0 µg L− 1 (4.5 µg L− 1 total), respectively), but nonetheless approximately 4-fold higher from ATCC/OE484/hy59_S2 incorporating the hybrid KS14/KS21 than from the full KS14 swap (3.7, 9.7 and 3.6 µg L− 1 (17 µg L− 1 total); 1500-fold lower yields than 1) (Fig. 4 and Supplementary Fig. 10). Although the low titers of these compounds precluded their structure elucidation by NMR, we obtained additional confirmatory evidence for their identities by inactivation of samR0479 (which introduces the hydroxyl used for macrocyclization), which resulted in exclusive production of linear dehydroxy mini-stambomycins 13 (Supplementary Fig. 10).
As observed previously, the strains also produced substantial quantities of the shunt products 2 − 5 (inactivation of samR0479 led correspondingly to the dehydroxy versions of these compounds 6 − 9 (Supplementary Figs. 7 and 10)). The yields were ca. 80-fold higher than those of the corresponding mini-stambomycins, with the highest titers observed in the strain incorporating the hybrid KS14/KS21. The amount of shunt metabolites was also approximately 123-fold higher than from strain CPN2/OE484 (which incorporates an ACP13-CDD4 swap/NDD9-KS21 interface) (Figs. 3a and 4, Supplementary Table 5). Thus, contrary to expectation, although using the KS as a fusion site improved communication between Pks4 and Pks9, it also substantially boosted TEI-mediated off-loading of stalled upstream intermediates.
In principle, such stalling could result from a slow rate of chain extension in the now hybrid acceptor module (for example, in the full KS swap construct, KS14 and ACP21 are completely mismatched for chain extension). To evaluate this idea, we modified ACP21 within ATCC/OE484/hy59_S1 incorporating the full-length KS14, targeting a sequence region previously identified as mediating intramodular communication between the KS and ACP during chain extension (Supplementary Fig. 11)19,26. Specifically, we exchanged loop 1 and the initial portion of helix αII of ACP21 for the corresponding sequence of ACP14, using CRISPR-Cas9 (Supplementary Fig. 11). As we anticipated that creation of this substantially hybrid ACP might engender structural perturbation, we also engineered a minimal mutant of ACP21 in which only one of the two most critical residues in the recognition motif was mutated to the corresponding amino acid in ACP14 (G1499 of Pks9 ◊ D; the second residue, R, of the motif is already common to the two ACPs) (Supplementary Fig. 10). Analysis of the loop/helix αII swap by HPLC-MS showed that all mini-stambomycin production had been abolished (Supplementary Fig. 10), consistent with the anticipated disruption to ACP14 structure. Production by the ACP site-directed mutant was not any better than by the full KS swap construct (Fig. 4 and Supplementary Fig. 10), as only metabolite 11 remained above the limits of detection.
In principle, the hybrid KS14/KS21 domain may have worked better than KS14 for chain extension due to improved interaction with ACP21, with stalling displaced to later modules. If this were the case, we might expect to see accumulation in the medium of shunt metabolites corresponding to the intermediate generated by module 21. Indeed, in the case of strain hy59_S2 (chimeric KS14/KS21) but not hy59_S1 (KS14), we detected masses consistent with the A/B and C/D forms of intermediate 14 generated by module 21, at yields comparable to those of the final mini-stambomycins (Fig. 4, Supplementary Fig. 10) (and correspondingly, 15, the dehydroxylated analogue of 14, was detected in the SamR0479 mutant (Supplementary Fig. 10)). The same metabolite 14 was identified from the ACP21 G ◊D mutant (Fig. 4 and Supplementary Fig. 11), consistent with interrupted chain transfer to KS22. Taken together, these data confirm module 22 as a new blockage point in the engineered systems.
Relative efficacy of PKS engineering using PCR-targeting and CRISPR-Cas9. As multiple of our core constructs were generated by both PCR-targeting and CRISPR-Cas9, we were able to directly compare the efficiency of the two techniques (Fig. 3 and Supplementary Fig. 4). Globally, our results confirm that both approaches can be employed to introduce large-scale modifications to PKS biosynthetic genes (i.e. deletions of single or multi-gene regions)32,44−46. We have also demonstrated, for only the second time to our knowledge, that CRISPR-Cas9 can be leveraged to specifically modify modular PKS domains47. Of the two methods, CRISPR-Cas9 was the more rapid, as the corresponding constructs were engineered in approximately half of the time. In addition, while CRISPR-Cas9 allowed for direct modification of the host genome, PCR-targeting relied on the availability of suitable cosmids housing the target genes, and resulted in a 33 bp attB-like ‘scar’ sequence in the genome (Supplementary Fig. 4)48. In addition to hampering iterative use of the approach, the scar apparently provoked a moderate reduction in stambomycin yields in mutant K7N6 compared to the wild type, an effect also noted upon comparison of several analogous mutant strains (e.g. K7N4 vs. CPN4, Fig. 3). Nonetheless, we did encounter certain difficulties with use of CRISPR-Cas9 (i.e. failure to obtain construct CPN3, occasional reversions to wild type, etc.), observations motivating ongoing efforts in other laboratories to further enhance the suitability of CRISPR-Cas9 for editing PKS pathways47,49−54.