Skeletal editing via multi-step engineering of a modular polyketide synthase

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: minor_revision

Abstract
Assembly line biosynthesis creates numerous structurally diverse natural products using a
common modular synthetic strategy. The collinearity between the architectures of modular
polyketide synthases (PKS) and the structures of their polyketide products would seem to
render these biosynthetic machineries excellent platforms for designer biosynthesis, yet
reliable strategies to reprogram these assembly lines without diminishing their activities have
not been identified. Here, as a best practice for PKS engineering, we demonstrate the
reprogramming of the mediomycin PKS without significant loss of productivity. Using in
vitro CRISPR/Cas9 gene editing followed by heterologous expression, we reconstruct an
inaccessible drug lead of the fibrinogen receptor, tetrafibricin, at 82 ± 3 mg/L yield, retaining
26% productivity after five-step module editing using an evolution-supported cut site,
downstream of the acyltransferase domain. A macrocyclic aminopolyol is also accessed
through thioesterase swapping. These results pave the way toward the rational
reprogramming of PKSs to access desired complex organic molecules.

Introduction
Modular polyketide synthases (PKSs) are multidomain enzyme complexes that biosynthesize a
wide variety of polyketide natural products, including the clinically important medicines
erythromycin, tacrolimus (FK506), and ivermectin (a semi-synthetic derivative of avermectin)1.
The reaction cycle of a PKS module is equivalent to that of the metazoan fatty acid synthase
(FAS)2-4 - a carbon building block that is passed from an acyltransferase (AT) domain to an acyl
carrier protein (ACP) domain and a growing acyl chain that is bound to a ketosynthase (KS)
domain are fused to generate an elongated chain that can be processed by accompanying
ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains. However, the
multimodular nature of PKS assembly lines enables differential processing of each intermediate5-
8. In theory, PKSs can be engineered to access designer polyketides; however, in practice9-14 the
efficient (i.e., retaining production titre) and flexible reprogramming of PKSs is challenging due
to our limited understanding of domain-domain interactions and substrate recognition10,15-17.
The PKS module is the set of domains responsible for one round of chain extension and processing.
It was originally defined with an upstream KS and a downstream ACP18. However, studies have
shown that KSs can act as gatekeepers that select between intermediates bound to the upstream
ACP19-22, and that interactions between these two domains are important23,24. Additionally, our
previous evolutionary analysis of aminopolyol PKSs revealed that KSs genetically co-migrate with
upstream processing enzymes19 and we expanded this idea to other types of PKSs (Supplementary
Fig. 1). This co-migration is apparent in modular PKSs both with and without embedded ATs,
known as cis-AT and trans-AT PKSs19,25. How often AT domains co-migrate with neighbouring
enzymatic domains in cis-AT PKSs is likely related to the chemistry they perform together. Sets
of processing enzymes+ACP+KS that incorporate an -methyl substituent in a stereocontrolled
manner collaborate with methylmalonyl-specific upstream ATs21. KSs that extend polyketides with
ethylmalonyl extender units collaborate with ethylmalonyl-specific downstream ATs26. How well
such sets of enzymatic domains tolerate AT swaps is the subject of active investigation27. While
the genetic co-migration of processing enzymes and KSs within cis-AT PKSs was studied by
comparing related aminopolyol PKSs, the co-migration of its predominantly malonyl-specific AT
domains could not be studied due to gene conversion, which overwrites their evolutionary
history19,28. Thus, to reprogram aminopolyol PKSs, we evaluated two alternative strategies to
determine which was superior, AT+processing enzymes+ACP+KS units (exchange unit or “XU”)
or processing enzymes+ACP+KS+AT units (“XUA”). “XU” was originally introduced in the
context of nonribosomal peptide synthetase engineering29 and later adopted for PKS engineering14.
Because KS and AT are structurally connected, engineering modular PKSs by XUA-based module
editing might be a reasonable strategy (Fig. 1)30-32.

Here, we demonstrate the skeletal editing of aminopolyol polyketide structures through PKS
reprogramming. Using a previously established heterologous expression system for mediomycin
biosynthetic gene cluster19 as a platform, an artificial PKS was engineered through successive
multidomain manipulations to produce tetrafibricin, a fibrinogen receptor inhibitor33,34, with a
yield of 104 ± 4 µM (82 ± 3 mg/L) [mean ± s.e.m. (n = 3), the same applies below], demonstrating
the most complex and efficient multistep PKS engineering achieved to date to the best of our
knowledge. Furthermore, we produced an artificial macrolactone using the ACP-TE co-
transplantation strategy developed in this study.

Results
Reconstructing the carbon scaffold of tetrafibricin
Tetrafibricin is an aminopolyol polyketide with potent fibrinogen receptor inhibition activity that
was first isolated in 199333,34. Subsequent attempts to obtain tetrafibricin from the producing strain
have been unsuccessful19, dropping this non-peptidyl antiplatelet drug candidate out of further
development. Interestingly, the “producer” strain, S. neyagawaensis NR0557, instead produces a
larger polyketide, neotetrafibricin. Based on the domain architecture of the neotetrafibricin PKS,
misdocking between tfbORF5 and tfbORF9, premature hydrolysis by TE, or spontaneous
degradation during the isolation process may have led to tetrafibricin formation. As an alternative
approach, tetrafibricin has been targeted by chemical synthesis. While many fragment syntheses
have been reported, none has yet yielded the final natural product35.

We previously reported that the structural diversification of aminopolyols is naturally driven by
the recombination of the genes encoding PKSs19. Thus, we envisioned performing similar
recombinations in the lab to transform the mediomycin PKS (medPKS) into a tetrafibricin-
producing PKS (Supplementary Fig. 2a). To this end, the mediomycin biosynthetic gene cluster
was cloned into a bacterial artificial chromosome (BAC) for in vitro gene editing using
CRISPR/Cas9 (Supplementary Fig. 2b)13. The heterologous expression of this BAC in
Streptomyces lividans TK23 ΔredD–X, an engineered host lacking the undecylprodigiosin
biosynthetic gene cluster, was demonstrated in our previous work to enable the production of
clethramycin and desulfoclethramycin (guanidinylated mediomycin and desulfomediomycin)19.
When this work was initiated, we measured a combined yield of these polyketides of 533 ± 6 µM
(635 ± 7 mg/L). Given that this level of productivity was sufficiently high, we selected this host
for subsequent experiments. The guanidino group is not hydrolysed to an amine to provide
mediomycin and desulfomediomycin since medX, the gene encoding the amidinohydrolase, was
not originally included in the BAC because it is located outside of the mediomycin biosynthetic
gene cluster36.

We first attempted to swap XUA7 and XUA9 within medPKS, as in a natural recombination event
that occurred in the evolution of the aminopolyol PKSs19. In this campaign, XUA9 was first
duplicated (PKSv1), but then the doubling was resolved (PKSv2), which had the benefit of
minimizing opportunities for internal recombination. The cut site for the XUA boundary was set
C-terminal to the first tyrosine of the conserved LPTY(A/P)FQ(H/R)xRYWL motif in the last half
of the post-AT linker,37 referred to as PAL227 (Supplementary Figs. 2b and 2d). This evolution-
guided engineering19 preserves the interactions between KS, the upstream ACP, and the upstream
processing enzymes. The genes encoding the engineered assembly line, PKSv1, were expressed in
S. lividans TK23 ΔredD–X19. The anticipated products (Fig. 1b) 1a + 1b were generated at a
combined yield of 436 ± 55 µM (524 ± 66 mg/L), 82% that of clethramycin and
desulfoclethramycin (Fig. 1c and Supplementary Fig. 3a). Next, the downstream XUA9 was
replaced by XUA7 to create PKSv2 (Fig. 1a), which generated 2a + 2b at a combined yield of 298
± 33 µM (347 ± 38 mg/L) (Fig. 1c and Supplementary Fig. 3a). The structures of 1b and 2b were
confirmed by HR-MS analyses (Supplementary Table 1 and Supplementary Figs. 10–24) and a
series of NMR analyses (Supplementary Fig. 4, Supplementary Tables 2 and 3) (see also
Supplementary Figs. 25–55 for 1D and 2D NMR spectral data).

In the third step, we sought to trim the carboxy end of the polyketide product. It has been well-
documented that such a truncation can be obtained through the appropriate positioning of a
thioesterase (TE) domain38. Thus, we first positioned the mediomycin TE (medTE) downstream
of the ACP domain of XUA19 to create PKSv3_medTE (Supplementary Fig. 2c and 2f). However,
only a trace amount of the expected product 3 was detected by mass spectrometry (Supplementary
Fig. 5). Introduction of the pikromycin TE (pikTE) was also attempted (Supplementary Fig. 5);
however, PKSv3_pikTAE (Supplementary Fig. 2c and 2f) only generated 3 at a yield of 59 ± 7 µM
(49 ± 6 mg/L), 20% that of 2a + 2b. Inspired by the recent ACP-centric view of PKS modules,
we
hypothesized that the natural ACP cognate of medTE might be necessary for efficient TE-catalysed
hydrolysis. With the ACP+TE didomain from the medPKS positioned downstream of the KR
domain of XUA19 (Supplementary Fig. 1e), PKSv3 (Fig. 1a) generated 3 at a yield of 249 ± 15
µM (205 ± 12 mg/L), 84% that of 2a + 2b (Fig. 1c and Supplementary Fig. 3b). The sulfonated
analogue of 3 was not observed.

In the fourth step, XUA11 was removed from PKSv3 (Fig. 1a). The resulting PKSv4 generated 4
at a yield of 265 ± 26 µM (211 ± 21 mg/L), 106% that of 3 (Fig. 1c and Supplementary Fig. 3b).
Finally, a copy of XUA4 was inserted downstream of XUA2 (Fig. 1a). The resulting PKSv5
generated 5 at a yield of 140 ± 2 µM (115 ± 2 mg/L) (Fig. 1c and Supplementary Fig. 3b). The
yield of 5 was 53% that of 4.

XUA vs. XU strategy
At this stage, we had successfully created the polyketide skeleton of tetrafibricin through five steps
of PKS engineering using the XUA strategy. We then compared the XUA strategy with the XU
strategy, which employs a domain set with KS as the most downstream domain (AT+processing
enzymes+ACP+KS) (Fig. 2a). The XU strategy has been effectively applied in recent years to
modify several cis-AT PKSs and has been shown to be more effective than swapping using the
traditional module boundary9,12,14,39.

For Steps 1, 2, 4 and 5, we generated PKSv1_IXU, PKSv2_XU, PKSv4_XU and PKSv5_XU from
medPKS, PKSv1, PKSv3 and PKSv4, respectively (Fig. 2b and Supplementary Fig. 2f), using the
region 10 aa downstream of the GTNAH motif located at the C-terminal of the KS domain as the
cut site for the XU strategy. This cut site was employed and shown to work in a previous study40.
Step 3, which involves the migration of ACP-TE units, is not relevant to the comparison of the
XUA and XU strategies.

As a result, PKSv1_XU, PKSv2_XU, PKSv4_XU, and PKSv5_XU showed 93%, 82%, 59%, and
51% yields of PKSv1, PKSv2, PKSv4, and PKSv5, respectively (Fig. 2c). The differences in yield
between PKSv4 and PKSv4_XU (p < 0.001) and PKSv5 and PKSv5_XU (p = 0.002) were
statistically significant, while those between PKSv1 and PKSv1_XU (p = 0.383) and PKSv2 and
PKSv2_XU (p = 0.484) were not. These findings suggest that, at least in the context of
aminopolyol PKS engineering, the XUA strategy appears more robust against productivity loss.

Production of tetrafibricin
The PKSv5 product 5 differs from tetrafibricin in its guanidino group. To convert this group to an
amine, we aimed to co-express an amidinohydrolase gene from the aminopolyol producing strains
with PKSv5. To identify a promiscuous amidinohydrolase, tfbX1, tfbX2, ecoX, and medX were
each co-expressed with PKSv4 (Supplementary Fig. 6a). As TfbX1 and EcoX efficiently converted
4 to 7, tfbX1 and ecoX were each expressed with PKSv5, and both of them successfully converted
5 to tetrafibricin. The more efficient combination, tfbX1 and PKSv5, produced tetrafibricin at a
yield of 104 ± 4 µM (82 ± 3 mg/L) (Fig. 3b and Supplementary Fig. 6b). The yield of tetrafibricin
was 75% that of 5, demonstrating the efficiency of TfbX1. Notably, ecoX co-expression with
PKSv3 or PKSv4 gave 6 (227 ± 15 µM; 178 ± 12 mg/L) or 7 (188 ± 39 µM; 142 ± 29 mg/L),
respectively (Fig. 3b and Supplementary Fig. 6b), showcasing the promiscuity of EcoX. The
structures of 6, 7 and tetrafibricin were determined by HR-MS analyses (Supplementary Table 1)
and a series of NMR analyses (Supplementary Fig. 4, Supplementary Tables 4–6). Tetrafibricin
produced through module editing here showed potent fibrinogen receptor antagonistic activity, as
previously reported (see Supplementary Discussion).

Notably, a transformant carrying PKSv5, cultured before monospore isolation (see Methods),
generated 9 (Supplementary Fig. 7), a two-module-skipped version of 5. Co-expression of tfbX1
without isolating spores resulted in 10 (Supplementary Table 7) as the major product (tetrafibricin
was observed as a minor product). Plausibly, in vivo recombination between two direct repeat
regions encoding XUA4 (spanning 5,322 bp) occurred. On the other hand, we observed that the
productivity of 5 or tetrafibricin was not abolished even after a subculture, suggesting that the
recombination event occurs during transformation. Interestingly, we did not observe an equivalent
recombination for PKSv1, which possesses 2 copies of XUA9, for reasons that are unknown.

Production of an aminopolyol macrolide and its biological activity
Macrocyclic polyketides often exhibit potent bioactivities, as exemplified by clinical drugs such
as erythromycin and amphotericin. By applying the ACP+TE didomain transplantation strategy
that efficiently yielded 3, we further exploited TEs from other macrolactone PKS systems to
engineer an aminopolyol macrolide. We transplanted the ACP+TE didomains from the PKSs that
produce the macrocycles desertomycin and amphotericin for the ACP+TE didomain of medPKS
(Fig. 4a and Supplementary Fig. 2f). Although the amphotericin ACP+TE transplant only provided
trace quantities of cyclic product 8, the desertomycin ACP+TE transplant, PKSv6, yielded 8 at 9.6
± 0.4 µM (11.5 ± 0.5 mg/L) (Fig. 4b and Supplementary Fig. 8). The ester moiety of 8 was observed
by NMR, confirming the production of an artificial 54-membered macrolide (Fig. 4c and
Supplementary Table 8). The desertomycin TE was also transplanted without its cognate ACP,
generating PKSv6(TE) (Fig. 4a), but the yield was half that of PKSv6 (5.1 ± 0.3 µM; 6.1 ± 0.4
mg/L) (Fig. 4b).

The antibacterial and cytotoxic activities of 8 were evaluated. This cyclized clethramycin did not
inhibit the growth of Micrococcus luteus and Staphylococcus aureus, while clethramycin showed
weak activity against both strains (IC for M. luteus was 32.1 µM, agreeing with a previous report)
50
(Supplementary Fig. 9Aa). Cytotoxicity was measured on T lymphoma Jurkat cells, human ovarian
adenocarcinoma SKOV-3 cells, and malignant pleural mesothelioma MESO-1 cells. Whereas
clethramycin showed cytotoxicity (IC 0.32, 6.0 and 20 µM, respectively) equivalent to that of
50
etoposide (0.28, 2.3 and 16 µM, respectively), 8 was only active against Jurkat cells and ~100-
times less potent (23 µM) (Supplementary Fig. 9b).

Methods
Plasmids and bacterial strains
Plasmids and bacterial strains used in this study are listed in Supplementary Tables 9 and 10,
respectively.

The culturing procedures of bacterial strains followed our previous paper54. Specifically, the
growth of Escherichia coli strains DH5α and NEB10β (New England Biolabs, Ipswich, MA) used
Luria broth (LB), which contained (per L of deionized water) 10 g of tryptone, 5 g of yeast extract,
and 5 g of NaCl, and was adjusted to pH 7.5 following formulation. The corresponding solid
medium (LA) was prepared by supplementing LB with agar at 15 g/L. For the production of
engineered products, each transformant of Streptomyces lividans TK23 ΔredD-X was inoculated
into a 50-mL test tube containing 15 mL of seed medium, containing (per L of deionized water)
5 g of glucose, 15 g of soya flour, and 5 g of yeast extract; the cells were cultured with reciprocal
shaking at 320 rpm and 27°C for 2 days. A 375-μL aliquot of the resulting vegetative culture was
used to inoculate a 125-mL flask containing 15 mL of the production media. The production media
contained (per L of deionized water) 20 g of glycerol, 10 g of molasses, 5 g of casein, 1 g of
Polypepton, and 4 g of CaCO ; the pH was adjusted to 7.2 following formulation. The fermentation
3
cultures were grown on a rotary shaker at 180 rpm and 27°C for 6 days. For quantification or large-
scale preparation of the products, cells were grown under the same conditions in baffled 500-mL
flasks, each containing 100 mL of the production media.

In vitro module editing
The principle of in vitro module editing is originally described in ref. 6. In brief, a parent BAC
was digested in vitro by Cas9, and then a donor DNA was ligated by Gibson assembly.
sgRNAs (see Supplementary Data 1 for target sequences) were prepared using the EnGen sgRNA
Synthesis Kit, S. pyogenes (New England Biolabs), following the manufacturer’s instructions. The
Cas9 (New England Biolabs) reaction mixture containing the specific combination of a parent
BAC and sgRNAs (Supplementary Table 11) was incubated at 37°C for 120 min. The digested
BAC and the donor DNA (Supplementary Data 2) were subjected to Gibson assembly. After 50
min-incubation at 50°C, the ligated DNA (edited BAC) was recovered by
phenol/chloroform/isoamyl alcohol treatment followed by 2-propanol precipitation, then
transformed into E. coli NEB10β by electroporation. The transformants were selected on LA
containing 100 µg mL−1 ampicillin and 50 µg mL−1 kanamycin. The obtained plasmids were
further confirmed by PCR (Supplementary Data 1) and restriction digestion.

Cloning of amidinohydrolases
medX was amplified by PCR using the genomic DNA of S. blastmyceticus NBRC12747 as a
template. ecoX (AAX98207), tfbX1 (WP_190130807) and tfbX2 (WP_190128809) were
synthesized and cloned into pUC-GW-Kan by GENEWIZ (South Plainfield, NJ), which were used
as PCR templates. The primers used for PCR are listed in table S11. The PCR fragments were
subcloned into the XbaI-HindIII site of pKU565T_ermE*p.

Transformation of Streptomyces lividans TK23 ΔredD-X
The BACs and expression vectors for amidinohydrolases were introduced into S. lividans TK23
ΔredD-X by PEG-assisted protoplast transformation. Subsequently, the transformants carrying
pKU518med_PKSv5 were subjected to monospore isolation as follows. Mature spores were
harvested from transformants grown on SFM agar plates containing (per L of tap water) 20 g of
mannitol, 20 g of soya flour, and 20 g of agar. A dense spore suspension was serially diluted and
plated onto fresh SFM agar. Well-separated single colonies were then picked and patched to new
SFM agars for further analysis.

UHPLC/MS analysis of culture extract
One millilitre of culture broth was lyophilized and then extracted with 1 ml of methanol. Debris
were removed by centrifugation and the supernatant was filtrated through 0.22 µm PTFE
membrane to give an analytical sample for UHPLC. Analytical UHPLC and HR-ESI-MS (positive
mode) were performed using a ACQUITY UPLC System (Waters, Framingham, MA) in
conjunction with a ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 i.d. × 100 mm) (Waters), a
ACQUITY UPLC PDA eλ detector (Waters), and a XevoG2 ToF system (Waters). Mobile phase
A was water + 0.1% formic acid, and mobile phase B was acetonitrile + 0.1% formic acid. The
flow rate was 0.8 mL min−1, and the column temperature was kept at 55˚C by a column oven. The
gradient elution program was 5−100% B over 5 min and 100% B for 1 min. The isocratic elution
was performed by 37% B for clethramycin, desulfoclethramycin, 1a, 1b, 2a, 2b and 8, 17% B for
3, 4, 6 and 7, and 15% for 5, tetrafibricin, 9 and 10 (n=1 for each sample). Data acquisitions and
analyses were performed by using MassLynx V4.1 software (Waters). The standard curve for
clethramycin was obtained based on the peak area of UV chromatogram at 358 nm, and applied
for quantifying clethraymcin and the compounds sharing the same conjugated double bond system
with clethramycin (i.e. desulfoclethramycin, 1a, 1b, 2a, 2b and 8). Similarly, the standard curve
for tetrafibricin was obtained based on the peak area of UV chromatogram at 337 nm, and applied
for quantifying tetrafibricin and the compounds sharing the same conjugated double bond system
with tetrafibricin (i.e. 3, 4, 5, 6, 7, 9, and 10).

Isolation of compounds
Isolation of 1b
0.25 litres of fermentation broth of S. lividans TK23 ΔredD-X/pKU518med_PKSv1 were
centrifuged to separate a mycelial cake and supernatant. The mycelial cake was extracted twice
with CHCl :MeOH = 1:1, and the organic solvent was removed in vacuo. The residual extract was
3
loaded on a Sephadex LH20 column and eluted with MeOH. The fractions containing 1b were
combined and dried up in vacuo. The residues were dissolved in MeOH, then combined and
filtrated. The crude extract containing 1b was purified by preparative reversed-phase HPLC using
a YMC-Triart C18 column (5 µm, 20 i.d. × 250 mm, YMC, Co. Ltd, Kyoto, Japan) with 50 mM
ammonium formate solution/acetonitrile as the mobile phase, with a gradient of separation: 0–25
min 30–45% B, 25–30 min 45–55% B, to afford 1.5 mg of 1b.

Isolation of 2b
0.47 litres of fermentation broth of S. lividans TK23 ΔredD-X/pKU518med_PKSv2 were
centrifuged to separate a mycelial cake and supernatant. The mycelial cake was extracted twice
with CHCl :MeOH = 1:1, and the organic solvent was removed in vacuo. The residual extract was
3
loaded on a Sephadex LH20 column and eluted with MeOH. The fractions containing 2b were
combined and dried up in vacuo. The residues were dissolved in MeOH, then combined and
filtrated. The crude extract containing 2b was purified by preparative reversed-phase HPLC using
a YMC-Triart C18 column (5 µm, 20 i.d. × 250 mm, YMC, Co. Ltd, Kyoto, Japan) with 50 mM
ammonium formate solution/acetonitrile as the mobile phase, with a gradient of separation: 0–25
min 30–45% B, 25–30 min 45–55% B, to afford 1. 2 mg of 2b.

Isolation of 6
Two litres of fermentation broth of S. lividans TK23 ΔredD-
X/pKU565T_ecoX/pKU518med_PKSv3 were centrifuged to separate a mycelial cake and
supernatant. The mycelial cake was extracted twice with 500 mL of acetone, and the acetone was
removed in vacuo. The residual aqueous extract and the supernatant of culture broth were
separately loaded on a DIAION HP20 (Mitsubishi Chemical Co., Tokyo, Japan) column (100mL)
and eluted with a stepwise gradient of H O–MeOH (0, 50, 100, 100% MeOH; 5 C.V. each). The
2
50% MeOH and the first 100% MeOH fractions were combined and dried up in vacuo. The
residues were dissolved in MeOH and filtrated to give 0.42 and 1.26 g, respectively, of crude
extract. These crude extracts were combined and subjected to reversed-phase MPLC using a Purif-
Pack ODS column (30 µm, SIZE:60) (Shoko Science Co., Ltd., Kanagawa, Japan) with an isocratic
elution by H O. The fractions containing 6 were combined, lyophilized and dissolved in MeOH.
2
The MeOH solution was subjected to gel filtration chromatography using a Sephadex LH-20
(Cytiva, Uppsala, Sweden) column developed with MeOH. The fraction containing 6 (706 mg)
was further separated by Toyopearl HW-40F (Tosoh Corporation, Tokyo, Japan) developed with
MeOH. The fraction containing 6 (65 mg) was purified by preparative reversed-phase HPLC using
a CAPCELL PAK MG-II C18 column (5 µm, 20 i.d. × 250 mm) (Shiseido Co., Ltd., Tokyo, Japan)
with 50 mM ammonium formate solution/acetonitrile (84:16) as the mobile phase to afford 1.4 mg
of 6.

Isolation of 7
Two litres of fermentation broth of S. lividans TK23 ΔredD-
X/pKU565T_ecoX/pKU518med_PKSv4 were centrifuged to separate a mycelial cake and
supernatant. The mycelial cake was extracted twice with 500 mL of acetone, and the acetone was
removed in vacuo. The residual aqueous extract and the supernatant of culture broth were
separately loaded on a DIAION HP20 column (100mL) and eluted with a stepwise gradient of
H O–MeOH (0, 50, 100, 100% MeOH; 5 C.V. each). The 50% MeOH and the first 100% MeOH
2
fractions were combined and dried up in vacuo. The residues were dissolved in MeOH, then
combined and filtrated to give 0.92 g of crude extract. The crude extract was further partitioned
between H O and ethyl acetate/n-butanol (1:1). The aqueous layer (660 mg) was subjected to
2
reversed-phase MPLC using a Purif-Pack ODS column (100 µm, SIZE:20) with an isocratic
elution by H O. The fractions containing 7 were combined, lyophilized and dissolved in MeOH.
2
The MeOH solution was subjected to gel filtration chromatography using a Toyopearl HW-40F
column developed with H O and then 50% MeOH. The fraction containing 7 (301 mg) was
2
purified by preparative reversed-phase HPLC using a CAPCELL PAK MG-II C18 column (5 µm,
20 i.d. × 250 mm) with 50 mM ammonium formate solution/acetonitrile (85:15) as the mobile
phase to afford 8.5 mg of 7.

Isolation of tetrafibricin and 10
One litre of fermentation broth of S. lividans TK23 ΔredD-
X/pKU565T_ecoX/pKU518med_PKSv5 (before monospore isolation) was centrifuged to separate
a mycelial cake and supernatant. The mycelial cake was extracted twice with 500 mL of acetone,
and the acetone was removed in vacuo. The residual aqueous extract and the supernatant of culture
broth were separately loaded on a DIAION HP20 column (100mL) and eluted with a stepwise
gradient of H O–MeOH (0, 50, 100, 100% MeOH; 5 C.V. each). The 50% MeOH and the first
2
100% MeOH fractions were combined and dried up in vacuo. The residues were dissolved in
MeOH, then combined and filtrated. The crude extract (1.07 g) was subjected to hydrophilic
interaction MPLC using a Purif-Pack NH column (60 µm, SIZE:20) (Shoko Science) with a
stepwise gradient of H O–acetonitrile (100, 90, 70, 50% acetonitrile). The fractions containing
2
both of tetrafibricin and 10 (106 mg) was purified by preparative reversed-phase HPLC using a
CAPCELL PAK MG-II C18 column (5 µm, 20 i.d. × 250 mm) with 50 mM ammonium formate
solution/acetonitrile (85:15) as the mobile phase to afford 3.6 mg of tetrafibricin and 2.3 mg of 10.

Isolation of 8
Two litres of fermentation broth of S. lividans TK23 ΔredD-
X/pKU565T_ecoX/pKU518med_PKSv6 were centrifuged to remove supernatant. The mycelial
cake was extracted twice with 500 mL of acetone, and the acetone was removed in vacuo. The
residual aqueous extract was loaded on a DIAION HP20 column (100mL) and eluted with a
stepwise gradient of H O–MeOH (0, 50, 100, 100% MeOH; 5 C.V. each). The first 100% MeOH
2
fraction was concentrated in vacuo, and the residual aqueous extract was lyophilized. The crude
extract dissolved in MeOH was subjected to gel filtration chromatography using a Sephadex LH-
20 column developed with MeOH. The fractions containing 8 were combined and concentrated in
vacuo and then further purified by preparative reversed-phase HPLC using a CAPCELL PAK MG-
II C18 column (5 µm, 20 i.d. × 250 mm) with 50 mM ammonium formate solution/acetonitrile
(50:50) as the mobile phase to afford 4.2 mg of 8.

NMR spectroscopy
NMR spectra were recorded on Bruker AVANCE III 900 (Bruker Daltonics, Bremen, Germany)
for 1b and 2b, and an NMR 600 NB CL spectrometer (Varian, Palo Alto, CA) for 6, 7, tetrafibricin,
8 and 10 at room temperature. Chemical shifts (δ) are reported in ppm with the residual solvent
signals as the internal standards (methanol-d
4
at δ
H
3.31 and δ
C
49.0 ppm). Coupling constants
(J) are reported in Hertz. The data are reported as (s = singlet, d = doublet, t = triplet, m =
multiplet, or unresolved; coupling constant(s)). 13C-NMR spectra were recorded with complete
1H decoupling.

The structures of 1b, 2b, 6, 7, tetrafibricin, and 10 were determined based on 1D and 2D NMR
spectral analyses (See Supplementary Figs. 25–55 provided as a separate file). Key 2D NMR
(COSY, HMBC, and HSQC-TOCSY) correlations established the substructures intended to be
modified in each step (coloured in red) (Supplementary Fig. 13). As the almost identical chemical
shifts and the 2D NMR correlations indicate, the remaining substructures are the same as in the
previous step.

Antibacterial Assay
The procedure of the antibacterial assay followed our previous paper55. Specifically,
Staphylococcus aureus and Micrococcus luteus (laboratory stocks) were precultured in LB
medium until the optical density at 620 nm became 0.5. The bacteria suspension was diluted 2000-
fold with LB medium and inoculated into each well of a 384-well plate, followed by addition of
serially diluted test compounds. After incubation for 24 h at 37 °C, the optical density for each
well was measured at 620 nm using an EnVision Multilabel Plate Reader (PerkinElmer, Inc.,
Waltham, MA).

Cytotoxicity Assay
The procedures of the cytotoxicity assay followed our previous paper13. Specifically, the
cytotoxicities against T lymphoma Jurkat cells, human ovarian adenocarcinoma SKOV-3 cells and
malignant pleural mesothelioma MESO-1 cells were examined. Jurkat cells were cultured in
RPMI1640 medium supplemented with 10% fetal bovine serum, penicillin (50 U/mL),
streptomycin (50 μg/mL), and Glutamax. SKOV-3 cells were cultured in DMEM medium
supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (50 μg/mL).
MESO-1 cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum,
penicillin (50 U/mL), and streptomycin (50 μg/mL). All cell lines were seeded in a 384-well plate
at a density of 1,000 cells/well in 20 μL of media and incubated at 37°C in a humidified incubator
with 5% CO . After 4 h, 2-fold dilution samples dissolved in DMSO were added to the cell
2
culture at the concentration of 0.5% (0.1 µL) and incubated for 72 h. Cell viabilities were
measured using a CellTiter-Glo luminescent cell viability assay and EnVision multilabel plate
reader.

Platelet Assay
To prepare a fibrinogen-coated plate, 20 µL of 30 µg/mL fibrinogen (from human plasma)
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in PBS was added to a high-binding
384-well microplate (781061, Greiner Bio-One) and kept at 4°C overnight. After washing twice
with 50 µL PBS, 20 µL of 5 times-diluted Blocking One (Nacalai tesque, Inc., Kyoto, Japan) in
PBS was added to each well then kept at room temperature for 30 min. The blocking reagent was
discarded, and wells were washed twice with 50 µL PBS.

To prepare pCMV3-C-OFPSpark-ITGA2B(G991C), site-directed mutagenesis on pCMV3-C-
OFPSpark-ITGA2B was achieved by PCR using Q5 Hot Start High-Fidelity 2X Master Mix (New
England Biolabs). The mutated linear dsDNA was amplified with primers ITGA2B(G991C)-F and
ITGA2B(G991C)-R (table S11) using pCMV3-C-OFPSpark-ITGA2B as a template. The PCR
product was treated with KLD Enzyme Mix (New England Biolabs), and the ligated DNA was
introduced into NEB5α (New England Biolabs).

HEK293 cells cultured in DMEM medium supplemented with 10% fetal bovine serum were
transfected by pCMV3-C-ITGA2B(G991C)-OFP and pCMV3-C-ITGA3(wt)-GFP with the
assistance of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). The transfected cells
were seeded in an aforementioned fibrinogen-coated plate at the density of 10,000 cells/well in 19
µL of media and immediately treated with 1 µL of the tetrafibricin (produced in this study) or
RGDS peptide (positive control) (abcam, Cambridge, UK) solution. After incubating at 37°C in a
humidified incubator with 5% CO for 24 h, 5 µL of Hoechst 33342 (Dojindo Laboratories,
2
Kumamoto, Japan) was added and incubated for 60 min. The supernatant was removed by
decantation, and each well was washed twice with 50 µL of PBS. The number of cells bound to
the plate was measured using Opera Phenix (PerkinElmer).

Bioinformatic analysis
Sequences used for phylogenetic analysis (Supplementary Fig. 1) were obtained from NCBI
GenBank database (Supplementary Table 12) and were processed on Geneious Prime 2025.1.3
(https://www.geneious.com) platform. Sequence alignments were created by MAFFT v7.49056
plugin using G-INS-i algorithm, BLOSUM45 scoring matrix, 1.7 gap open penalty, 0.103 offset
value. Phylogenetic trees were constructed by FastTree 2.1.1157 plugin using WAG model,
Gamma20 likelihood, and pseudocounts enabled. The amino acid sequences of five
amidinohydrolases, TfbX1, TfbX2, EcoX, MedX, and DstH (BCB17038), were aligned by using
Clustal Omega 1.2.258 with the default settings.

Statistics
A two-tailed Welch’s t-test was performed (n = 5 per group) with a significance level of 0.01.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Overall, I am satisfied with the authors’ responses to my previous comments. Nonetheless, there are a few remaining points to address (in textual order):\nPage 3, lines 51-55. The text at this point would seem to suggest that KSs only co-migrate with certain up- and downstream AT domains – i.e. those that incorporate building blocks other than malonate. The authors then state that, in the case of their system, co-migration with malonate-specific ATs could not be evaluated due to gene conversion. There would thus seem to be a contradiction between these two observations, as the latter statement clearly implies that co-migration with malonate-specific ATs is possible.\nPage 3, line 60. The phrasing here is odd. Suggest, ‘…we chose to evaluate two alternative strategies to determine which was superior…’\nPage 3, line 63. XUAs not XUA’s\nPage 3, line 70. Do ACP and TE actually co-migrate evolutionarily, or is it rather that the authors employed a co-transplantation approach?\nPage 4, line 89. Suggest, ‘When this work was initiated, we measured a combined yield of these polyketides of…’. And line 93, …included in the BAC because it is located outside\nPage 4, line 97. Suggest, ‘…which had the benefit of minimizing opportunities…’\nPage 5, line 99. Suggest, ‘(equivalent to the last half of the post-AT linker,37 referred to as PAL227).\nPage 6, line 117. ‘Percentage’ not ‘percentile’\nPage 7, line 123. Suggest, ‘Introduction of the pikromycin TE…’\nPage 7, line 136. ‘we had successfully…’; Line 140. ‘…using the traditional module boundary.’\nPage 7, Line 143. Suggest, ‘…downstream of the GTNAH motif…’ The site 10 residues downstream of the GTNAH was that employed previously by Keatinge-Clay, et al. (e.g. in doi: 10.1038/s41467-024-50844-6), a fact that should be stated explicitly, as evidently, the precise choice of fusion point can have important functional consequences for the resulting hybrid PKSs.\nPage 9, line 174. Remove the , after tfbX1; Line 175. Remove ‘the’ after Plausibly, Line 179. Add a , after XUA9\nPage 12, line 241. This statement would seem incorrect. The type of collaboration referred to here (between KS domains and their downstream ACP partners) is characteristic of classical modules.\nPage 12. I must once again take issue with the concept of an ‘updated module’. A.T. Keatinge-Clay originally proposed that modules should be redefined as starting with the AT domain and ending after the KS, because this domain set yields improved engineering results. The present work demonstrates that a DIFFERENT domain set (XUA) can give even better results (or in other words, the updated module needs updating!). Overall, the message emerging from this type of research is NOT that modules need redefining (indeed, ONLY ‘classical’ modules are competent for both chain extension and chemical processing), but that alternative domain sets (i.e. XUs and/or XUAs) are preferable as BioBricks for synthetic biology. References in the Discussion section to ‘updated modules/boundaries’ should therefore be removed from the text.\nPage 12, line 254. The authors have demonstrated the utility of the XUA BioBrick, and state here that it has already been applied to engineer additional systems. However, the precise cut site employed in this work within the PAL2 region (lptY at the beginning of PAL2) differs from that used earlier (...rywL at the end of PAL2). This is an important detail, and so should be noted explicitly, and it dovetails with independent findings by the Keasling group that several sites within the post-AT linker can give rise to functional hybrids (doi: 10.1038/s41467-023-40464-x).\nPage 13, line 258. It would be appropriate here to cite the original reference (doi: 10.1016/j.chembiol.2010.05.017) in addition to the review.", "reviewer_2": "Title: Review of “Skeletal editing via multi-step engineering of a modular polyketide synthase”\nSubmitted to: Nature Communications\nOpinion: Accept after minor revisions\nSummary:\nAssembly-line biosynthesis enables the production of a wide array of structurally diverse natural products through a shared modular synthetic strategy. In this manuscript, Shin-ya and colleagues investigated rational reprogramming of cis-AT type I modular polyketide synthases (PKSs), whose intrinsic collinearity between enzymatic architecture and product structure has made them theoretical attractive yet practically elusive platforms for designer biosynthesis.\nUsing an integrated approach combining in vitro CRISPR/cas9 editing with heterologous expression, the authors present a technically impressive demonstration of an example of mediomycin PKS reprogramming without significant loss in productivity. Through a five-step module-editing strategy targeting an evolutionarily supported engineering site located downstream of acyltransferase (AT) domain, the authors reconstructed the previously inaccessible drug lead tetrafibricin – a fibrinogen receptor antagonist – achieving a yield of 82 mg/L. The work then expands further to compare XUA and XU strategies, demonstrates the importance of ACP-TE comigration for efficient chain release and cyclization, and extends the approach to produce an aminopolyol macrolide.\nOverall, this manuscript shows a sophisticated, rational PKS reprogramming study with productive examples. The experimental design is careful and clear, and the results are convincing and supported by analytical data. The manuscript will be of a broad interest to the fields of natural product biosynthesis and synthetic biology.\nMajor Comments:\n1. It is strongly recommended to clarify why experimental decisions were made – especially in the introduction section. The\nmanuscript shows successful reprogramming of mediomycin PKS, but it does not explicitly justify why this particular system\nwas selected over other aminopolyol PKS(s).\n2. Regarding generality and scope of the engineering strategy: While the authors convincingly demonstrate the power of the\ndownstream AT linker/XUA strategy in the context of aminopolyol PKSs, the broader applications and generality of this\napproach are still not completely clear.\n3. The manuscript claims the technique chosen is a “best practice” for PKS engineering, however, it does not clearly\ncompare how this approach improves upon previous methods.\n4. The retention of productivity is extremely impressive in this study. However, the mechanistic reason for this is only\nsomewhat descriptive. The authors attribute the success to preservation of domain-domain interactions and evolutionary\ncomigration, further expansion and detail on this could be added.\n5. The manuscript demonstrates biosynthesis access to tetrafibricin, but the biological implications remain slightly\nunderdeveloped. It is also recommended to briefly contextualize the pharmacological relevance of tetrafibricin.\nMinor Comments:\nThere are some additional questions that are outlined in the points below that I hope will benefit the manuscript:\no In the Abstract portion of the paper or elsewhere appropriate, specify the percentage retention of productivity relative to the\nprevious/native system.\no The manuscript alternates between “engineering step,” “module editing” and “domain swapping.” While all are fine to use,\nmore consistency would improve readability.\no The comparison in Fig. 2 is convincing, but it would be helpful to state whether to observed differences are statistically\nsignificant.\no The bi-products (9) and (10) are interesting. Would they pose a general risk for large-repeat engineering strategies? How\nwould it be mitigated?\no The term “engineering efficiency” is used to describe yield retention. Please clarify if this is strictly referring to product titre\nor also incorporates success rate, stability, etc.\no Please explain why a geometric mean was used instead of an arithmetic mean in this context.", "reviewer_3": "The manuscript describes an excellent piece of work that will serve as blueprint for PKS engineering approaches in the coming years. It consolidates insights gained over the past few years and successfully translates them into application. Expectations, such as extensive testing of bioactivity, go beyond the scope of this work. The presentation of this work in the journal Nature would have been both justified and highly desirable.\nThe manuscript has been revised with great care. There are two points: (i) As the authors themselves state (Response 1 to Reviewer 3) that “it is not necessary to detail the concept of an ‘updated module’ or ‘XU’ for introducing the XUA strategy,” the term “updated module boundary” should be removed from the manuscript. It is sufficient to define the elements of PKSs using the terminology of modules, exchange units (XUs), and XU assembly (XUA). (ii) The authors should also refrain from referring to future work with statements such as “A more systematic study is currently underway.”"}, "author_response": "Point-by-point response to Reviewers’ comments\nReviewer #1 (Remarks to the Author):\nOverall, I am satisfied with the authors’ responses to my previous comments. Nonetheless, there are a\nfew remaining points to address (in textual order):\nWe sincerely appreciate your previous comments, which helped us improve the quality of the\nmanuscript. We have addressed the remaining concerns you raised below.\nPage 3, lines 51-55. The text at this point would seem to suggest that KSs only co-migrate with certain\nup- and downstream AT domains – i.e. those that incorporate building blocks other than malonate. The\nauthors then state that, in the case of their system, co-migration with malonate-specific ATs could not\nbe evaluated due to gene conversion. There would thus seem to be a contradiction between these two\nobservations, as the latter statement clearly implies that co-migration with malonate-specific ATs is\npossible.\nThe co-migration of ATs with upstream or downstream domains seems to depend on the specific\nchemistry performed by the set of co-migrating domains. We prefer to leave the reader with a reference\nto a study that partially addresses this (ref. 27) rather than discuss the possible chemistries further.\nPage 3, line 60. The phrasing here is odd. Suggest, ‘…we chose to evaluate two alternative strategies\nto determine which was superior…’\nWe have revised accordingly.\nPage 3, line 63. XUAs not XUA’s\nWe have corrected accordingly.\nPage 3, line 70. Do ACP and TE actually co-migrate evolutionarily, or is it rather that the authors\nemployed a co-transplantation approach?\nThe latter case here. We have replaced ‘co-migration’ with ‘co-transplantation’ for clarity.\nPage 4, line 89. Suggest, ‘When this work was initiated, we measured a combined yield of these\npolyketides of…’. And line 93, …included in the BAC because it is located outside\nWe have revised accordingly.\nPage 4, line 97. Suggest, ‘…which had the benefit of minimizing opportunities…’\nWe have revised accordingly.\nPage 5, line 99. Suggest, ‘(equivalent to the last half of the post-AT linker,37 referred to as PAL227).\nWe have revised accordingly.\nPage 6, line 117. ‘Percentage’ not ‘percentile’\nWe have corrected accordingly.\nPage 7, line 123. Suggest, ‘Introduction of the pikromycin TE…’\nWe have revised accordingly.\nPage 7, line 136. ‘we had successfully…’; Line 140. ‘…using the traditional module boundary.’\nWe have corrected accordingly.\nPage 7, Line 143. Suggest, ‘…downstream of the GTNAH motif…’ The site 10 residues downstream\nof the GTNAH was that employed previously by Keatinge-Clay, et al. (e.g. in doi: 10.1038/s41467-\n024-50844-6), a fact that should be stated explicitly, as evidently, the precise choice of fusion point\ncan have important functional consequences for the resulting hybrid PKSs.\nAccording to your comment, we have added, “This cut site was employed and shown to work in a\nprevious study40.”\nPage 9, line 174. Remove the , after tfbX1; Line 175. Remove ‘the’ after Plausibly, Line 179. Add a ,\nafter XUA9\nWe have corrected accordingly.\nPage 12, line 241. This statement would seem incorrect. The type of collaboration referred to here\n(between KS domains and their downstream ACP partners) is characteristic of classical modules.\nWe have rephrased the sentence as \"The non-native interactions between KSs and downstream ACPs\ndid not disrupt the function of engineered PKSs.\"\nPage 12. I must once again take issue with the concept of an ‘updated module’. A.T. Keatinge-Clay\noriginally proposed that modules should be redefined as starting with the AT domain and ending after\nthe KS, because this domain set yields improved engineering results. The present work demonstrates\nthat a DIFFERENT domain set (XUA) can give even better results (or in other words, the updated\nmodule needs updating!). Overall, the message emerging from this type of research is NOT that\nmodules need redefining (indeed, ONLY ‘classical’ modules are competent for both chain extension\nand chemical processing), but that alternative domain sets (i.e. XUs and/or XUAs) are preferable as\nBioBricks for synthetic biology. References in the Discussion section to ‘updated modules/boundaries’\nshould therefore be removed from the text.\nWe have removed the term throughout the text to avoid confusion. Thank you again for your fair\ndiscussion.\nPage 12, line 254. The authors have demonstrated the utility of the XUA BioBrick, and state here that\nit has already been applied to engineer additional systems. However, the precise cut site employed in\nthis work within the PAL2 region (lptY at the beginning of PAL2) differs from that used earlier (...rywL\nat the end of PAL2). This is an important detail, and so should be noted explicitly, and it dovetails with\nindependent findings by the Keasling group that several sites within the post-AT linker can give rise\nto functional hybrids (doi: 10.1038/s41467-023-40464-x).\nWe really appreciate you for pointing this out. According to your suggestions, the exact cut sites have\nbeen discussed in more detail, as follows (revisions underscored):\nWe observed that editing using the post-AT cut site outperformed editing using the KS-AT linker cut\nsite. The post-AT linker cut site has been employed to swap ATs27 and reductive loops44. We show that\nthe cut site (i.e., after the LPTY motif) can be used to swap XUAs in aminopolyol PKSs. Notably, a\nrecent study from our lab illustrated another post-AT cut site at the end of PAL2 (i.e., after the RYWL\nmotif) predominantly functioned in other PKSs45. This flexibility in acceptable cut sites within PAL2\ndoes not conflict with the results of an AT-swapping study that compared the use of several cut sites46.\n*ref. 46 = doi: 10.1038/s41467-023-40464-x\nPage 13, line 258. It would be appropriate here to cite the original reference (doi:\n10.1016/j.chembiol.2010.05.017) in addition to the review.\nWe have corrected accordingly.\nReviewer #2 (Remarks to the Author):\nSummary:\nAssembly-line biosynthesis enables the production of a wide array of structurally diverse natural\nproducts through a shared modular synthetic strategy. In this manuscript, Shin-ya and colleagues\ninvestigated rational reprogramming of cis-AT type I modular polyketide synthases (PKSs), whose\nintrinsic collinearity between enzymatic architecture and product structure has made them theoretical\nattractive yet practically elusive platforms for designer biosynthesis.\nUsing an integrated approach combining in vitro CRISPR/cas9 editing with heterologous expression,\nthe authors present a technically impressive demonstration of an example of mediomycin PKS\nreprogramming without significant loss in productivity. Through a five-step module-editing strategy\ntargeting an evolutionarily supported engineering site located downstream of acyltransferase (AT)\ndomain, the authors reconstructed the previously inaccessible drug lead tetrafibricin – a fibrinogen\nreceptor antagonist – achieving a yield of 82 mg/L. The work then expands further to compare XUA\nand XU strategies, demonstrates the importance of ACP-TE comigration for efficient chain release\nand cyclization, and extends the approach to produce an aminopolyol macrolide.\nOverall, this manuscript shows a sophisticated, rational PKS reprogramming study with productive\nexamples. The experimental design is careful and clear, and the results are convincing and supported\nby analytical data. The manuscript will be of a broad interest to the fields of natural product\nbiosynthesis and synthetic biology.\nThank you for your positive evaluation. Showcasing a practical example presented here would raise\nbroad expectations for PKS engineering.\nMajor Comments:\n1. It is strongly recommended to clarify why experimental decisions were made – especially in the\nintroduction section. The manuscript shows successful reprogramming of mediomycin PKS, but it\ndoes not explicitly justify why this particular system was selected over other aminopolyol PKS(s).\nThis is initially because the heterologous expression system for mediomycin BGC had previously been\nestablished. Then, as described in the main text, we quantified the production yields of clethramycin\nand desulfoclethramycin, and they were sufficient even if the engineered PKS reduced yield by less\nthan a hundredth. To make this experimental background clearer in the introduction section, we have\nmentioned as follows (revision underscored):\nUsing a previously established heterologous expression system for mediomycin biosynthetic gene\ncluster19 as a platform, an artificial PKS was engineered …\n2. Regarding generality and scope of the engineering strategy: While the authors convincingly\ndemonstrate the power of the downstream AT linker/XUA strategy in the context of aminopolyol PKSs,\nthe broader applications and generality of this approach are still not completely clear.\nOne of our challenges is to use PKS engineering as an alternative synthetic strategy for complex\nmolecules, such as medicines. This point is briefly mentioned in the discussion section as follows:\n“Our results encourage using PKS module editing as a reasonable and alternative strategy for\nobtaining tetrafibricin and analogues thereof in yields appropriate for medicinal chemistry. The\nefficient programming of modular PKSs can be transformative in the synthesis of high yields of\nvaluable natural product-based medicines.”\nAs to generality, the concept of the post-AT cut site is supported by complementary work by one of us\n(ref. 45). While there may be different approaches or necessary optimisations for individual targets,\nour report as one piece of practical evidence will guide future engineering efforts.\n3. The manuscript claims the technique chosen is a “best practice” for PKS engineering, however, it\ndoes not clearly compare how this approach improves upon previous methods.\nGiven the approach previously considered a strong candidate, we employed the XU strategy, which\nwas not superior to the XUA strategy (Fig. 2c). Since the products of each comparative experiment\nwere the same, retaining the intact KS-AT didomain architecture had a greater impact on yield than\nnative AT-ACP interactions.\n4. The retention of productivity is extremely impressive in this study. However, the mechanistic reason\nfor this is only somewhat descriptive. The authors attribute the success to preservation of domain-\ndomain interactions and evolutionary comigration, further expansion and detail on this could be added.\nA mechanistic study to dissect all of domain-domain contributions (KS/AT, AT/ACP, KS/ACP,\nprocessing enzymes/ACP, etc.) that make XUA-swapping better than XU-swapping falls outside the\nfocus of this paper. As Reviewer #3 remarked, we also consider this work an integration of recent\ninsights into a practical reference, rather than a leap of an idea. The comparison between XUA and\nXU strategies led to a consistent conclusion (i.e., the retention of the KS-AT didomain has a reduced\nnegative impact on engineering results).\n5. The manuscript demonstrates biosynthesis access to tetrafibricin, but the biological implications\nremain slightly underdeveloped. It is also recommended to briefly contextualize the pharmacological\nrelevance of tetrafibricin.\nUnfortunately, the tetrafibricin derivatives obtained through this work did not show remarkable anti-\nplatelet activity (Supplementary Discussion). At this point, we do not further explore or continue\nderivatisation, as a structure-activity relationship study is not the focus of this work.\nThe context of tetrafibricin has been added to the main text as follows (revision underscored):\nSubsequent attempts to obtain tetrafibricin from the producing strain have been unsuccessful19,\ndropping this non-peptidyl antiplatelet drug candidate out of further development.\nMinor Comments:\nThere are some additional questions that are outlined in the points below that I hope will benefit the\nmanuscript:\no In the Abstract portion of the paper or elsewhere appropriate, specify the percentage retention of\nproductivity relative to the previous/native system.\nWe have revised the Abstract as follows (revision underscored):\n… tetrafibricin, at 82 ± 3 mg/L yield, retaining 26% productivity after five-step …\no The manuscript alternates between “engineering step,” “module editing” and “domain swapping.”\nWhile all are fine to use, more consistency would improve readability.\nWe have thoroughly edited the text to improve consistency.\no The comparison in Fig. 2 is convincing, but it would be helpful to state whether to observed\ndifferences are statistically significant.\nWe have assessed the statistical significances and described them in the main text as follows:\nThe differences in yield between PKSv4 and PKSv4_XU (p < 0.001) and PKSv5 and PKSv5_XU (p\n= 0.002) were statistically significant, while those between PKSv1 and PKSv1_XU (p = 0.383) and\nPKSv2 and PKSv2_XU (p = 0.484) were not.\no The bi-products (9) and (10) are interesting. Would they pose a general risk for large-repeat\nengineering strategies? How would it be mitigated?\nGenerally, avoiding a large or even a short tandem repeat can reduce the risk of an unintentional\nrecombination event (such as removing a module before introducing it elsewhere or using alternate\ncodons). In the current case, it’s unclear why the PKSv1 appears recombination-proof, whereas the\nPKSv5 gene does not (both have a complete, large tandem repeat), making the ‘risk’ evaluation\ncomplex. Thus, we consider that the mechanistic and quantitative interpretations of this genetic event\nfall outside our focus. Notably, the phenotype observed after monospore isolation indicates that even\na repeat of more than 5,000 bp can be stably carried by the host strain during subculture.\no The term “engineering efficiency” is used to describe yield retention. Please clarify if this is strictly\nreferring to product titre or also incorporates success rate, stability, etc.\nThe word ‘efficient’ refers to a retention of production titre. We have clarified this in the Introduction\nsection as follows (revision underscored):\n… in practice2-7 the efficient (i.e., retaining production titre) and flexible reprogramming of PKSs …\no Please explain why a geometric mean was used instead of an arithmetic mean in this context.\nWe have added the description as follows (revision underscored):\n(76% geometric mean, representing the step-by-step average of productivity loss)\nReviewer #3 (Remarks to the Author):\nThe manuscript describes an excellent piece of work that will serve as blueprint for PKS engineering\napproaches in the coming years. It consolidates insights gained over the past few years and\nsuccessfully translates them into application. Expectations, such as extensive testing of bioactivity, go\nbeyond the scope of this work. The presentation of this work in the journal Nature would have been\nboth justified and highly desirable.\nThank you for understanding and supporting the importance of our work. We are happy if our results\nhelp others and encourage PKS engineering efforts.\nThe manuscript has been revised with great care. There are two points: (i) As the authors themselves\nstate (Response 1 to Reviewer 3) that “it is not necessary to detail the concept of an ‘updated module’\nor ‘XU’ for introducing the XUA strategy,” the term “updated module boundary” should be removed\nfrom the manuscript. It is sufficient to define the elements of PKSs using the terminology of modules,\nexchange units (XUs), and XU assembly (XUA). (ii) The authors should also refrain from referring to\nfuture work with statements such as “A more systematic study is currently underway.”\n(i) Completely agree. We have removed the term “updated module boundary”.\n(ii) We have removed the sentence accordingly."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Dear Authors,\nThank you for accepting the suggestions made and highlighting the edits made to the paper in your response to the peer review. There is one minor suggestion to be made post-revision. Overall, the paper is strong in both quality and style, and only minor edits need be made before it is published. The authors did a wonderful job of presenting their research, and their conclusions are novel. Furthermore, the work is credible, ethical, and maintains academic integrity.\n1. In Supplementary Figure 2, it would be beneficial to the reader to further explain and describe panel (f) of the diagrams. Further distinguishing between PKSs is important"}, "author_response": "Point-by-point response to Reviewers’ comments\nReviewer #2 (Remarks to the Author):\nThank you for accepting the suggestions made and highlighting the edits made to the paper in your\nresponse to the peer review. There is one minor suggestion to be made post-revision. Overall, the paper\nis strong in both quality and style, and only minor edits need be made before it is published. The\nauthors did a wonderful job of presenting their research, and their conclusions are novel. Furthermore,\nthe work is credible, ethical, and maintains academic integrity.\nWe sincerely appreciate your positive and encouraging comments. We have addressed the remaining\nconcern you raised below.\n1. In Supplementary Figure 2, it would be beneficial to the reader to further explain and describe panel\n(f) of the diagrams. Further distinguishing between PKSs is important.\nWe agree that more detail should be provided in the legend for Supplementary Figure 2 (f).\nAccordingly, we have added the underscored sentences to clarify the points to be compared:\nf … The dashed boxes indicate the difference to be compared. For PKSv1, v2, v4 and v5 variants, the\nPKSs ended with “_XU” are generated using the XU cut sites instead of the XUA cut sites. For PKSv3\nvariants, PKSv3_medTE and PKSv3_pikTE embed the indicated TE without its native, adjacent ACP.\nFor PKSv6 variants, PKSv6(TE) has DstTE like PKSv6 but not its adjacent ACP, while\nPKSv6_amphACP-TE was generated by transplanting both ACP and TE from amphotericin PKS."}]

Decision letter