The PurR family transcriptional regulator promotes butenyl-spinosyn production in Saccharopolyspora pogona

Abstract

Butenyl-spinosyn, derived from Saccharopolyspora pogona, is a broad-spectrum and effective bioinsecticide. However, the regulatory mechanism affecting butenyl-spinosyn synthesis has not been fully elucidated, which hindered the improvement of production. Here, a high-production strain S. pogona H2 was generated by Cobalt-60 γ-ray mutagenesis, which showed a 2.7-fold increase in production compared to the wild-type strain S. pogona ASAGF58. A comparative transcriptomic analysis between S. pogona ASAGF58 and H2 was performed to elucidate the high-production mechanism that more precursors and energy were used to synthesize of butenyl-spinosyn. Fortunately, a PurR family transcriptional regulator TF00350 was discovered. TF00350 overexpression strain RS00350 induced morphological differentiation and butenyl-spinosyn production, ultimately leading to a 5.5-fold increase in butenyl-spinosyn production (141.5 ± 1.03 mg/L). Through transcriptomics analysis, most genes related to purine metabolism pathway were downregulated, and the butenyl-spinosyn biosynthesis gene was upregulated by increasing the concentration of c-di-GMP and decreasing the concentration of c-di-AMP. These results provide valuable insights for further mining key regulators and improving butenyl-spinosyn production.

Key points
• A high production strain of S. pogona H2 was obtained by⁶⁰Co γ-ray mutagenesis.
• Positive regulator TF00350 identified by transcriptomics, increasing butenyl-spinosyn production by 5.5-fold.
• TF00350 regulated of butenyl-spinosyn production by second messengers.

Full text

Vol.:(0123456789)
Applied Microbiology and Biotechnology (2025) 109:14
https://doi.org/10.1007/s00253-024-13390-1
APPLIED MICROBIAL ANDCELL PHYSIOLOGY
The PurR family transcriptional regulator promotes butenyl‑spinosyn
production inSaccharopolyspora pogona
XinyingLi1,2· JingnanWang2· ChangSu2· ChaoGuo2· ZhouqinXu2· KehuiWang2· JianPang3,5· BoLv1·
ChaoWang2· ChunLi1,3,4
Received: 11 September 2024 / Revised: 11 December 2024 / Accepted: 18 December 2024
© The Author(s) 2025
Abstract
Butenyl-spinosyn, derived from Saccharopolyspora pogona, is a broad-spectrum and effective bioinsecticide. However, the
regulatory mechanism affecting butenyl-spinosyn synthesis has not been fully elucidated, which hindered the improvement
of production. Here, a high-production strain S. pogona H2 was generated by Cobalt-60 γ-ray mutagenesis, which showed a
2.7-fold increase in production compared to the wild-type strain S. pogona ASAGF58. A comparative transcriptomic analy-
sis between S. pogona ASAGF58 and H2 was performed to elucidate the high-production mechanism that more precursors
and energy were used to synthesize of butenyl-spinosyn. Fortunately, a PurR family transcriptional regulator TF00350 was
discovered. TF00350 overexpression strain RS00350 induced morphological differentiation and butenyl-spinosyn produc-
tion, ultimately leading to a 5.5-fold increase in butenyl-spinosyn production (141.5 ± 1.03mg/L). Through transcriptomics
analysis, most genes related to purine metabolism pathway were downregulated, and the butenyl-spinosyn biosynthesis gene
was upregulated by increasing the concentration of c-di-GMP and decreasing the concentration of c-di-AMP. These results
provide valuable insights for further mining key regulators and improving butenyl-spinosyn production.
Key points
• A high production strain of S. pogona H2 was obtained by 60Co γ-ray mutagenesis.
• Positive regulator TF00350 identified by transcriptomics, increasing butenyl-spinosyn production by 5.5-fold.
• TF00350 regulated of butenyl-spinosyn production by second messengers.
Keywords Butenyl-spinosyn· PurR family transcriptional regulator· Saccharopolyspora pogona· Transcriptomic
Introduction
Butenyl-spinosyn, discovered from the actinomycete Sac-
charopolyspora pogona, is a 26-membered ring macrolide
with broad-spectrum insecticidal activities (Hahn etal. 2006;
Lewer etal. 2009). The structure of butenyl-spinosyn is sim-
ilar to that of spinosad, with a distinct tetra-cyclic macrolide
with forosamine and tri-O-methylrhamnose (Santos and
Pereira 2020). The main difference is a 2-butenyl group at
the C21 carbon position of an ethyl group. Butenyl-spinosyn
* Bo Lv
lv-b@bit.edu.cn
* Chao Wang
wc@ags.ac.cn
* Chun Li
lichun@mail.tsinghua.edu.cn
1 Key Laboratory ofMedical Molecule Science
andPharmaceutics Engineering, Ministry ofIndustry
andInformation Technology, Institute ofBiochemical
Engineering, School ofChemistry andChemical
Engineering, Beijing Institute ofTechnology,
Beijing100081, China
2 Academy ofNational Food andStrategic Reserves
Administration, Grain andOils Processing Research Institute,
Beijing100037, China
3 Key Lab forIndustrial Biocatalysis, Ministry ofEducation,
Department ofChemical Engineering, Tsinghua University,
Beijing100084, China
4 Center forSynthetic andSystems Biology, Department
ofChemical Engineering, Tsinghua University,
Beijing100084, China
5 Key Laboratory forNorthern Urban, Agriculture ofMinistry
ofAgriculture andRural Affairs, Beijing University
ofAgriculture, Beijing102206, China
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Applied Microbiology and Biotechnology (2025) 109:14 14 Page 2 of 14
has the same insecticidal mechanism of action as spinosad
and exhibits a broader spectrum of insecticidal activity in
a constantly overstimulated insect central nervous system
by binding to nicotinic acetylcholine receptors (nAChR)
or γ-aminobutyric acid receptors (GABARs). In addition,
butenyl-spinosyn is rapidly degraded by sunlight and micro-
organisms in soils without environmental pollution (Liu and
Li 2004; Zhao etal. 2015). Therefore, butenyl-spinosyn as
an environmentally friendly insecticide shows promising
prospects for further development. However, the limited pro-
ductivity of wild-type strains has hindered its wider adop-
tion and utilization. Overcoming the challenge of increasing
the yield of butenyl-spinosyn is a critical issue that requires
immediate attention. Various approaches including mutagen-
esis, ribosome engineering, and genetic engineering have
been employed to develop high-yielding strains.
A variety of mutagenic methods, including chemical
mutagenesis (N-methyl-N’-nitro-N-nitrosoguanidine, NTG),
physical mutagenesis (cobalt-60 (60Co) γ-ray, ultraviolet)
as well as atmospheric room temperature plasma (ARTP)
mutations are remarkable in improving strain characteristics.
Compound mutagenesis and iterative random mutagenesis
are efficient methods that could overcome the limitations of
single mutagenesis. The butenyl-spinosyn production in S.
pogona was improved by fivefold through ribosome engi-
neering combined with ARTP/UV (Zhao etal. 2024). How-
ever, it is important to note that mutagenesis methods are
characterized by their stochastic nature, lengthy processes
and potential for genetic instability. As research into micro-
bial metabolic pathways and genetic laws deepened, rational
design and genetic modification were applied to microbial
genetic breeding. Fortunately, 23 genes were well character-
ized in the spinosad and its derivatives biosynthesis path-
way, including 19 genes located within the bus cluster and
an additional 4 genes responsible for the biosynthesis of
the rhamnosyl moiety distributed elsewhere in the genome
(Hahn etal. 2006; Guo etal. 2020). Thus, metabolic engi-
neering strategies have been utilized to improve production
by upregulating the expression of structural genes, increas-
ing the supply precursors, and eliminating competing path-
ways (Rang etal. 2020c; He etal. 2021; Tang etal. 2021a).
Currently, the combined regulation of polyketide synthase
(PKS) and succinic semialdehyde dehydrogenase (GabD)
genes has improved butenyl-spinosyn production (Rang
etal. 2020b, 2022a). Several transcriptional regulators, such
as Bfr, LytS-L, pII, RegX3, Sp1418 and SP2854, have also
been identified for their ability to increase production by
globally affecting the carbon or nitrogen metabolism (He
etal. 2020, 2021; Rang etal. 2020a, 2021, 2022b; Tang
etal. 2021b; Hu etal. 2022) and in addition to the effects
on butenyl-spinosyn synthesis, PNPase and AfsR (a tran-
scriptional regulator from the SARP family) also affected
strain morphological characteristics (Li etal. 2018, 2019).
However, although many studies have been conducted on
S. pogona, a PurR family transcriptional regulator on strain
metabolism and regulation is still unpublished.
In this study, 20 candidate transcriptional regulators were
identified by comparative transcriptomic analysis between
a high-performance mutant designated S. pogona H2 and
the wild-type strain S. pogona ASAGF58. Among these
factors, TF00350, a member of the PurR family transcrip-
tional regulators, was characterized to enhance the produc-
tion of butenyl-spinosyn. Then, the mechanism of TF00350
involving the second messengers cyclic dimeric (3′−5′)
GMP (c-di-GMP) and c-di-AMP, stimulating the synthesis
of more precursors for secondary metabolites, influencing
the growth and morphological changes of the mycelium was
analyzed. This study has provided insight into how the regu-
latory factor TF00350 facilitates the biosynthesis of target
products and also offers a strategy for identifying key regu-
latory factors.
Materials andmethods
Strains, primers, andmedium
All strains and primers used in the studies are summarized
in Supplementary TableS1. Escherichia coli was cultured
in Luria–Bertani medium (tryptone 10g/L, NaCl 10g/L,
yeast extract 5g/L) or on LB agar medium at 37°C with
antibiotics (50mg/L of apramycin).
S. pogona ASAGF58 (accession number: CCTCC M
20241920) was cultivated on GYM (glucose 4g/L, yeast
extract 4g/L, CaCO3 2g/L, agar 20 g/L) plates for sporu-
lation. Then, fresh spores were collected and cultured in
Tryptone Soya Broth (TSB, 30 g/L) for 48h. Next, a 10%
(vol/vol) seed culture was used to inoculate the fermenta-
tion medium (glucose 60g/L, soluble starch 20g/L, corn
steep solid 10g/L, peptonized milk 20g/L, CaCO3 5g/L,
MgSO4·7H2O 1g/L, NaCl 1g/L, 1 g/L vegetable oil, pH
7.2), and cultivated at 30°C at 200 rpm for 168 h. The
96-well plate used for mutation screening was inoculated at
220 rpm for 216 h. The filling volume was 600 μL per well
of each well-plate.
60Co γ‑ray mutagenesis
Fresh harvest spores of S. pogona were resuspended in ster-
ile water in a 0.02% sterile glucose solution and incubated
for 12 h at 30°C. The spores were removed into a sterile 1.5
mL tube, wrapped in tinfoil, and mutagenized by 60Co γ-ray
at different doses (10Gy, 30Gy, 60Gy, 90Gy, 120Gy, and
150 Gy). Then, the spore suspensions were diluted to dif-
ferent concentrations (10–4, 10–5, 10–6, and 10–7) and spread
on GYM plates for 168 h at 30℃. Three replicates were
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Applied Microbiology and Biotechnology (2025) 109:14 Page 3 of 14 14
measured for each concentration. The spores were screened
randomly at different concentrations for subsequent 96-well
plate fermentation as the primary screen. After the primary
screening, 40 high-production mutants were further screened
for re-screening by shake-flask fermentation. Then, the high-
performance strain H2 was obtained after three consecutive
rounds of subculturing from the 40 mutants.
Morphological andfermentation characteristics
ofS. pogona
Mycelia of S. pogona were grown on a GYM plate for 72h
and 120h, scraped from the agar surface, and lyophilized
(vacuum lyophilization). Subsequently, morphological char-
acteristics were examined using Jeol JSM-IT700HR (Jeol
Ltd., Tokyo, Japan).
Butenyl-spinosyn was analyzed by high-performance liq-
uid chromatography (HPLC), and the analysis conditions
were as follows: Waters 2998 HPLC system (Waters, Mil-
ford, MA, USA) equipped with an Agilent reversed-phase
TC-C18 column (100 × 4.6 mm, 3.5μm) (Agilent Tech-
nologies, Santa Clara, CA, USA). The elution solvent was
45% acetonitrile, 45% methanol, and 10% water with 0.05%
ammonium acetate. The samples were analyzed at a flow
rate of 1.0 mL/min for 10 min at a UV detector detection
wavelength of 244 nm.
The biomass was determined by the wet weight method,
biomass relative quantity (%) = (M3-M1)/(M2-M1) × 100%.
M1 was the weight of Eppendorf tube. M2 was the weight
of the tube with the fermentation broth. M3 was the weight
of the tube after centrifugation (4000rpm for 10min) and
the supernatant was discarded. The SBA-40E biosensor
analyzer (Biology Institute of Shangdong Academy of Sci-
ences, China) was used to determine the glucose concen-
tration. Acyl-CoAs and second messengers were measured
by using commercial enzyme-linked immunosorbent assay
(ELISA) kits (Shanghai Enzyme-linked Biotechnology Co.,
Ltd., China).
Construction ofgenetically engineered strains
Plasmids pSET159-TFs containing genes encoding tran-
scriptional regulators were constructed by Gibson Assem-
bly (Pang etal. 2023). Using pSET159-permE as a tem-
plate, 6kb fragments were amplified by V159-F/R primer
pairs to be used in subsequent cloning as a vector back-
bone. Each transcriptional regulator gene fragment was
amplified from the S. pogona ASAGF58 genome (acces-
sion number: PRJNA534337) with a 25 bp homologous
arm and cloned into the vector backbone mentioned above.
Then, these recombinant plasmids were transformed into
E. coli DH5α (Song etal. 2015) and verified by sequenc-
ing. The correct plasmids were transformed to E. coli
S17-1 (Jiang etal. 2023), which was used as a donor for
the intergeneric conjugation into S. pogona (Bierman etal.
1992).
Transcriptome sequencing ofS. pogona ASAGF58
andH2
S. pogona samples were harvested at 72 h, 120 h, and 192
h for transcriptome sequencing. The fermentation broth
was centrifuged at 12,000 rpm at 4°C and the supernatant
was discarded. The cell precipitate was frozen with liquid
nitrogen and then stored at −80℃ while awaiting tran-
scriptome sequencing. Samples were prepared from two
biological replicates. Transcriptome sequencing Library
preparation and sequencing Transcriptome sequencing
was performed by Novogene Bioinformatics Technology
Co., Ltd. (Beijing, China). Differentially expressed genes
(DEGs) were screened based on log2|fold change|> 1 and
p < 0.05. Then, gene ontology (GO) and Kyoto Encyclo-
pedia of Genes and Genomes (KEGG) enrichment path-
way analyses were performed on the differential expressed
genes.
RNA extraction andquantitative real‑time PCR
(qRT‑PCR) analysis
Total RNA was extracted from S. pogona using the RNAp-
rep Pure Cell/Bacteria Kit or RNAprep Pure Tissue Kit
(Tiangen Biotech., Beijing, China). The RNA was reverse
transcribed to cDNA using the First Strand cDNA Max-
ima Synthesis kit (TOYOBO, Shanghai, China). Real-
time qPCR amplification was carried out using Power
SYBR Green PCR Master Mix (Thermo Fisher Scientific,
Waltham, USA) under previously documented procedures.
The primer sequences used in qRT-PCR are listed in Sup-
plementary TableS1. The 16S rRNA gene was utilized
as an internal reference to assess the relative expression
levels of the target genes.
Statistical analysis
All experiment data were presented as the mean val-
ues ± standard deviation (SD). The statistical analysis and
most of the graphs were generated in Origin (Origin Lab,
Northampton, MA, USA) or GraphPad Prism software
(GraphPad Software, Inc. La Jolla, CA, USA). Statisti-
cal significance was analyzed by Student’s t-test (two-tail)
with ***p < 0.001, **p < 0.01, and *p < 0.05.
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Applied Microbiology and Biotechnology (2025) 109:14 14 Page 4 of 14
Results
60Co mutagenesis andscreening ofhigh
butenyl‑spinosyn producing strains
The S. pogona spores were exposed to different doses of
60Co γ-ray (10 Gy, 30 Gy, 60 Gy, 90 Gy, 120 Gy, and 150
Gy). A total of 500 mutants were screened in a 96-well
plate, and lethality and positive mutation rates were calcu-
lated. When spores were treated with 60 Gy, the lethality
rate reached 90%, and the positive mutation rate reached a
maximum of 60%. A high-performance strain designated
H2 was obtained by rescreening from 40 mutations (Fig.1a
and 1b). Subsequently, the fermentation performance of
H2 was investigated in batch culture. Butenyl-spinosyn
was extracted from the fermentation broth, and the titer of
butenyl-spinosyn was determined by HPLC and LC–MS.
Butenyl-spinosyn was found at m/z 758.48076 at a retention
time of 7.8min (Supplemental Fig.S1). As shown in Fig.1c,
butenyl-spinosyn was not detected at 24h cultivation in S.
pogona ASAGF58 and H2, but it was detected at 48 h and
was efficiently and rapidly synthesized up to 192h of cultiva-
tion in strain H2. At 192h, butenyl-spinosyn production by
H2 was 2.7 times higher than that in ASAGF58. Similarly, a
Fig. 1 Fermentation profiles of ASAGF58 and high-yield strain H2.
a The process of mutant strain screening and validation. b Lethality
rate and mutation rate of S. pogona. c Comparison of butenyl-spino-
syn production in ASAGF58 and H2. d Comparison of glucose con-
sumption between ASAGF58 and H2. e The biomass of ASAGF58
and H2
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Applied Microbiology and Biotechnology (2025) 109:14 Page 5 of 14 14
faster rate of glucose consumption and biomass production
was observed in H2 compared to ASAGF58, with a difference
of 16.7g/L glucose consumed at 192 h (Fig.1d). The biomass
of H2 exceeded that of ASAGF58, with H2 reaching its maxi-
mum growth level at 144h, whereas ASAGF58 reached its
plateau at only 96h (Fig.1e). Meanwhile, the transcript levels
of five polyketide synthase genes (busA/B/C/D/E) respon-
sible for the synthesis of the carbon chain backbone were
upregulated, especially at 120h, demonstrating that butenyl-
spinosyn production was enhanced in strain H2 (Hahn etal.
2006) (Supplemental Fig.S2). It was hypothesised that high
biomass accumulation and glucose consumption promoted
the synthesis of butenyl-spinosyn.
Identification PurR family transcriptional regulator
TF00350
To gain a deeper insight into the underlying mechanisms of
high yield production, transcriptomics was performed on
wild-type ASAGF58 and the high yield mutant strain H2.
The differences between ASAGF58 and H2 were analyzed
at the logarithmic phase (72h), stationary phase (120h),
and decline phase (192 h). The number of upregulated and
downregulated DEGs in these strains was counted. DEGs
(Log2FC ≥ 1 or ≤ −1) were mainly concentrated at 72h, with
2,140 upregulated and 1,265 downregulated genes (Fig.2a).
The results of the KEGG pathway enrichment analysis
showed that numerous DEGs were involved in pathways
such as ribosome, oxidative phosphorylation, glycolysis/
gluconeogenesis and biosynthesis of amino acids (Fig.2b).
Changes in primary and secondary metabolism provided
more precursors and energy for cell growth and second-
ary metabolism. At 120h and 192h, DEGs were mainly
involved in the biosynthesis of secondary metabolites and
signal transduction (Fig.2c and 2d). It was hypothesized
that some potential primary signaling molecules were able
to globally regulate secondary metabolite production.
The efficient synthesis of butenyl-spinosyn suggested that
the key precursors and energy were being synthesized and
used to synthesize secondary metabolites. However, most
genes involved in the ribosome, oxidative phosphorylation,
and purine metabolism pathways exhibited downregulated
72 120192
0
500
1000
1500
2000
2500
3000
sGEDforebmuN
Time (h)
Up Down
a
b
d
c
0.00.5 1.01.5
KEGG enrichment analysis (72h)
Rich factor
Count
6.00
24.74
102.00
Methanemetabolism
Chlorocyclohexane andchlorobenzene degradation
Biotin metabolism
Two-componentsystem
Nitrogen metabolism
Chloroalkane andchloroalkenedegradation
Fattyacidbiosynthesis
Biosynthesis of siderophoregroup nonribosomal peptides
Microbialmetabolismindiverse environments
Biosynthesisof12-,14- and16-membered macrolides
Prodigiosinbiosynthesis
Tryptophan metabolism
Fattyacidmetabolism
Steroiddegradation
Type Ipolyketidestructures
0.00.5 1.01.5
KEGG enrichment analysis (120 h)
Rich factor
Count
1.00
5.48
30.00
Methanemetabolism
Chlorocyclohexaneand chlorobenzenedegradation
Biotin metabolism
Two-componentsystem
Nitrogen metabolism
Chloroalkane andchloroalkenedegradation
Fattyacidbiosynthesis
Biosynthesis of siderophoregroup nonribosomal peptides
Microbialmetabolismindiverse environments
Biosynthesis of 12-, 14-and 16-memberedmacrolides
Prodigiosinbiosynthesis
Tryptophan metabolism
Fattyacidmetabolism
Steroiddegradation
Type Ipolyketidestructures
0.00.5 1.01.5
KEGG enrichment analysis (192 h)
Rich factor
Count
2.00
8.94
40.00
Methanemetabolism
Chlorocyclohexaneand chlorobenzenedegradation
Biotin metabolism
Two-componentsystem
Nitrogen metabolism
Chloroalkane andchloroalkenedegradation
Fattyacidbiosynthesis
Biosynthesis of siderophoregroup nonribosomal peptides
Microbialmetabolismindiverse environments
Biosynthesisof12-,14- and16-membered macrolides
Prodigiosinbiosynthesis
Tryptophan metabolism
Fattyacidmetabolism
Steroiddegradation
Type Ipolyketidestructures
Fig. 2 Transcription analysis of ASAGF58 and H2. a Comparative transcriptome analysis of the DEGs between ASAGF58 and H2. b-d Bubble
plot of the significant pathways with KEGG enrichment of DEGs at 72, 120, and 192 h, respectively
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Applied Microbiology and Biotechnology (2025) 109:14 14 Page 6 of 14
expression, affecting crucial processes such as DNA repli-
cation, transcription, and protein synthesis (Fig.3 and Sup-
plemental Fig.S3). It was not conducive to the growth and
metabolism of S. pogona. In fact, the growth and metabo-
lism of H2 were better than that in ASAGF58 throughout
the fermentation process, suggesting that intermediates and
energy may have been redistributed and effectively utilized
to enhance the synthesis of secondary metabolites. The syn-
thesis of secondary metabolites is an extremely complex
process. It is highly controlled by nutrient conditions, sig-
nal molecular or pathway-specific and global transcriptional
regulators rather than the gene alone.
Fig. 3 The differences in pivotal metabolic pathways between
H2 and ASAGF58 in the ribosome, oxidative phosphoryla-
tion, and purine metabolism pathways. The heatmap colors rep-
resent gene expression levels (Log2FC) for each sample, as
indicated in the legend on the right. PRPP: phosphoribosyl
pyrophosphate, PRA: 5-phosphoribosylamine, GAR: 5’-phospho-
ribosylglycinamide, FGAR: 5’-phosphoribosyl-N-formylglycina-
mide, FGAM: 2-(formamido)-N1-(5’-phosphoribosyl) acetamidine,
AIR: aminoimidazole ribotide, CAIR: 1-(5-phospho-D-ribosyl)−5-
amino-4-imidazolecarboxylate, SAICAR: 1-(5’-phosphoribosyl)−5-
amino-4-(N-succinocarboxamide)-imidazole, AICAR: 1-(5’-
phosphoribosyl)−5-amino-4-imidazolecarboxamide, FAICAR: 1-(5’-
phosphoribosyl)−5-formamido-4-imidazolecarboxamide, IMP:
inosine monophosphate, XMP: xanthosine monophosphate, GMP:
guanosine monophosphate, GDP: guanosine diphosphate, GTP:
gnosine 5’-triphosphate, AMP: adenosine monophosphate, ADP:
adenosine 5’-diphosphate, ATP: adenosine 5’-triphosphate, purF:
gutamine phosphoribosyl pyrophosphate amidotransferase, purD:
phosphoribosylglycinamide synthetase, purN: phosphoribosylgly-
cinamide formyltransferase, purS/L/Q: phosphoribosylformylgly-
cinamidine synthase, purM: phosphoribosylaminoimidazole syn-
thetase, purC: phosphoribosylaminoimidazole-succinocarboxamide
synthetase, purB: adenylosuccinate lyase, purH: phosphoribosylami-
noimidazolecarboxamide formyltransferase, guaB: IMP dehydro-
genase, guaA: guanylate synthetase, gmk: guanylate kinase, adk:
adenylate kinase, pyk: pyruvate kinase, hpt: hypoxanthine phospho-
ribosyltransferase, apt: adenine phosphoribosyltransferase, rpsA/B
/C/D/E/F/G/H/I/J/K/L/M/N/O/P/Q/R2/S/T: 30S ribosomal protein,
atpA/B/C/D/E/F/G/H: ATP synthase, sdhA/B/C/D: succinate dehy-
drogenase, nuoA/B/C/D/E/F/G/H/I/J/K/L/M/N: NADH dehydro-
genase, cydA/B: cytochrome d oxidase subunit, cyoE: heme o syn-
thase, qcrB/C: cytochrome c reductase, coxA/B/C: cytochrome c
oxidase
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Applied Microbiology and Biotechnology (2025) 109:14 Page 7 of 14 14
To confirm the above speculation, 20 candidate regula-
tory factors with high transcript levels were identified, and
the engineered strains, overexpressing the regulatory fac-
tors, were constructed to validate the role of these genes in
the synthesis of butenyl-spinosyn (Supplementary TableS2
and Fig.4a). The results showed that 14 regulatory factors
had a positive influence on the yield of butenyl-spinosyn.
The overexpression strains RS00350 and RS35905, corre-
sponding to the regulatory factors TF00350 and TF35905,
exhibited a 5.5-fold and 3.6-fold increase in yield compared
to the wild-type strain, respectively.
Sequence analysis revealed that TF035905, which
belongs to the TetR repressor family, was similar to the MftR
repressor responsible for regulating the synthesis of myco-
factsin (MFT). MFT had been shown to be important for
maintaining cellular redox balance in the absence of oxygen
(Mendauletova and Latham 2022). Redox homeostasis was
critical for maintaining energy and metabolism balances.
TF00350, a PurR family transcription factor, negatively
regulated the purine pathway. Additional of adenine and
hypoxanthine to the medium of ASAGF58 did not affect
the synthesis of butenyl-spinosyn (Supplemental Fig.S4).
However, DEGs associated with purine metabolism were
significantly downregulated in H2 (Fig.3). We speculated
that purine played an important role in metabolic pathways
involved in nitrogen sources, signaling molecules, energy
sources, and maintenance of purine homeostasis.
Effects ofTF00350 onbutenyl‑spinosyn production
andstrain growth
To investigate the effect of TF00350 on butenyl-spinosyn
production, a butenyl-spinosyn cumulative curve was estab-
lished. The production capacity of ASAGF58 and RS00350
was determined by HPLC. The butenyl-spinosyn could
be detected on the second day and rapidly accumulated in
RS00350. Conversely, the ASAGF58 started to produce
butenyl-spinosyn at 72 h (Fig.5a). The transcription of
twenty-three butenyl-spinosyn biosynthesis genes was ana-
lyzed at 72, 120, and 192 h. The results showed that the
expression level of most of the bus gene cluster was signifi-
cantly upregulated (Fig.5b). To study the effect of RS00350
on growth in a fermentation medium, the growth charac-
teristics of the ASAGF58 and RS00350 were measured at
different time points. During the first 120h of cultivation,
the biomass of the engineered strain RS00350, characterized
by a smooth surface, was lower than that of ASAGF58. As
time progressed, the mycelial surface of RS00350 remained
smooth and did not develop any spike-like protrusions, in
contrast to ASAGF58 (Fig.5c). From the perspectives of
biomass accumulation and glucose consumption, it was also
shown that the growth rate was accelerated in the logarith-
mic phase and more glucose was consumed for the biosyn-
thesis of secondary metabolites (Fig.5d and 5e). Therefore,
it was speculated that altering the metabolic properties pro-
moted the production of butenyl-spinosyn.
Elucidation oftheregulatory mechanism ofTF00350
function
In order to understand the regulatory mechanism of
TF00350, transcriptomic analysis was performed on
ASAGF58 and TF00350 overexpression strain RS00350 at
72 and 120 h, respectively. Firstly, focusing on the transcrip-
tion levels of genes involved in the butenyl-spinosyn bio-
synthesis pathway, it was found that the genes were strongly
upregulated at 72 and 120 h, which was consistent with the
qRT-PCR results (Supplemental Fig.S5). The expression
Fig. 4 The function validation of 20 transcriptional regulators. a Flowchart of the functional validation of transcriptional regulators. bThe pro-
duction of butenyl-spinosyn in transcriptional regulator overexpression strain
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Applied Microbiology and Biotechnology (2025) 109:14 14 Page 8 of 14
levels of genes encoding several key precursors involved
in the polyketide chain were significantly changed, such as
branched-chain amino acid aminotransferase (BCAT), dihy-
drolipoyl dehydrogenase (OVDH) and acyl-CoA dehydroge-
nase (ADD) (Fig.6a). Meanwhile, the improved production
in RS00350 consumed more propionyl-CoA, malonyl-CoA,
and methylmalonyl-CoA, which are the important precur-
sors in the biosynthesis of polyketide chain (Fig.6b). The
high expression level of the bus biosynthesis cluster and the
high consumption of precursors may promote the synthesis
of butenyl-spinosyn. Furthermore, 817 DEGs were found
between 72 and 120h, with 485 upregulated DEGs and 332
downregulated DEGs (Log2FC ≥ 2 or ≤ −2) (Fig.6c and
6d). GO functional enrichment analysis revealed no signifi-
cant enrichment of upregulated molecular functions other
than catalytic activity. However, the downregulated path-
ways were mainly enriched in transcription activity, protein
kinase activity and signal transducer related activity (Fig.6e
and Supplemental Fig.S6a). KEGG analysis also revealed
that DEGs were mainly associated with enriched nitrogen
metabolism, two-component system (TCS), and macrolides
biosynthesis (Supplemental Fig. S6b and S6c). The changes
in signaling molecules and the two-component system
probably suggested that TF00350 may indirectly regulate
the synthesis of butenyl-spinosyn via signaling molecules.
A homology to TF00350, as a regulator of purine metabo-
lism, a homolog to was shown in Streptomyces to regulate
intracellular concentrations of purines, including second
messenger c-di-GMP and c-di-AMP (Sivapragasam and
Grove 2019). In TCS, external stimuli are sensed by a mem-
brane-anchored histidine kinase (HK), which is then phos-
phorylated. The stimuli are then transduced via the phos-
phoryl group to the REC (receiver) domain of the response
regulator (RR) (Alvarez and Georgellis 2023). Second, mes-
sengers play a key role in converting the output signaling of
the HK-RR system into intracellular responses (Zschiedrich
etal. 2016). It has been reported that changes in second mes-
senger levels could regulate morphological differentiation
and secondary metabolite production (Wang etal. 2024; You
etal. 2024). The transcript levels of second messenger syn-
thetase related genes, dgc (for diguanylate cyclases) and xdh
(for xanthine dehydrogenase) responsible for the synthesis
of c-di-GMP, showed a significant upregulation, and disA
(endoding diadenylate cyclase), responsible for the synthesis
of c-di-AMP, showed a significant downregulation (Supple-
mental Fig.S7). To demonstrate the changes in c-di-GMP
Fig. 5 The effect of TF00350 on the growth and metabolism of S.
pogona. a Comparison of butenyl-spinosyn production between
ASAGF58 and RS00350. b Gene expression levels of the butenyl-
spinosyn biosynthetic gene. busA/B/C/D/E: polyketide synthase,
busJ: dehydrogenase, busM/L: cyclase, busF: [4 + 2]-carbocyclases,
busG: rhamnosyltransferase, busH/I/K: O-methyl-transferase, busN:
3-ketoreductase, busO: 2,3-dehydratase, busP: forosamyltransferase,
busQ: 3,4-dehydratase, busR: transaminase, busS: dimethyltrans-
ferase, epi: 3′5’-epimerase, gdh: NDP-glucose-dehydratase, gtt:
NDP-glucose synthase, kre: 4’-ketoreductase. c Mycelial morphol-
ogy comparison between ASAGF58 and RS00350 at 72 and 120 h.
d Growth curve analysis and (e) Glucose consumption of ASAGF58
and RS00350
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Applied Microbiology and Biotechnology (2025) 109:14 Page 9 of 14 14
and c-di-AMP, the concentrations were detected during the
fermentation processes. The concentration of c-di-GMP in
H2 showed an increasing trend from 48 to 120h, reaching a
maximum at 120h (Fig.7a). The concentration of c-di-AMP
remained stable throughout the process. However, it was sig-
nificantly lower than that of ASAGF58 (Fig.7b). Therefore,
it was speculated that TF00350 inhibited the purine pathway
and reduced the concentration of c-di-AMP, but improved
c-di-GMP through purine salvage pathways. Indeed, c-di-
GMP and c-di-AMP were global transcription factors that
affect cell metabolism by binding to their specific recep-
tor and altering protein function. At the same time, the
improvement of butenyl-spinosyn production is the result
of the upregulated transcript levels of biosynthetic gene and
the use of precursors with the proven metabolic remodeling
(Fig.5a and 5b). In addition, the downregulated transcript
levels of growth genes (bldC, whiA, whiB, whiG, ftsK, ftsW,
and ssgA) and the changes in mycelium morphology also
showed that the metabolism was regulated by second mes-
sengers (Fig.7c).
Discussion
The lack of a well-established genetic manipulation method
in S. pogona made modifying the metabolic reaction net-
works difficult. Therefore, iterative rounds of random
mutagenesis were used to further increase the production.
60Co γ-ray can readily induce DNA breakage and base dam-
age with a high mutation rate and a low revertant mutation
Fig. 6 Transcription analysis between ASAGF58 and RS00350. a
Schematic illustration of gene expression in precursor synthesis, pro-
tein name colored as red corresponds to upregulated genes. BCAT:
branched-chain amino acid aminotransferase, BCKDHA: 2-oxois-
ovalerate dehydrogenase E1 component, BCKDHB: 2-oxoisovaler-
ate dehydrogenase E2 component, ACAD: acyl-CoA dehydrogenase,
OXCT1: 3-oxoacid CoA-transferase subunit, ACAA: acetyl-CoA
acyltransferase, HIBADH: 3-hydroxyisobutyrate dehydrogenase,
GPI: glucose-6-phosphate isomerase, PFK: ATP-dependent phos-
phofructokinase, ALDO: 1,6-diphosphofructose aldolase, G: glu-
cose-1P, G6P: glucose 6-phosphate, F6P: fructose 6-phosphate,
FBP: fructose 1,6-bisphosphate, G3P: glyceraldehyde 3-phosphate,
4-MO: 4-methyl-2-oxopentanoate, 3-ME: S-(3-methylbutanoyl)-
dihydrolipoamide-E, 3-MA: 3-methyl-1-hydroxybutyl-ThPP, 3-MeA:
3-methylcrotonyl-CoA, 3-MgA: 3-methylglutaconyl-CoA, 3-M-2-OP:
3-methyl-2-oxopentanoic acid, 2-ME: S-(2-methylbutanoyl)-dihy-
drolipoamide-E, 2-MBC: 2-methylbutanoyl-CoA, T-2-MA: 2-meth-
ylbut-2-enoyl-CoA, 3-H-2-MA: 3-hydroxy-2-methylbutyryl-CoA,
2-MAA: 2-methylacetoacetyl-CoA, 3-MO: 3-methyl-2-oxobutanoic
acid, 3-HA: 3-hydroxyisobutyryl-CoA, MS: methylmalonate semi-
aldehyde. b The concentration of propionyl-CoA, malonyl-CoA, and
methylmalonyl-CoA in ASAGF58 and RS00350 at 72 and 120h. c–d
Upregulated and downregulated genes were detected in RS00350 and
ASAGF58. e GO functional enrichment analysis of downregulated
genes
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Applied Microbiology and Biotechnology (2025) 109:14 14 Page 10 of 14
rate. Tylosin production increased by 2.7 ± 0.22-fold in
Streptomyces fradiae NRRL-2702 after UV and gamma
irradiation mutagenesis (Khaliq etal. 2009). The micro-
algae mutant Scenedesmus sp. Z-4 increased lipid content
by 1.13-fold by 60Co γ-ray mutation (Liu etal. 2015). The
mutant strain Paecilomyces hepiali ZJB18001 improved
cordycepin production by 2.3-fold through 60Co γ-ray and
ultraviolet irradiation (Cai etal. 2021). In this work, S.
pogona has been used at different doses of 60Co γ-ray to
improve the production. With increased dose levels, lethality
is raised with increasing doses of treatment. When treated
with 60Gy, the positive mutation rate reached a maximum
but decreased under other conditions. Finally, a high-yield
strain, S. pogona H2, was screened with a 2.7-fold increase
in butenyl-spinosyn production.
To explore the mechanism of high production, com-
parative transcriptomics was performed between S. pogona
ASAGF58 and H2. Transcriptome analysis showed that
gene expression levels of the glycolysis and tricarboxylic
acid cycle pathways were low in the logarithmic phase and
increased in the stationary phase in the high-yield strain S.
pogona H2 (Supplemental Fig.S3). On the contrary, almost
all genes had a high expression level in the early stage of fer-
mentation and remained unchanged or decreased in the mid-
dle and late stages in S. pogona ASAGF58, indicating that it
was very important to provide sufficient energy or precursors
for the synthesis of butenyl-spinosyn (Fig.2). The synthesis
of secondary metabolites was controlled by complex regula-
tory networks. LysR family regulatory factor has been iden-
tified as a regulator of spinosyn synthesis, but the specific
transcription factor responsible for regulating butenyl-spi-
nosyn synthesis remains unknown (Mu etal. 2024). Mining
regulators with significantly altered transcript levels through
transcriptomics were a rapid and crucial strategy to guide
the discovery of new transcription factor that specifically
regulate the biosynthetic gene cluster. In our work, 20 can-
didate transcriptional regulators were identified by compara-
tive transcriptomics. Of all the transcriptional regulators,
Fig. 7 The regulatory mechanism of PurR on S. pogona. The concen-
tration of (a) c-di-GMP and (b) c-di-AMP in H2 and ASAGF58. c
Schematic illustration of gene expression related to purine metabolic
pathway and strain growth. whiA/B: sporulation regulatory protein,
whiG: RNA polymerase sigma factor, ftsK/W: cell division protein,
ssgA: spore division protein, bldC: developmental transcriptional
regulator
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Applied Microbiology and Biotechnology (2025) 109:14 Page 11 of 14 14
TF00350, a PurR family transcriptional regulator, has never
been reported to be involved in secondary metabolism syn-
thesis and could dramatically improve butenyl-spinosyn
yield in this study. The PurR family transcriptional repres-
sor is widely distributed in bacterial genomes (Xiao etal.
2023). PurR regulates de novo purine nucleotide biosyn-
thesis and transport by sensing various small signals (such
as PRPP (phosphoribosyl pyrophosphate), hypoxanthine,
guanine, and (p)ppGpp) from the external environment
(Anderson etal. 2022; Houlberg and Jensen 1983; Travis
and Schumacher 2021). PurR is crucial for maintaining the
balance of purine concentration in microorganisms. Purines
are essential components of nucleic acids and perform vari-
ous functions such as acting as cofactors (e.g., FAD, NAD,
NADPH, CoA), signaling molecules (e.g., cAMP, c-di-AMP,
and c-di-GMP), phosphate donors, and primary carriers of
cellular energy (Cho etal. 2011). Purines serve as a nitrogen
source for certain microorganisms (Papakostas etal. 2013).
In addition to its primary functions, PurR has been impli-
cated in a number of biological activities, such as increasing
virulence and promoting adaptability to stress conditions
(Goncheva Mariya etal. 2020; Sause etal. 2019; Xiao etal.
2023). Therefore, enzymes or metabolites involved in the
purine metabolic pathways play a crucial role in maintain-
ing cellular homeostasis and responding to changes in the
surrounding environment.
It was speculated that TF00350 exerted global regula-
tory functions mediated by second messenger. By tran-
scriptomic analyses, we proposed a molecular mechanism
in which TF00350 could transmit a pseudosignal that
upregulated the survival gene associated with nutrient
acquisition and downregulated the growth gene associ-
ated with ribosome synthesis and oxidative phosphoryla-
tion pathway by directly regulating the content of purines.
Then, the resources in the cell were redistributed to cope
with the pseudosignal of environmental changes. Sub-
sequently, S. pogona underwent morphological differ-
entiation and produced antibiotics. TF00350 indirectly
promoted the energy and precursors toward the synthesis
of butenyl-spinosyn. The mycelium morphologies of S.
pogona were changed from a smooth to a spike-like state
by unknown genes. c-di-GMP or c-di-AMP may be the
pseudosignal that TF00350 indirectly affected morpholog-
ical differentiation and antibiotics production. The second
messenger c-di-GMP, abundant in actinomycetes, is syn-
thesized from two GTPs by diguanylate cyclases (DGCs)
and degraded by phosphodiesterases (PDEs) (Chan etal.
2004; Christen etal. 2005). Alteration of c-di-GMP lev-
els significantly affects antibiotic biosynthesis and mor-
phological differentiation (Liu etal. 2019; Makitrynskyy
etal. 2020). In Streptomyces, aerial mycelium formation
and spore germination are regulated by the bld (bald) loci
and whi (white) genes through c-di-GMP binding (Bignell
etal. 2014; Willemse etal. 2011). c-di-GMP forms a com-
plex with BldD or RsiG-σWhiG to inhibit the expression
of differentiation genes (Schumacher etal. 2022). Some
studies have shown that high levels of c-di-GMP hinder
the bacteria in vegetative growth, while low levels of c-di-
GMP lead to accelerated hypersporulation (Gallagher
etal. 2020; Tschowri etal. 2014). Interestingly, secondary
metabolites were now also be associated with morpho-
logical differentiation. However, the role of c-di-GMP in
regulating antibiotic production is not understood. Overex-
pression of DGCs can increase toyocamycin production in
Streptomyces diastatochromogenes and erythromycin pro-
duction in Saccharopolyspora erythraea, while decreasing
actinorhodin production in Streptomyces coelicolor and
moenomycin A production in Streptomyces ghanaensis
ATCC14672 (Liu etal. 2019; Nuzzo etal. 2021; Wang
etal. 2024; Xu etal. 2019). c-di-AMP is another com-
mon second messenger in Streptomyces, synthesized from
two ATPs by diadenylate cyclases (DACs) and degraded
by a c-di-AMP-specific phosphodiesterase (PDE) (Yin
etal. 2020). c-di-AMP plays an important role in stress,
potassium transport, biofilm formation, spore formation,
and antibiotic production (Commichau etal. 2018; Xiong
etal. 2020; You etal. 2024). In S. erythraea, as c-di-AMP
concentration improves, erythromycin production also
increases. BldD (c-di-GMP effector) is a target of DasR
(a c-di-AMP effector). It has been suggested that there is
a regulatory association between c-di-GMP and c-di-AMP
(You etal. 2024). In our work, the concentration of c-di-
GMP and c-di-AMP also showed a highly negative cor-
relation. However, the reason for the c-di-GMP and c-di-
AMP concentration trend is unknown. PurR suppressed
the de novo purine synthesis pathway, but purines could
be obtained from the medium, degradation of nucleo-
tides, and a salvage pathway to maintain cellular survival.
Although TF00350 has been shown to affect second mes-
sengers of synthesis, the mechanisms involved still need to
be tested and proven in many more experiments.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00253- 024- 13390-1.
Author contribution XL and JW conducted the experiments, ana-
lyzed data, and wrote the manuscript. KW and CG performed the
mutant strain construction. XL, JW, CS, and ZX performed HPLC
and MS analyses. All authors discussed the results. BL and JP pro-
vided assistance in data analysis and manuscript revision. CW, BL,
and CL designed and strictly supervised the project. All authors read
and approved the manuscript.
Funding This research was funded by the National Key Research and
Development Program of China (2023YFA0914700), the National
Natural Science Foundation of China (22108153), and the Foundation
of Key Laboratory of Industrial Biocatalysis. Ministry of Education.
Tsinghua University (No.2023003).
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Applied Microbiology and Biotechnology (2025) 109:14 14 Page 12 of 14
Data availability The raw datasets of time-course transcriptome and
comparative transcriptome between wild-type ASAGF58, H2, and
TR00350 used in this study have been deposited in the NCBI SRA
database (accession number: PRJNA1145573). All data analyzed in
the study are included in this published article and its additional infor-
mation files.
Declarations
Ethical approval This article does not contain any studies with human
participants performed by any of the authors.
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons
Attribution-NonCommercial-NoDerivatives 4.0 International License,
which permits any non-commercial use, sharing, distribution and repro-
duction in any medium or format, as long as you give appropriate credit
to the original author(s) and the source, provide a link to the Creative
Commons licence, and indicate if you modified the licensed material.
You do not have permission under this licence to share adapted material
derived from this article or parts of it. The images or other third party
material in this article are included in the article’s Creative Commons
licence, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons licence and
your intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://crea-
tivecommons.org/licenses/by-nc-nd/4.0/.
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