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Metabolic engineering strategies based on secondary messengers (p)ppGpp and c-di-GMP to increase erythromycin yield in Saccharopolyspora erythraea Zhen Xu, Di You, Li-Ya Tang, Ying Zhou, and Bang-Ce Ye ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00372 • Publication Date (Web): 11 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019
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ACS Synthetic Biology
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Metabolic engineering strategies based on secondary
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messengers (p)ppGpp and c-di-GMP to increase
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erythromycin yield in Saccharopolyspora erythraea
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Zhen Xu1,2, Di You2, Li-Ya Tang2, Ying Zhou2, Bang-Ce Ye1,2*
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1Institute
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Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of
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Technology, Hangzhou 310014, Zhejiang, China.
of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River
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2Lab
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University of Science and Technology, Shanghai 200237, China.
of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China
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Corresponding author
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Bang-Ce Ye
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Professor, Lab of Biosystems and Microanalysis,
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State Key Laboratory of Bioreactor Engineering,
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East China University of Science and Technology, Shanghai 200237, China
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Tel/Fax: 0086-21-64252094
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Email:
[email protected] 21 22
Running title: Metabolic engineering based on secondary messengers
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Keywords: second messengers, nitrogen limitation, (p)ppGpp, c-di-GMP, relA, antibiotics
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biosynthesis
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Abstract
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Secondary messengers (such as (p)ppGpp and c-di-GMP) were proved to play
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important roles in antibiotic biosynthesis in actinobacteria. In this study, we found
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that transcription levels of erythromycin-biosynthetic (ery) genes were upregulated
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in nutrient-limitation, which depended on (p)ppGpp in Saccharopolyspora erythraea.
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Further study demonstrated that the expression of ery genes and intracellular
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concentrations of (p)ppGpp showed synchronization during culture process. The
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erythromycin yield was significantly improved (about 200%) by increasing
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intracellular concentration of (p)ppGpp through introduction of C-terminally
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truncated (p)ppGpp synthetase RelA (1.43 kb of the N-terminal segment) from
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Streptomyces
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WT/pIB-PBAD-relA1-489). As the intracellular concentration of (p)ppGpp in an industrial
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erythromycin-high-producing strain E3 was greatly higher (about 10- to 100-fold)
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than WT strain, the applications of the above-described strategy didn’t work in E3
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strain. Further research revealed that low concentration of 2-oxoglutarate in E3
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strain exerted a “nitrogen-rich” pseudo-signal, leading to the downregulation of
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nitrogen metabolism genes which limited the use of nitrogen sources and thus the
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high intracellular (p)ppGpp concentration. Furthermore, the secondary messenger,
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c-di-GMP, was proved to be able to activate ery genes transcription by enhancing
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binding of BldD to promoters of ery genes. Overexpressing the diguanylate cyclase
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CdgB from S. coelicolor in S. erythraea increased the intracellular c-di-GMP
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concentration, and improved erythromycin production. These findings demonstrated
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that increasing the concentration of intracellular secondary messengers can activate
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ery genes transcription, and provided new strategies for designing metabolic
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engineering based on secondary messengers to improve antibiotics yield in
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actinobacteria.
coelicolor
into
S.
erythraea
strain
NRRL2338
(named
as
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Graphical abstract
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Transcription levels of erythromycin-biosynthetic (ery) genes were upregulated by
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increasing intracellular concentration of (p)ppGpp and c-di-GMP in S. erythraea. Our
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study demonstrated new strategies for designing metabolic engineering based on
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secondary messengers to improve antibiotics yield in actinobacteria.
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In all domains of life, nucleotide-based second messengers allow an almost
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immediate cellular response to changing environmental conditions. In prokaryotes,
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nucleotide-based
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pentaphosphate/tetraphosphate
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monophosphate (c-di-GMP), play an important and ubiquitous role in regulating
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relevant life-style transition. The accumulation of (p)ppGpp that induces the
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stringent response is in response to nutritional stresses 1. When cells are starved for
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amino acids, the (p)ppGpp synthetase RelA is directly activated by binding to
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ribosomes blocked by uncharged tRNA at the A site 2. Cellular processes regulated by
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secondary messenger (p)ppGpp are various and conserved in bacteria, such as amino
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acid biosynthesis, rRNA and tRNA synthesis, antibiotic resistance, virulence of
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pathogens, long-term persistence, competence and biofilm formation 3-11. (p)ppGpp
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also exhibits regulatory effects on antibiotic biosynthesis in actinobacteria
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prokaryotes, c-di-GMP has emerged as an important and ubiquitous second
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messenger regulating many important bacterial processes. In general, c-di-GMP
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modulates bacteria motility
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production
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effectors and RNA effectors 27. BldD, which controls the progression of multicellular
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differentiation in actinomycetes was also reported to be a c-di-GMP receptor 28.
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Actinomycetes are known to produce a large number of secondary metabolites, such
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as antibiotics or other pharmaceutically active compounds. The production of
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secondary metabolites is generally linked to nutrient limitation and morphological
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differentiation, and (p)ppGpp have been proved to play a crucial role in these
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processes
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nitrogen limitation requires the presence of the (p)ppGpp synthetase gene, relA 15, 29,
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30.
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C-terminally deleted derivative (relA1-489) of S. coelicolor relA (1.46-kb of N-terminal
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segment) under the control of the thiostrepton-inducible tipA promoter could result
26,
second
21,
messengers, ((p)ppGpp)
virulence
22-24,
such and
as cyclic
cell cycle progression
guanosine di-guanosine
25,
12-20.
In
antibiotic
and so on. C-di-GMP receptors are highly diverse, including protein
12, 15, 18-20.
In S. coelicolor, accumulation of (p)ppGpp under conditions of
It was proved that, in a relA null mutant of S. coelicolor, expression of a
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in (p)ppGpp accumulation in a ribosome-independent mode and restore antibiotic
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production 12, 14, 31, 32. Previously, BldD was reported to regulates transcription of key
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developmental genes in Streptomyces 33 and binds to the promoter of ery clusters in
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S. erythraea
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tetramer that mediates the effective dimerization of BldD, which activated the DNA
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binding activity of BldD. C-di-GMP is synthesized from GTP by diguanylate cyclases
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(DGCs) that share a conserved domain containing the amino acid motif GGDEF and
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degraded by phosphodiesterases (PDEs) that contain one of two conserved domain
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families: EAL motif and HD-GYP motif 35. Overexpressing the active DGC CdgB from S.
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coelicolor blocked development, and overexpression of the PDE YhjH from E. coli
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induced precocious hyper-sporulation without formation of aerial hyphae of S.
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venezuelae. These data suggested that intracellular levels of c-di-GMP binding to
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BldD controlled the developmental switch between vegetative growth and
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sporulation 28.
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In this study, we investigated the relationship between the above two
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nucleotide-based second messengers ((p)ppGpp and c-di-GMP) and erythromycin
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biosynthesis in S. erythraea. The results demonstrated that up-regulation of ery
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genes induced by nitrogen-limitation required (p)ppGpp and BldD-mediated
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regulation of ery genes was activated by c-di-GMP. The erythromycin yield was
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significantly improved by increasing intracellular concentration of (p)ppGpp and
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c-di-GMP. The findings revealed a link between erythromycin biosynthesis and
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secondary messengers, and provided a new metabolic engineering strategy based on
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second messengers to improve antibiotics yield in actinobacteria.
34.
Recently, C-di-GMP was proved to be able to assemble into a
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Results and Discussion
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Up-regulation of erythromycin biosynthetic genes induced by nitrogen-limitation
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depended on (p)ppGpp.
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Erythromycin production was decreased when S. erythraea was grown on YSA
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medium supplemented with 3 mM ammonium compared with no ammonium
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addition
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(SACE_0721) was up regulated when the utilization of nitrogen sources is limited 37.
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Here, we further investigated the transcription of five erythromycin synthetic genes
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(belonging to five transcription units of ery clusters) (SACE_0713, SACE_0717,
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SACE_0720, SACE_0721, and SACE_0731) of S. erythraea cultivated in nitrogen-rich
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(N+) and nitrogen-limited (N-) sources.
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As shown in Figure 1, transcripts of the erythromycin synthetic genes were
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up-regulated 2- to 34-fold in nitrogen-limited compared with nitrogen-rich in three
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different growth stages (early-exponential phase, late-exponential phase, and
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stationary phase). These results indicated that the expression of the erythromycin
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biosynthetic genes are induced by nitrogen limitation.
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(p)ppGpp synthesized in response to nutrient-limitation was proved to exhibit
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regulatory effects on antibiotic biosynthesis in Streptomyces
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investigate the effect of (p)ppGpp on erythromycin biosynthetic genes of S.
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erythraea, we constructed a relA-deleted mutant (ΔrelA). As shown in Figure 2A, no
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(p)ppGpp was detected in the ΔrelA strain, suggesting that RelA protein was
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responsible for (p)ppGpp synthesis in S. erythraea. The effect of the relA mutation on
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morphological differentiation was examined. The results showed that deletion of
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relA delayed morphological differentiation compared with the wild-type strain (WT)
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(Fig. 2B). Scanning electron microscopy (SEM) further revealed that the WT strain
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had clear aerial hyphae (Fig. 2C), whereas the development (aerial hyphae growing
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into the air) of ΔrelA was blocked (Fig. 2D). The pigment production of ΔrelA was also
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affected (Fig. 2E).
36.
Previous study showed that erythromycin synthetic gene eryA1
12, 13, 15, 16.
In order to
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Figure 1. The expression of the erythromycin biosynthetic genes was promoted in nitrogen limitation. The transcriptional levels of erythromycin biosynthetic genes (SACE_0713, SACE_0717, SACE_0720, SACE_0721, and SACE_0731) of S. erythraea NRRL2338 cultivated in nitrogen-rich 7 / 40
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(N+) and nitrogen-limited (N-) Evans medium in early-exponential phase (A), late-exponential phase (B), and stationary phase (C). Three independent replicates were used to calculate the standard deviation. *P < 0.05, **P < 0.01, ***P
