Metabolic Engineering 39 (2017) 29–37

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Metabolic Engineering
journal homepage: www.elsevier.com/locate/ymben

Engineering of an Lrp family regulator SACE_Lrp improves erythromycin MARK

production in Saccharopolyspora erythraea
Jing Liua, Yunfu Chena, Weiwei Wanga, Min Rena, Panpan Wua, Yansheng Wanga, Changrun Lia,
⁎ ⁎ ⁎
Lixin Zhanga,b, Hang Wua, , David T. Weavera, , Buchang Zhanga,
a
Institute of Health Sciences, School of Chemistry and Chemical Engineering, School of Life Sciences, Anhui University, Hefei 230601, China
b
CAS Key Laboratory of Pathogenic Microbiology & Immunology, Drug Discovery Center for Tuberculosis, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China

A R T I C L E I N F O A BS T RAC T

Keywords: Leucine-responsive regulatory proteins (Lrps) are a group of transcriptional regulators that regulate diverse
Saccharopolyspora erythraea cellular processes in bacteria and archaea. However, the regulatory role of Lrps in antibiotic biosynthesis
Leucine-responsive regulatory protein remains poorly understood. In this study, we show that SACE_5388, an Lrp family regulator named as
Branched-chain amino acid SACE_Lrp, is an efficient regulator for transporting and catabolizing branched-chain amino acids (BCAAs),
Erythromycin
playing an important role in regulating erythromycin production in Saccharopolyspora erythraea. SACE_Lrp
Engineering
directly controlled the expression of the divergently transcribed SACE_5387-5386 operon putatively encoding a
BCAA ABC transporter by interacting with the intergenic region between SACE_Lrp and SACE_5387
(SACE_Lrp-5387-int), and indirectly controlled the expression of ilvE putatively encoding an aminotransferase
catabolizing BCAAs. BCAA catabolism is one source of the precursors for erythromycin biosynthesis. Lysine and
arginine promoted the dissociation of SACE_Lrp from SACE_Lrp -5387-int, whereas histidine increased their
binding. Gene disruption of SACE_Lrp (ΔSACE_Lrp) in S. erythraea A226 resulted in a 25% increase in
erythromycin production, while overexpression of SACE_5387-5386 in A226 enhanced erythromycin produc-
tion by 36%. Deletion of SACE_Lrp (WBΔSACE_Lrp) in the industrial strain S. erythraea WB enhanced
erythromycin production by 19%, and overexpression of SACE_5387-5386 in WBΔSACE_Lrp
(WBΔSACE_Lrp/5387-5386) increased erythromycin production by 41% compared to WB. Additionally,
supplement of 10 mM valine to WBΔSACE_Lrp/5387-5386 culture further increased total erythromycin
production up to 48%. In a 5-L fermenter, the erythromycin accumulation in the engineered strain
WBΔSACE_Lrp/5387-5386 with 10 mM extra valine in the industrial culture media reached 5001 mg/L, a
41% increase over 3503 mg/L of WB. These insights into the molecular regulation of antibiotic biosynthesis by
SACE_Lrp in S. erythraea are instrumental in increasing industrial production of secondary metabolites.

1. Introduction frequently been used to increase the erythromycin production, and S.

erythraea has been studied with respect to polyketide biosynthesis
Saccharopolyspora erythraea is an important filamentous actino- pathway (Adrio and Demain, 2006; Carata et al., 2009; Mironov et al.,
mycete utilized for production of erythromycin (Butler, 2008). 2004).
Erythromycin and its derivatives are broad-spectrum polyketide anti- The engineering of relevant regulatory elements to modulate
biotics that are widely used clinically against pathogenic Gram-positive transcription repression or activation has improved the production of
bacteria (McDaniel et al., 2001). Extensive genetic and biochemical target metabolites (Lu et al., 2007; Niu and Tan, 2013; Santos-
studies have identified the genes involved in erythromycin biosynthesis Aberturas et al., 2011). For example, by tandem deletion of γ-
(Donadio and Staver, 1993; Weber et al., 1990). The erythromycin gene butyrolactone receptor genes, the validamycin production was in-
cluster (ery cluster) contains 20 genes arranged in four major poly- creased by 55% in Streptomyces hygroscopicus (Tan et al., 2015),
cistronic units, but lacks regulatory genes unlike many other antibiotic- and by tandemly overexpressing the TetR regulator SACE_7301 in a
producing actinomycetes (Reeves et al., 1999). Over the past 60 years, high erythromycin-producing S. erythraea WB, the erythromycin
the traditional methods of random mutagenesis and selection have production was enhanced by 27% (Wu et al., 2014a). Recently,

⁎
Corresponding authors.
E-mail addresses: wuh2007@gmail.com (H. Wu), David.weaver.t@gmail.com (D.T. Weaver), zhbc@ahu.edu.cn (B. Zhang).

http://dx.doi.org/10.1016/j.ymben.2016.10.012
Received 1 August 2016; Received in revised form 5 October 2016; Accepted 25 October 2016
Available online 26 October 2016
1096-7176/ © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
J. Liu et al. Metabolic Engineering 39 (2017) 29–37

Rogers et al. developed an MphR biosensor system which could 2.3. Heterologous expression and purification of SACE_Lrp
monitor erythromycin concentration based on GFP reporter, and that
is helpful to explore erythromycin biosynthesis regulation and screen For heterologous expression of SACE_Lrp protein in E. coli, the
high-yield erythromycin producers (Rogers et al., 2015). SACE_Lrp gene was amplified by PCR from the genome of S.
The family of Leucine-responsive Regulatory Proteins (Lrps) is erythraea A226 using the primer pair 5388-28a-F/R, and cloned into
widely distributed among prokaryotes and controls diverse cellular pET28a generating an N-terminal His-tag fusion. The constructed
processes (Brinkman et al., 2003; Peeters and Charlier, 2010). The Lrp plasmid pET28a-Lrp was introduced into E. coli BL21 (DE3), and
from Escherichia coli is the most extensively studied regulator directly protein expression was induced with IPTG at a final concentration of
or indirectly controlling expression of about 400 genes (Ernsting et al., 0.4 mM at 22 °C for 8–10 h. His6-tagged SACE_Lrp protein was
1992; Tani et al., 2002) involved in diverse processes such as amino extracted and purified on a Ni2+-NTA spin column (BIO-RAD). The
acid metabolism and pili synthesis (Calvo and Matthews, 1994; quality of the purified protein was estimated by sodium dodecyl sulfate
Newman and Lin, 1995). Lrp family proteins are also regulators of polyacrylamide gel electrophoresis (SDS-PAGE). The protein concen-
other biological processes, including peptide transport, energy, central tration was determined using the Bradford assay.
metabolism, DNA repair and recombination, bacterial persistence and
virulence (Brinkman et al., 2003; Deng et al., 2011; Peeters and 2.4. Electrophoretic mobility shift assays (EMSAs)
Charlier, 2010). These Lrp family members are site-specific DNA-
binding proteins with molecular masses of around 15–18 kDa consist- The EMSAs were performed as described previously (Hellman and
ing of two principle domains, an N-terminal DNA-binding domain with Fried, 2007). The intergenic segment between SACE_Lrp and
a helix-turn-helix motif and a C-terminal domain, folding into an αβ SACE_5387 (SACE_Lrp-5387-int), and the promoter regions of
sandwich, that is involved in oligomerization and effector binding eryAI and ermE were amplified by PCR with their respective primers.
(Deng et al., 2011; Kumarevel et al., 2008; Peeters and Charlier, 2010). The DNA probes were incubated individually with various concentra-
Lrp homologs from several genera are responsive to a variety of amino tions of His6-tagged SACE_Lrp in binding buffer [10 ml Tris-HCl (pH
acids, for example, the well-studied Lrp of E. coli has been reported to 7.5), 5 mM MgCl2, 60 mM KCl, 10 mM DTT, 50 mM EDTA and 10%
respond to Leu and Ala (Chen and Calvo, 2002; Hart and Blumenthal, glycerol] at 30 °C for 20 min in 20 μl reaction mixture. After incuba-
2011). tion, the samples were fractionated on 6% native PAGE gels in ice-cold
As one of the largest bacterial genera, actinomycetes are a 1× TAE buffer at 60 mA for 30–40 min
particularly abundant source of antibiotics and related compounds,
producing more than half of medically important antimicrobial and 2.5. Verification of the interaction between SACE_Lrp and the
antitumor agents. However, the regulatory role of Lrp regulators in effectors using GFP reporter assay in E. coli
antibiotic biosynthesis of actinomycetes is unclear. Recently, an Lrp
regulatory gene SCO2140 has been reported to be involved in For construction of the reporter plasmid, pUPW-EGFP (Wu et al.,
antibiotics production and morphological differentiation of 2007) was digested with HindIII/NdeI to obtain the enhanced green
Streptomyces coelicolor A3(2), but the mechanism of SCO2140 fluorescent protein gene (egfp) fragment. Also, a fragment containing
regulation in antibiotic production remains unknown (Yu et al., 2016). both SACE_Lrp and the SACE_Lrp-5387-int was amplified, using the
Biosynthesis of erythromycin requires two precursors, propionyl- primer pair Lrp-DE-F and DE-R with NdeI and EcoRI restriction sites.
CoA and methylmalonyl-CoA to provide a starter unit and extender The PCR product and the egfp fragment were together joined into
units for biosynthesis of 6-deoxyerythronolide B (Cortes et al., 1990). corresponding HindIII/EcoRI sites of pKC1139, creating pKC-Lrp-DE.
In S. erythraea, propionyl-CoA and methylmalonyl-CoA are supplied The fragment only containing the SACE_Lrp-5387-int was amplified
from fatty acid catabolic pathways, glycolysis/citrate cycle pathways, using the primer pair DE-F/R and cloned into corresponding NdeI/
and branched-chain amino acid (BCAA) degradation pathways (Li EcoRI sites of pKC-Lrp-DE, creating the pKC-DE as the control. The
et al., 2013; Oliynyk et al., 2007). Increasing the flux of precursor two plasmids were respectively transformed into DH5α, detecting
metabolites strongly influences the erythromycin production (Reeves green fluorescence (excitation at 485 nm; emission at 510 nm,
et al., 2006, 2004). In this study, we identified an Lrp-family protein Molecular Devices) for estimating the interaction of SACE_Lrp and
SACE_5388, named as SACE_Lrp, which divergently transcribed to its effectors. All fluorescence values were normalized to growth rates
SACE_5387-5386 operon putatively encoding a BCAA ABC transporter (OD600).
in S. erythraea (Marcellin et al., 2013; Oliynyk et al., 2007).
Furthermore, using SACE_Lrp approach to regulate erythromycin 2.6. Gene deletion, complementation and overexpression
biosynthesis, we demonstrated a significant improvement in erythro-
mycin production. Gene deletion, complementation and overexpression in S. ery-
thraea were performed as previously described (Wu et al., 2014b).
2. Materials and methods With S. erythraea A226 genomic DNA as a template, two 1.5-kb DNA
fragments flanking the SACE_Lrp were amplified by PCR using the
2.1. Strains, plasmids and growth conditions primer pairs 5388-up-F/R and 5388-down-F/R. The two PCR products
were respectively digested with EcoRI/KpnI and XbaI/HindIII, and
All strains and plasmids used in this study are listed in Table S1. S. ligated into the corresponding sites of pUCTSR (Han et al., 2011),
erythraea and its derivatives were grown with appropriate antibiotics obtaining pUC-ΔSACE_Lrp. By the homologous chromosomic recom-
at 30 °C on solid R3M medium for protoplast regeneration and bination with linearized fragments, a 381-nt fragment of the
phenotypic observation, or in liquid TSB medium for seed culture, SACE_Lrp gene was replaced by thiostrepton resistance gene (tsr) in
DNA extraction and protoplast preparation. The E. coli strains DH5α S. erythraea A226. The desired thiostrepton-resistant mutant, named
and BL21 (DE3) were grown in LB liquid medium or on LB solid plates as S. erythraea ΔSACE_Lrp, was further confirmed by PCR analysis
at 37 °C, for DNA cloning and heterologous SACE_Lrp production, using the primers 5388-C-F/R.
respectively. Using the primers 5388-C-F/R, a 441-bp SACE_Lrp was amplified
by PCR with the genomic DNA of A226 as a template. The PCR product
2.2. Primers was cleaved with NdeI/XbaI, and inserted into the corresponding sites
of integrative expression plasmid pIB139 (Wilkinson et al., 2002),
All primers used in this study are listed in Table S2. yielding pIB-Lrp. By PEG-mediated protoplast transformation, pIB-

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J. Liu et al. Metabolic Engineering 39 (2017) 29–37

Fig. 1. SACE_Lrp directly binds to the Probe PLrp-5387. (A) Genetic locus organization of the SACE_Lrp. (B) Purification of His6-tagged SACE_Lrp. (C) EMSA of binding of SACE_Lrp to
the Probe PLrp-5387.

Lrp was introduced into the ΔSACE_Lrp mutant and the parental intervals). Cell debris was removed by centrifugation at 12,000 rpm
strain A226, respectively. The complemented strain ΔSACE_Lrp/pIB- for 10 min, and the supernatant was treated for the quantification of
Lrp and overexpression strain A226/pIB-Lrp were obtained by apra- intracellular amino acid concentration as above.
mycin resistance screening and confirmed by PCR analysis with the
primers Apr-F/R. 2.9. Fermentation and measurement of erythromycin
For SACE_5387-5386 overexpression in S. erythraea A226, a
1039-bp DNA fragment containing a full-length SACE_5387-5386 For flask fermentation of S. erythraea and its derivatives, spores
was amplified with the primers 5387-F and 5386-R, inserted into the were inoculated into 30 ml of TSB seed medium and grown for 48 h.
corresponding NdeI/XbaI sites of pIB139, and the constructed pIB- 3 ml seed culture was transferred into 30 ml R5 liquid medium. All
5387-5386 was introduced into A226 to obtain the overexpressed fermentation cultures were grown at 220 rpm, 30 °C for 6 days.
strain A226/pIB-5387-5386. For bioreactor cultures, S. erythraea WB and its derivatives were
In accord with above procedures, we constructed WBΔSACE_Lrp, incubated in a 5-L fermenter using industrial culture media (Baoxing,
WBΔSACE_Lrp/5387-5386 and WBΔSACE_Lrp/pIB139 from the Shanghai, China). Samples (50 ml) were taken every 24 h in terms of
industrial S. erythraea WB strain. previously reported method (Wu et al., 2014a).
Erythromycin A was extracted from the fermentation cultures and
2.7. RNA isolation and quantitative real-time PCR assay analyzed by Waters 1500-series HPLC and Waters ACQuity UPLC
(Agilent extend-C18 column, 5 µm, 250×4.6 mm) as previously de-
Total RNA was isolated from S. erythraea A226 and ΔSACE_Lrp scribed (Wu et al., 2014b).
after 2 days grown on R5 liquid medium by the RNA extraction/
purification kit (SBS), and the RNA concentration was determined 2.10. Statistical analysis
using a microplate reader (BioTek). Isolated RNA (400 ng) was treated
with DNase I (MBI Fermentas), and reverse transcription was achieved All the data in this study were stated as means ± standard error of
using a cDNA synthesis kit (MBI Fermentas). Quantitative real-time mean (SEM), and analyzed by Student's t-test, with *P < 0.05 and ** P
PCR reactions were performed on the Applied Biosystems Step-One < 0.01 indicating significant differences.
Plus system with Maxima™ SYBR Green/ROX qPCR Master Mix (MBI
fermentas). The hrdB gene from S. erythraea A226 was used as the 3. Results
internal control to normalize samples.
3.1. SACE_5388 is the Lrp homolog in S. erythraea
2.8. Measurement of free amino acid concentration
The genomic sequence of S. erythraea predicts that SACE_5388 is
A HITACHI l-8900 amino acid analyzer was used for the quanti- an Lrp family homolog (Fig. 1A) (Marcellin et al., 2013; Oliynyk et al.,
fication of free amino acids from S. erythraea A226 and ΔSACE_Lrp 2007), exhibiting 22–56% amino acid identity with previously reported
with the ninhydrine colorimetric method (Wu et al., 2012). The Lrp homologs (Fig. S1), so named as SACE_Lrp. SACE_Lrp shares the
supernatant and the mycelia were separated from the fermentation highest similarity to the Lrp of Corynebacterium glutamicum (56%
medium by centrifugation at 12,000 rpm for 10 min. The supernatant identity, Fig. S1), known to directly control the expression of its
was mixed with 10% salicylsulfonic acid for 20 min at −20 °C, divergently transcribed brnFE encoding a BCAA ABC transporter
centrifuged to discard sediments at 12,000 rpm for 60 min and filtered (Lange et al., 2012). In S. erythraea, SACE_Lrp is divergently oriented
with 0.22 µm water-phase filter for extracellular amino acid measure- from SACE_5387-5386 that is also annotated as a putative BCAA ABC
ment. The mycelia were suspended in 1 ml ddH2O, and disrupted by transporter (Fig. 1A). The similarity between these two species implies
sonication in an ice-bath (20 cycles of 5 s sonication with 10 s that SACE_Lrp may directly control the expression of SACE_5387-

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Fig. 2. SACE_Lrp responds to lysine, arginine and histidine in vitro and in vivo. (A) Effector screen of SACE_Lrp by EMSAs, with lysine, arginine, histidine, etc. All amino acid-effector
concentrations are in mM. (B) An illustration of the reporter plasmids. Addition of the respective amino acid-effectors affects egfp expression in the reporter system. (C) Detection of the
relative fluorescence units (RFUs) after adding lysine, arginine and histidine in E. coli DH5α/pKC-Lrp-DE and DH5α/pKC-DE. The Mean values of three replicates are shown, with the
standard deviation indicated by error bars. *P < 0.05.

5386. Thus, we expressed His6-tagged SACE_Lrp in E. coli BL21 (DE3) 3.3. Inactivation of SACE_Lrp enhances erythromycin production
(Fig. 1B), and examined its affinity to PLrp-5387 (SACE_Lrp-5387-int)
with electrophoretic mobility shift assays (EMSAs). A SACE_Lrp-PLrp- To explore the effect of SACE_Lrp on erythromycin production,
5387 complex formed in a concentration-dependent manner (Fig. 1C), SACE_Lrp was disrupted with tsr replacement in S. erythraea A226
indicating SACE_Lrp is similar to the Lrp from C. glutamicum, and is (Fig. 3A) (Wu et al., 2014b), and the mutant S. erythraea ΔSACE_Lrp
most likely the Lrp family regulator. was confirmed by PCR analysis (Fig. 3B). In comparison with the
parent strain S. erythraea A226, S. erythraea ΔSACE_Lrp improved
erythromycin A production by 25% (P < 0.05). Complementation of the
3.2. SACE_Lrp is responsive to lysine, arginine and histidine SACE_Lrp gene in the deletion mutant recovered erythromycin A
production to the original level (Fig. 3C). ΔSACE_Lrp showed similar
Lrp family regulators are responsive to a variety of amino acids growth rates in R5 culture by the mycelium dry weight and similar
(Peeters and Charlier, 2010). To determine the potential amino acid sporulation rates on R3M agar medium to the parent strain S.
effectors of SACE_Lrp, a series of EMSA analyses were performed with erythraea A226, indicating that SACE_Lrp was not involved in cell
SACE_Lrp binding to the PLrp-5387 probe. Only lysine, arginine and growth and morphological differentiation of S. erythraea (Fig. S2).
histidine from the 20 amino acids influenced the affinity, with lysine To further confirm the negative regulatory role of SACE_Lrp in
and arginine reducing the binding affinity, whereas histidine increasing erythromycin production, pIB139 and pIB-Lrp (Table S1) were respec-
it (Fig. 2A). tively introduced into A226. The results showed that the erythromycin
To investigate whether SACE_Lrp interacted with the three amino A yield of A226/pIB-Lrp was decreased by 23% (P < 0.01) compared to
acids in vivo, we constructed a biosensor system in E. coil DH5α A226/pIB139 (Fig. 3C). Taken together, these findings demonstrated
(Mustafi et al., 2012). The system used two plasmids, pKC-Lrp-DE that SACE_Lrp negatively regulates erythromycin production, and has
expressing the reporter egfp gene and the SACE_Lrp gene both under no effect on cell growth or morphological differentiation in S. ery-
the control of SACE_Lrp-5387-int but in different direction, and pKC- thraea.
DE only expressing the reporter egfp gene under the control of
SACE_Lrp-5387-int (Fig. 2B). When 20 mM lysine or arginine was 3.4. SACE_Lrp regulates erythromycin biosynthesis indirectly
added to the culture media, the green fluorescence was significantly
enhanced by 27% or 43% in DH5α/pKC-Lrp-DE, while the green To investigate the regulatory mode of SACE_Lrp, we measured
fluorescence was reduced by 33% when 20 mM histidine was added transcripts of eryAI (SACE_0721, encoding polyketide synthase I) and
into DH5α/pKC-Lrp-DE culture (Fig. 2C). As the control, the green ermE (SACE_0733, encoding rRNA methyltransferase) by qRT-PCR.
fluorescence showed no difference in DH5α/pKC -DE after the addition The transcriptional levels of eryAI and ermE in ΔSACE_Lrp after two
of 20 mM lysine, arginine or histidine in the culture media. These days of growth were increased by 4- and 2- folds respectively compared
results indicated that in vivo lysine, arginine and histidine are indeed with A226 (Fig. 4A), indicating that SACE_Lrp represses the transcrip-
the effectors of SACE_Lrp, similar to the effects in vitro. tion of eryAI and ermE. However, EMSA tests revealed that SACE_Lrp

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Fig. 3. SACE_Lrp negatively regulates erythromycin production in S. erythraea A226. (A) Schematic deletion of SACE_Lrp by linearized fragment homologous recombination in S.
erythraea A226. (B) PCR confirmation of the SACE_Lrp deletion mutant by the primers 5388-C-F/R. Lanes: M, 5000-bp DNA ladder; 1, the positive control, 1810 bp amplified from
pUC-ΔSACE_Lrp; 2, the negative control, 800 bp amplified from A226; 3, the sample, 1810 bp amplified from mutant ΔSACE_Lrp. (C) Erythromycin A production in S. erythraea A226
and its derivatives by HPLC analysis. Mean values of three replicates are shown, with the standard deviation indicated by error bars. *P < 0.05, **P < 0.01.

could not bind to the promoter regions of eryAI and ermE even up to 3.5. Overexpression of SACE_5387-5386 improves erythromycin
2 µM concentration (Fig. 4B), indicating SACE_Lrp may regulate production
erythromycin biosynthesis indirectly in S. erythraea A226.
Interestingly, the transcripts of the key gene of BCAA metabolism Given the fact that SACE_5387-5386 putatively encoded the BCAA
ilvE (SACE_1646, putative encoding BCAA aminotransferase) and ABC transporter, and qRT-PCR analysis showed transcription of
SACE_5387-5386 in ΔSACE_Lrp were increased by 3- and 6- folds SACE_5387-5386 was increased in ΔSACE_Lrp (Fig. 4A), it was
in comparison to A226 respectively, suggesting that SACE_Lrp may reasonable to explore whether overexpressing SACE_5387-5386 would
regulate BCAA's transport and metabolism. affect erythromycin production in S. erythraea. We therefore intro-
duced pIB-5387-5386 (Table S1) into S. erythraea A226, and as
expected, the A226/pIB-5387-5386 strain enhanced production of
erythromycin A by 31% (P < 0.05) relative to A226/pIB139 (Fig. 5).

Fig. 4. SACE_Lrp indirectly regulates erythromycin biosynthesis. (A) Effects of SACE_Lrp disruption on transcriptional levels of eryAI, ermE, SACE_5387-5386 and ilvE. qRT-PCR
was used to quantify the amounts of transcripts in A226 and ΔSACE_Lrp cultured for 48 h in liquid R5 medium. Mean values of three replicates are shown, with the standard deviation
indicated by error bars. (B) EMSA assays of DNA-binding of SACE_Lrp to PeryAI and PermE. *P < 0.05, **P < 0.01.

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3.7. Engineering high-yield industrial S. erythraea WB enhances

erythromycin production

The above results indicated that deletion of SACE_Lrp, over-

expression of SACE_5387-5386 and supplementation of BCAAs were
efficient strategies to improve the erythromycin production in S.
erythraea A226, so we explored their practical applications in industry.
Firstly, the SACE_Lrp was deleted in a high-yield S. erythraea WB,
and erythromycin A production was increased by 19% (P < 0.01)
compared to its parent S. erythraea WB when cultured in 50 ml
industrial fermentation medium for 6 days (Fig. 7A). Secondly, pIB-
5387-5386 was introduced into WBΔSACE_Lrp, and the engineered
strain WBΔSACE_Lrp/5387-5386 increased erythromycin production
by 41% (P < 0.01) (Fig. 7A). Lastly, when 10 mM valine was added into
the industrial fermentation medium, S. erythraea WBΔSACE_Lrp/
Fig. 5. Effect of SACE_5387-5386 overexpression on erythromycin production in S. 5387-5386 totally increased erythromycin production by 48% (P <
erythraea A226. Mean values of three replicates are shown, with the standard deviation 0.01).
indicated by error bars. *P < 0.05.
We tested erythromycin production of S. erythraea
WBΔSACE_Lrp/5387-5386 in a 5-L fermenter with the industrial
3.6. SACE_Lrp influences BCAA's transport and catabolism medium. The yield of erythromycin A was upgraded from 3503 mg/L
with S. erythraea WB to 4780 mg/L with S. erythraea
To investigate the effect of deleting SACE_Lrp on the transport of WBΔSACE_Lrp/5387-5386, an increase of 36% (Fig. 7B).
BCAAs, we measured the extracellular and intracellular concentrations Furthermore, when 10 mM valine was added into the industrial
of free BCAAs in A226 and ΔSACE_Lrp. Compared to the control strain medium, the erythromycin A yield of S. erythraea WBΔSACE_Lrp/
A226, the extracellular concentrations of leucine, isoleucine and valine 5387-5386 reached 5001 mg/L, a total 41% increase (Fig. 7B).
were reduced by 21%, 14% and 18% with ΔSACE_Lrp respectively
(Fig. 6A), indicating that the deletion of the SACE_Lrp promoted the
transport of BCAAs into the cell. We also found that the intracellular 4. Discussion
concentrations of leucine, isoleucine and valine were markedly reduced
by 52%, 69% and 51% in ΔSACE_Lrp compared to A226 respectively Lrp family regulators are abundantly present in prokaryotes,
(Fig. 6B), indicating that the SACE_Lrp disruption increased intracel- responsive to a variety of amino acids, and involved in various
lular BCAA catabolism. These results demonstrated that more BCAAs biological processes (Lintner et al., 2008). Our present work has
were transported into cell and catabolized in ΔSACE_Lrp, thus identified a novel Lrp-like protein, SACE_Lrp that regulates BCAA
providing more precursors for erythromycin biosynthesis. transport and catabolism, and negatively controls erythromycin bio-
Furthermore, we examined the effect of adding BCAAs to the synthesis, but has no effect on morphological differentiation in S.
growth media on erythromycin production, and found that addition erythraea. In S. coelicolor, disruption of SCO2140 significantly de-
of valine, isoleucine or leucine increased erythromycin production by creased the yields of actinorhodin and calcium-dependent antibiotic,
47% (P < 0.01), 35% (P < 0.01) and 16% in S. erythraea A226, and delayed the aerial mycelium formation in R2YE medium, indicat-
respectively (Fig. 6C). We also examined the effect of the three amino ing SCO2140 positively regulate antibiotics production and morpholo-
acid-effectors of SACE_Lrp on erythromycin biosynthesis, and the gical differentiation (Yu et al., 2016). These results indicate that Lrp
results showed that erythromycin production was increased by 12% homologs may modulate antibiotic biosynthesis in different manners.
and 20% (P < 0.05) after adding 10 mM lysine and arginine to the In typical antibiotic-producing actinomycetes, SACE_Lrp shares
growth media respectively, while no difference in erythromycin pro- high sequence identities with Lrp homologs of Nocardia farcinica
duction was observed with the addition of 10 mM histidine (Fig. S3). (NFA_44740, 81%), Streptosporangium roseum (Sros_7861, 57%),
Streptomyces clavuligerus (SCLAV_5669, 51%), S. venezuelae
(SVEN_0224, 51%) and S. avermitilis (SAV_3764, 52%) (Fig. 8),
inferring that Lrp family regulators are widely distributed in antibiotic-

Fig. 6. SACE_Lrp regulates transport and catabolism of BCAAs and erythromycin production in S. erythraea. (A) Concentrations of the extracellular BACCs at 48 h in A226 and
ΔSACE_Lrp. (B) Concentrations of the intracellular BACCs at 48 h in A226 and ΔSACE_Lrp. (C) Erythromycin A production after adding BCAAs by HPLC analysis. Mean values of three
replicates are shown, with the standard deviation indicated by error bars. **P < 0.01.

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Fig. 7. Using SACE_Lrp pathway to improve erythromycin yields in the industrial S. erythraea WB. (A) Erythromycin A production in S. erythraea WB and its derivatives by HPLC
analysis. Mean values of 3 replicates are shown, with the standard deviation indicated by error bars. **P < 0.01. (B) Time course of erythromycin production of WB and its derivatives in
a 5 L fermenter.

producing actinomycetes. Therefore, there is likely to be a beneficial to different amino acids, such as Lrp from E. coli responding to Leu and
application of our study on Lrp regulation to the engineering of other Ala (Hart and Blumenthal, 2011), NMB0573 from Neisseria meningi-
industrial antibiotic-producers. tidis to Leu and Met (Ren et al., 2007), RV3291c from Mycobacterium
SACE_Lrp regulates the transport of BCAAs (Fig. 6A and B), and tuberculosis to Leu, Met, Ile, His, and Thr (Shrivastava and
supplementation of culture medium with BCAAs, especially valine, Ramachandran, 2007), LysM from Sulfolobus solfataricus to Lys, Arg
markedly increased the production of erythromycin in S. erythraea and Gln (Song et al., 2013). It is noteworthy that all the reported Lrp
A226 (Fig. 6C) as briefly summarized in Fig. S4. These findings are homologs show only one-way response to their effectors, either
consistent with the previous report that the highest yield of erythro- increasing or decreasing binding affinities to their target regulatory
mycin production in S. erythraea 23338 was achieved by supplement- elements. In our study, the DNA-binding affinity of SACE_Lrp
ing valine (Licona-Cassani et al., 2012). Valine has also been reported decreased in the presence of lysine and arginine, while increased in
to be the vital source of precursors for macrolide antibiotics biosynth- the presence of histidine based on in vitro EMSA and in vivo GFP
esis in other actinomycetes (Tang et al., 1994). reporter system (Fig. 2). To our knowledge, SACE_Lrp is the first
When SACE_Lrp was disrupted, intracellular BCAA concentration reported Lrp showing opposing responses to different effectors, and a
became lower, indicating that SACE_Lrp may regulate amino acid deeper understanding of the fundamental utility of this regulation is
metabolism in S. erythraea A226 (Fig. 6B). Indeed, Lrp homologs have warranted.
been found to regulate amino acid metabolism in E. coli, Some regulators have an auto-regulatory function, such as WhiA
Mycobacterium tuberculosis and hyperthermophilic archaeon from S. venezuelae, AveT from S. avermitilis and SprA from S.
Pyrococcus (Deng et al., 2011; Peeters and Charlier, 2010). qRT-PCR chattanoogensis (Bush et al., 2013; Liu et al., 2015; Zhou et al.,
results showed that inactivation of SACE_Lrp up-regulated ilvE which 2015). As shown in Fig. S6A, the intergenic segment SACE_Lrp-5387-
putatively encodes BCAA aminotransferase(Fig. 4A). However, the int was predicted as a bidirectional promoter, simultaneously control-
EMSA tests revealed that SACE_Lrp did not bind to the promoter ling the expression of SACE_Lrp and SACE_5387-5386. Given that
region of ilvE (Fig. S5), indicating that SACE_Lrp may mediate amino SACE_Lrp could directly regulate the expression of the SACE_5387-
transfer of BCAA metabolism indirectly, and it will be of interest to 5386, our work further indicated that SACE_Lrp also acts as an auto-
investigate these mechanisms in the future. regulator responding to three amino acid-effectors based on a biosen-
Lrp homologs utilize amino acids as effector molecules, but respond sor system (Fig. S6), but the detailed mechanism needs to be probed.

Fig. 8. Neighbor-joining (NJ) distance tree constructed using SACE_Lrp amino acid sequence from the known genome sequences of antibiotic-producing actinomycetes using MEGA6.
Percentages represent the identities between Lrp homologs and SACE_Lrp.

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J. Liu et al. Metabolic Engineering 39 (2017) 29–37

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Supplementary data associated with this article can be found in the S.F., Leadlay, P.F., 2007. Complete genome sequence of the erythromycin-producing
bacterium Saccharopolyspora erythraea NRRL23338. Nat. Biotechnol. 25, 447–453.
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