Regulatory genes and their roles for improvement of antibiotic biosynthesis in Streptomyces

Abstract

The numerous secondary metabolites in Streptomyces spp. are crucial for various applications. For example, cephamycin C is used as an antibiotic, and avermectin is used as an insecticide. Specifically, antibiotic yield is closely related to many factors, such as the external environment, nutrition (including nitrogen and carbon sources), biosynthetic efficiency and the regulatory mechanisms in producing strains. There are various types of regulatory genes that work in different ways, such as pleiotropic (or global) regulatory genes, cluster-situated regulators, which are also called pathway-specific regulatory genes, and many other regulators. The study of regulatory genes that influence antibiotic biosynthesis in Streptomyces spp. not only provides a theoretical basis for antibiotic biosynthesis in Streptomyces but also helps to increase the yield of antibiotics via molecular manipulation of these regulatory genes. Currently, more and more emphasis is being placed on the regulatory genes of antibiotic biosynthetic gene clusters in Streptomyces spp., and many studies on these genes have been performed to improve the yield of antibiotics in Streptomyces. This paper lists many antibiotic biosynthesis regulatory genes in Streptomyces spp. and focuses on frequently investigated regulatory genes that are involved in pathway-specific regulation and pleiotropic regulation and their applications in genetic engineering.

Introduction

Secondary metabolites of Streptomyces, including antibiotics, immunomodulators, enzyme inhibitors and other bioactive substances, often have significant medicinal value. However, wild strains usually produce low levels of antibiotics. There is a large and complicated regulatory network in many Streptomyces strains, and the biosynthesis of one antibiotic in one strain may be controlled by more than one regulatory mechanism. For example, the production of actinorhodin (ACT) in Streptomyces coelicolor is regulated by both the cluster-situated regulator (CSR) actII-ORF4 (Fernández-Moreno et al. 1991) and the pleiotropic regulatory gene cprB (Onaka et al. 1998). Further, there is also more than one regulatory gene that affects the biosynthesis of a single antibiotic. For example, dnrI, dnrO and dnrN (Kitani et al. 2008; Parajuli et al. 2005) all regulate the production of daunorubicin (DNR) in Streptomyces peucetius.

To better and more comprehensively understand the mechanisms of antibiotic biosynthesis regulation, in this paper, we classify the antibiotic biosynthetic regulators in various Streptomyces strains that are associated with antibiotic production into three types. This work will provide a theoretical basis for the molecular perturbation of regulatory genes and will help with manipulating the antibiotic biosynthetic pathways and accordingly improving antibiotic production.

Regulatory genes involved in pathway-specific regulation

The CSRs, located within the antibiotic biosynthetic clusters, can modulate the antibiotic biosynthetic genes of the clusters in which they are included (Martín and Liras 2010; Rodríguez et al. 2013). The CSRs can only affect the biosynthetic pathway of a single, specific antibiotic and act as a master switch for biosynthesis of that individual antibiotic; this regulation is called pathway-specific regulation (Bibb 1996; Novakova et al. 2011). Some CSRs encode proteins that belong to a family known as the Streptomyces antibiotic regulatory proteins (SARPs). SARPs contain two characteristic structural domains: an OmpR DNA-binding domain and a bacterial transcription activation domain (BTAD) (Tanaka et al. 2007). The SARPs include the positive regulators ActII-ORF4 (Fernández-Moreno et al. 1991) and RedD (Wilson et al. 2001) in S. coelicolor and DnrI (Parajuli et al. 2005) in S. peucetius. In addition, some CSRs encode proteins belonging to another family, called the LuxR family, which is often found in Gram-negative bacteria (Lei et al. 2007). LuxR-type regulators contain a nucleotide triphosphate (NTP) binding motif at the N-terminus and a helix-turn-helix (HTH) motif at the C-terminus; examples include the positive regulators PimR (Antón et al. 2004) in Streptomyces natalensis and PikD (Xue et al. 1998) in Streptomyces venezuelae. There are many additional families of regulatory proteins, including the LysR and TetR families. The CSRs are shown in Table 1. Huang et al. (2005) reported that some CSRs can also control the expression of pleiotropic genes, and some pleiotropic regulators can affect the expression of CSRs. In this paper, we will not describe the cross-regulation between various types of regulators.

Table 1.

Regulatory genes involved in pathway-specific regulation

Regulatory gene(s)	Gene(s) from	Product(s) of regulation	Function	Notes	References
actII-ORF4	S. coelicolor	ACT	+	Encodes a SARP	Fernández-Moreno et al. (1991)
mmyR	S. coelicolor	Methylenomycin	−	A gene adjacent to mmyR provides positive regulation	Arias et al. (1999)
cdaR	S. coelicolor	Mmy and CDA	+	Encodes a SARP	Bibb (2005)
redD and redX	S. coelicolor	RED	+	Encode SARPs, and the transcription of redX regulates the transcription of redD	Romero-Rodríguez et al. (2015), Takano et al. (1992) and Wilson et al. (2001)
gdmRI and gdmRII	S. hygroscopicus 179977	Geldanamycin	+	Regulate the transcription of pks, gdmF and gdnA, which are involved in biosynthesis of geldanamycin	(He et al. 2008)
tylR, tylS, tylP, tylQ tylT and tylU	S. fradiae	Tylosin	tylR, tylS, tyl T and tylU: + tylQ: −	tylT and tylS encode SARPs, and TylP is similar to γ-butyrolactone receptor proteins	Bate et al. (1999, 2006) and Stratigopoulos and Cundliffe (2002)
aveR, aveR1, aveR2 and aveT	S. avermitilis	Avermectin	+	aveR contains a HTH motif; AveT belongs to the TetR family and activates transcription of aveR	Ikeda et al. (2003)
alpT, alpU, alpV, alpW and alpZ	S. ambofaciens	Alpomycin	alpT, alpU, and alpV: +; alpW: −	alpT, alpU and alpV encode SARPs, alpW encodes a transcriptional repressor protein, and alpZ encodes a γ-butyrolactone receptor protein	Aigle et al. (2005)
ccaR	S. clavuligerus	Cephamycin C and clavulanic acid	+	Encodes a SARP	Pérez-Llarena et al. (1997)
pimR	S. natalensis	Pimaricin	+	Encodes a LuxR family protein, does not regulate its own transcription	Antón et al. (2004)
pikD	S. venezuelae	Pikromycin	+	Encodes a LuxR family protein	Xue et al. (1998)
monH, monRI and monRII	S. cinnamonensis	Monensin	+	MonH is similar to PikD, and monRI encodes a SARP	Oliynyk et al. (2003)
spbR	S. pristinaespiralis	Pristinamycin	+	SpbR is a γ-butyrolactone receptor	Mast et al. (2015)
papR1-R5	S. pristinaespiralis	Pritinamycin	papR1, papR2 and papR4: +; papR3 and papR5: −	PapR1, PapR2, and PapR4 are SARPs, and PapR3 and PapR5 belong to the TetR family	Mast et al. (2015)
jadR* and jadR3	S. venezuelae	Jadomycin B	−	JadR* is a TetR-like protein, and JadR3 represses jadR2 and jadR3 but activates jadR1	Yang et al. (2001), Zhang et al. (2013) and Zou et al. (2014)
nysRI-RIII	S. noursei ATCC	Nystatin	+	Deletion of nysRI abolishes the transcription of nysRII-III	Sekurova et al. (2004)
amph RI- RIII	S. nodosus	Amphotericin	+	All contain a HTH motif in C-terminal	Carmody et al. (2004)
fscRI-RIII	S. pp. FR008	Candicidin	+	Encodes a LuxR family protein	Chen et al. (2003)
dnrI, dnrO and dnrN	S. peucetius	Daunorubicin	dnrI: +; dnrO: −; dnrN: +	DnrN is a RR belonging to the Uhp-LuxR superfamily and activates the transcription of dnrI, and DnrO positively controls dnrN	Otten et al. (2000) and Parajuli and Moon (2002)
strR	S. griseus	Streptomycin	+	Regulates streptomycin by activating the expression of strA and strB	Distler et al. (1987)
rapH, rapG and rapY	S. hygroscopicus	Rapamycin	rapH, rapG: + rapY: −	RapG and RapY each contain a HTH motif, and RapH contains a DNA-binding motif and an ATP-binding site	Yoo et al. (2015)
srrX, srrY, srrZ and srrB	S. rochei	Lankamycin and lancadicin	srrX and ssrY: + for both ssrZ: + for lankamycin srrB: − for both	srrY and srrZ encode SARPs, and ssrY positively regulates ssrZ	Arakawa et al. (2007) and Suzuki et al. (2010)
scbR and scbR2	S. coelicolor	ACT, RED, CDA and yCPK	ACT, CDA and RED: + yCPK: −	ScbR is a γ-butyrolactone receptor protein, and ScbR2 is an antibiotic receptor protein	Li et al. (2015a)
polR and polY	S. cacaoi	Polyoxin	+	Both encode SARPs, and the transcription of polR is positively regulated by polY	Hwang et al. (2003)
aur1PR3 and aur1PR4	S. aureofaciens	Auricin	+	Both encode SARPs, aur1PR3 is controlled by Aur1R, and Aur1P directly regulates the expression of aur1PR4	Rehakova et al. (2013)
barA, barB and varR,	S. virginiae	Virginiamycin	−	BarA, BarB and VarR are TetR-like regulators, and the transcription of barB is tightly repressed by BarA	Matsuno et al. (2003), Nakano et al. (2000) and Namwat et al. (2001)
vmsS, vmsT and vmsR	S. virginiae	Virginiamycin M and virginiamycin S	vmsS and vmsR: + for virginiamycin M and virginiamycin S; vmsT: − for virginiamycin M	VmsS and VmsR are SARPs, and VmsT is a RR of a TCS	Pulsawat et al. (2009)
aur1R	S. aureofaciens	Auricin	−	aur1R encodes a homolog of the TetR family, and Aur1R represses the expression of aur1P	Novakova et al. (2010)
fdmR1	S. griseus	Fredericamycin	+	Encodes a homologue of SARPs	Chen et al. (2008)
thnI	S. cattleya	Thienamycin	+	ThnI resembles LysR-type transcriptional activators and contains a HTH motif	Rodríguez et al. (2008)
thnU	S. cattleya	Cephamycin C	+	Encodes a SARP	Rodríguez et al. (2008)
asuR1, asuR2, asuR4 and asuR6	S. nodosus	Asukamycin	+	AsuR1 and AsuR6 belong to the LuxR family, AsuR2 belongs to the TetR family, and asuR5 encodes a SARP	Xie et al. (2012)
farR3 and farR4	S. lavendulae FRI-5	Indigoidine	farR3: + farR4: −	Both encode SARPs, FarR3 positively controls the biosynthesis of indigoidine, and FarR4 negatively controls the expression of farX, farA, farR1 and farR2	Kitani et al. (2008) and Kurniawan et al. (2014)
papR6	S. pristinaespiralis	Pritinamycin II	+	PapR6 is an orphan RR	Dun et al. (2015) and Mast et al. (2015)
redZ	S. coelicolor	RED	+	Encodes a NarL-type RR, and the transcription of redD depends on redZ and the translation of redZ depends on bldA	Guthrie et al. (1998) and Wang et al. (2009)
ssaA	Streptomyces sp. strain SS	Sansanmycin	+	SsaA has a N-terminal fork head-associated (FHA) domain and a C-terminal LuxR-type HTH motif	Li et al. (2013)
vlmI	S. viridifaciens	Valanimycin	+	vlmI encodes a SARP and can complement a redD mutation;	Garg and Parry (2010)
nanR1, nanR2 and nanR4	S. nanchangensis	Nanchangmycin	nanR1, nanR2: + nanR4: −	nanR1 and nanR2 encode SARPs, and nanR4 is an AraC-family transcriptional regulator and represses the transcription of nanR1 and nanR2	Yu et al. (2012)

+ Represents positive regulation, − represents negative regulation

tylR, tylP, tylQ, tylS and tylT

Eli Lilly and Company obtained the tylosin biosynthetic gene cluster from Streptomyces fradiae. Five regulators of this cluster are all CSRs. Sequence analysis identified TylP as a γ-butyrolactone (GBL) signal receptor (Bate et al. 1999; Wilson et al. 2001), and there were several indications that TylP is an effector-binding regulator and a regulator of tylosin biosynthesis (Bignell et al. 2007). The protein TylQ was reported to be a transcriptional repressor that blocked tylosin biosynthesis by controlling the expression of tylR (Stratigopoulos and Cundliffe 2002). Studies revealed that tylT and tylS encode proteins belonging to the SARPs family (Bate et al. 1999). Bate et al. (2006) identified a new regulatory gene, tylU, in the tylosin biosynthetic gene cluster. Targeted disruption of tylU decreased tylosin yield by approximately 80%, demonstrating that tylU is a positive regulatory gene for tylosin biosynthesis. TylR, a CSR, was able to regulate the core polyketide genes, but it primarily affected tylosin biosynthesis (Bate et al. 1999). The tylosin biosynthetic gene cluster is shown in Fig. 1.

Fig. 1.

Tylosin biosynthetic gene cluster of S. fradiae. Five regulatory genes are shown: TylP is a GBL signal receptor. TylQ is a transcriptional repressor and blocks tylosin biosynthesis by controlling the expression of tylR. tylT and tylS encode cluster-situated regulatory proteins of the SARPs family. tylU is a positive regulatory gene of tylosin biosynthesis. TylR is also a CSR

dnrI, dnrO and dnrN

A previous report (Parajuli and Moon 2002) has demonstrated that the DNR producer S. peucetius possesses two DNA segments, dnrR1 and dnrR2. Sequence analysis of dnrR1 and the subsequent inactivation of dnrI, which is contained within dnrR1, suggested the involvement of dnrI in the transcription of the biosynthetic genes of DNR. The disruption of dnrI resulted in the absence of DNR production (Madduri and Hutchinson 1995), and the overexpression of dnrI under the control of the strong ermE* promoter increased the production of DNR (Malla et al. 2010). These results indicated that dnrI positively regulates DNR biosynthesis. dnrN encodes a response regulator (RR) of UhpA-LuxR superfamily of regulatory proteins and has a motif that is highly similar to the HTH DNA-binding motif. An earlier study showed that reintroduction of dnrN into a dnrI:aphII mutant failed to restore DNR production. This suggested that DnrN activates the transcription of dnrI in the regulatory cascade; dnrI, in turn, positively triggers the transcription of DNR biosynthetic genes (Otten et al. 1995). dnrO, which is a negative regulatory gene, encodes a DNA-binding protein. DnrO has been shown to regulate antibiotic yield in S. peucetius by positively controlling dnrN (Otten et al. 2000; Parajuli and Moon 2002).

rapH, rapG and rapY

DNA sequence analysis of rapH and rapG in Streptomyces hygroscopicus revealed that RapH and RapG share significant similarity with two positive transcriptional families, the LAL and AraC families, respectively (Kuščer et al. 2007). RapH contained a DNA-binding motif and an ATP-binding site, while RapG contained a HTH DNA-binding motif (Yoo et al. 2015). In one study, antibiotic production was increased by 50% only if copies of both rapH and rapG under the control of their native promoter regions were introduced. Further, the complementation of rapH and rapG deletion mutants under the control of their native promoters led to a restoration of rapamycin production to parental levels (Yoo et al. 2015). The overexpression of both genes led to an abundant rapamycin synthesis, while the deletion of rapG and rapH caused a total shutdown of antibiotic production, suggesting that rapG and rapH play active roles in antibiotic biosynthesis. Furthermore, these genes cannot exert regulatory effects on the rapamycin biosynthetic gene cluster independent of each other. Yoo et al. (2015) found that the overexpression of rapY caused a drastic reduction in antibiotic production, while deletion of rapY increased antibiotic production by approximately fivefold. RapY, which contains an HTH motif near its N-terminus, plays a negative role in rapamycin production. The antibiotic regulatory genes in the biosynthetic gene cluster of rapamycin are shown in Fig. 2.

Fig. 2.

The location of regulatory genes in rapamycin biosynthetic gene cluster of S. hygroscopicus. RapY inhibits the transcription of rapX which is an ABC-transporter gene. Together, rapS and rapR negatively regulate most of the rapamycin biosynthetic genes. RapS also represses the expression of rapY. RapH and RapG positively regulate rapamycin biosynthesis

polY and polR

Li et al. (2009) found that the deletion of polR completely blocked polyoxin (POL) biosynthesis, which was complemented by introducing a copy of polR into the mutant. Similarly, the yield of POL was found to increase with the presence of an additional copy of polR in the mutant. PolR is necessary for the transcription of many structural genes in the POL biosynthetic gene cluster. Another CSR, polY in S. cacaoi, positively controls the production of this antibiotic. Both polR and polY encode proteins belonging to the SARPs family, and the expression of polR depends on the activity of PolY (Kitani et al. 2001).

aur1R, aur1P, aur1PR3 and aur1PR4

Some studies (Novakova et al. 2010, 2011; Rehakova et al. 2013) described four genes regulating auricin production in Streptomyces aureofaciens. aur1R encodes a homologue similar to the members of the TetR family. Antibiotic production was much higher in the disrupted strain than in the parental strain, which suggested a negative regulatory effect of aur1R on auricin production (Novakova et al. 2010). Aur1R can specifically negatively control the expression of aur1P, and this repression is released by auricin or its intermediates. In another experiment (Rehakova et al. 2013), aur1P was deleted from the chromosome, and no auricin was produced in the mutant, which suggested that aur1P is critical for the biosynthesis of auricin and exerts a positive effect on the expression of the biosynthetic genes of auricin. Aur1P belongs to the OmpR subfamily, which is similar to the RRs of two-component systems (TCSs). aur1PR3 and aur1PR4 encode proteins that are highly similar to those belonging to SARP family. The disruption of aur1PR3, an activator of auricin, resulted in a dramatic decrease in antibiotic production compared to the wild-type parent. Additionally, the expression of aur1PR3 was controlled by Aur1R (Novakova et al. 2011). Aur1P directly regulated the expression of aur1PR4 because its promoter was dependent on Aur1P (Rehakova et al. 2013).

thnI and thnU

There are two regulators, thnI and thnU, located in the thienamycin gene cluster of Streptomyces cattleya. ThnI is similar to the members of LysR family, and they all have a highly conserved HTH DNA-binding domain. A deletion mutant constructed by gene replacement failed to produce thienamycin, thereby revealing the importance of thnI in thienamycin synthesis. Gene expression analysis of the thienamycin gene cluster demonstrated that ThnI is a positive regulator and that it can control the expression of several genes involved in the assembly and export of thienamycin (Rodríguez et al. 2008). thnU encodes a positive regulator that belongs to the SARPs family. HPLC–MS analysis of a thnU mutant constructed using the same method that was used for thnI indicated that inactivation of thnU resulted in a loss of the production of Cephamycin C, whereas thienamycin synthesis was not affected. These results revealed the positive role of ThnI in thienamycin biosynthesis and the relevance of ThnU in cephamycin C biosynthesis (Rodríguez et al. 2008).

Regulatory genes involved in pleiotropic regulation

Many regulatory genes, which are mostly located outside of biosynthetic gene clusters, have pleiotropic (or global) effects on the production of multiple secondary metabolites or on both secondary metabolites and morphological development. In Streptomyces, the most abundant pleiotropic regulators belong to the TCSs, which are the predominant signal transduction systems in bacteria (Stock et al. 2000; Hakenbeck and Stock 1996). Other pleiotropic regulators can regulate antibiotic production in association with many small molecules called γ-butyrolactones (GBLs). This paper divides pleiotropic regulators into two subtypes. One type includes TCSs, orphan response regulation [e.g., farR1 (Kitani et al. 2008)], orphan histidine kinase regulation [e.g., ohkA (Lu and Jiang 2013)] and other special TCSs [e.g., abrC1/C2/C3 (Yepes et al. 2011)]. This subtype of pleiotropic regulatory genes is shown in Table 2a. The other includes the pleiotropic regulators closely associated with GBLs. To date, approximately 13 GBLs have been discovered, including A-factor from S. griseus (Ohnishi et al. 1999), IM-2 from S. lavendulae (Sato et al. 1989), SCB1, SCB2 and SCB3 (Hsiao et al. 2009; Takano et al. 2005), VBs from S. virginiae (Yamada et al. 1987) and factor 1 from S. viridochromogenes (Sato et al. 1989). Moreover, a few pleiotropic regulators contain a TPR structural domain [such as nsdA (Yu et al. 2006)], a protein repeat sequence consisting of 34 amino acids, which encodes an HTH secondary structural fragment. This type of pleiotropic genes is shown in Table 2b.

Table 2.

Regulatory genes involved in pleiotropic regulation

Regulatory gene(s)	Gene(s) from	Product(s) of regulation	Function	Notes	References
a)
afsQ1/afsQ2	S. lividans	ACT	+		Ishizuka et al. (1992)
afsK/afsR	S. coelicolor and S. griseus	ACT	+	AfsR is the transcriptional activator of afsS, which can activate actII-ORF4	Atsushi et al. (1994)
afsR-p/afsS	S. peucetius	Adriamycin	+		Parajuli et al. (2005)
cutR/cutS	S. coelicolor and S. lividans	ACT	−		Chang et al. (1996)
ecrA1/ecrA2 and ecrE2/ecrE1	S. coelicolor	RED	+	Coordinates with the expression of redD	Huang et al. (2001)
orfX/orf41	S. avermitilis	Avermectin	+	orfX exerts regulation by itself or by the collaboration of orf41 with orfX	Hwang et al. (2003)
phoR/phoP	S. coelicolor and S. lividans	ACT and RED	−	PhoP belongs to the Ormp family	Sola-Landa et al. (2003)
valP/valQ	S. hygroscopicus 5008	Validamycin			Bai et al. (2006)
absA1/absA2	S. coelicolor	CDA, yCPK and albaflavenone	−		Sheeler et al. (2005)
rapR/rapS	S. hygroscopicus	Rapamycin	−	RapS represses the expression of rapY	Yoo et al. (2015)
rapA1/rapA2	S. coelicolor	ACT and yCPK	+	The regulation of RapA1/A2 depends on ActII-ORF4 and KasO	Lu et al. (2007)
draK/R	S. coelicolor	ACT, yCPK and RED	ACT: + RED and yCPK: −	DraR binds to the promoter regions of actII-ORF4 and cpkO	Rodríguez et al. (2013)
abrA1/A2	S. coelicolor	ACT, CDA and RED	−		Yepes et al. (2011)
SCO0203/0204	S. coelicolor	ACT	−		Wang et al. (2009b)
ohkA	S. coelicolor	ACT, CDA and RED	−	No identified RR matches OhkA	Lu et al. (2011)
aur1P	S. aureofaciens	Auricin	+	aur1P encodes a protein similar to the RRs	Novakova et al. (2005)
farR1	S. lavendulae FRI-5	Nucleoside antibiotics and indigoidine		FarR1 is an orphan RR	Kitani et al. (2008)
glnR	S. coelicolor	RED and ACT	+	GlnR belongs to the OmpR family and indirectly regulates the production of antibiotics in response to changes in nitrogen availability	Pullan et al. (2011)
SCO3818	S. coelicolor	ACT	−	SCO0203 can phosphosphorylate SCO0204 and SCO3818, and there is a functional correlation between SCO0203 and SCO3818	Wang et al. (2009b)
jadR1/jadR2	S. venezuelae	Jadomycin B	jadR1: + jadR2: −	The jadR1 and jadR2 genes represent a novel TCS linking antibiotic synthesis to stress; jadR1 encodes a RR; jadR2 encodes a TetR-like protein, and JadR2 is a pseudo γ-butyrolactone receptor	Yang et al. (2001)
abrC1/C2/C3	S. coelicolor	ACT, CDA and RED	+	AbrC1 and AbrC2 are HKs, and AbrC3 is a RR	Yepes et al. (2011)
b)
nsdA and nsdB	S. coelicolor	ACT and CDA	−	Each encodes a protein containing a TPR structure	Yu et al. (2006)
tcrA	S. coelicolor	All secondary metabolites	−		Liu and Yang (2006)
afsR2	S. lividans and S. coelicolor	ACT and RED	+		Lee et al. (2000)
afsB	S. lividans and S. coelicolor	ACT, methylenomycin, CDA and RED	+		Horinouchi et al. (1989)
barX	S. virginiae	Virginiamycin		BarX is an AfsA-like protein	Bate et al. (1999) and Pulsawat et al. (2007)
farA	S. lavendulae	Nucleoside antibiotics	−	IM-2 binds to the FarA receptor to regulate the signal transduction of secondary metabolism	Kitani et al. (2001)
arpA	S. griseus	Streptomycin	−	ArpA is an A-factor receptor protein	Hong et al. (2007) and Kato et al. (2004)
adpA	S. griseus	Streptomycin	+	Encodes an Arac/XylS family protein and has two HTH motifs at the C-terminal	Higo et al. (2012), Ohnishi et al. (1999, 2005) and Zhu et al. (2005)
bldD	S. coelicolor	ACT, indigoidine, CDA and methylenomycin	+	BldD has a C-terminal domain of unknown function and an N-terminal domain that mediates DNA binding and dimerization	Den Hengst et al. (2010)
bldA	S. coelicolor	ACT	+	BldA regulates the production of antibiotics by controlling the activator ActII-ORF4	Fernández-Moreno et al. (1991)
crp	S. coelicolor	ACT, RED and CDA	+	Crp is a member of the cAMP receptor protein/fumarate-nitrate-reductase family of regulators	Gao et al. (2012)
wblA	S. coelicolor	ACT, RED and CDA	−	WblA is a protein of the WhiB family	Kang et al. (2007)
atrA	S. coelicolor	ACT	+	AtrA is a TetR-like protein, AtrA positively controls the transcription of actII-ORF4	Li et al. (2015b)
rrdA	S. coelicolor	RED and ACT	RED: − ACT: +	RrdA belongs to the TetR family, and RrdA negatively regulates RED by controlling the abundance of RedD mRNA	Ou et al. (2009)
avaR3	S. avermitilis	Avermectin and filipin	Avermectin: +; filipin: −	AvaR3 is a γ-butyrolactone autoregulator receptor homologue	Miyamoto et al. (2011)
cprA	S. coelicolor	ACT and RED	+	Encodes an AprA analogue	Onaka et al. (1998)
cprB	S. coelicolor	ACT	−	CprB shows high sequence similarity to CprA	Onaka et al. (1998)

+ Represents positive regulation, − represents negative regulation

For TCSs, there are two types of kinase super-families in Streptomyces that regulate secondary metabolites. One is the histidine kinase family. This family consists of two proteins, a phosphate donor—HK (histidine kinase) and a phosphate receptor—RR. Most of the HKs that belong to membrane-spanning proteins have three functional structural domains: a sensing domain, a transmitter domain and an ATPase domain. RRs contain two domains: a receiver domain and an effector domain. A HK activates itself by first phosphorylating its conserved histidine residues and then transferring the phosphate groups to the conserved asparagine acid residues of the RR. The phosphorylated RR then regulates the expression of other genes (Lu and Jiang 2013). This signal transduction process is shown in Fig. 3. Genes belonging to this family include afsQ1/afsQ2 (Ishizuka et al. 1992), cutR/cutS (Tanaka et al. 2007) and ecrA1/ecrA2 (Huang et al. 2001). The other type is the serine/threonine and tyrosine kinase family, whose members transmit signals to regulate secondary metabolism via a series of cascade reactions, such as phosphorylation or dephosphorylation of proteins triggered by external environmental changes. AfsR/AfsS (Tanaka et al. 2007) belongs to the serine/threonine and tyrosine kinase family. The genomic sequence analysis of S. coelicolor which is a model strain was performed in 2002. Bioinformatic analysis revealed that S. coelicolor contains many TCSs, encompassing 84 HKs and 80 RRs. Of these, 67 HKs have been matched with 67 RRs and are adjacent to genes encoding RRs, while the remaining TCSs are orphan HKs and orphan RRs (Hutchings et al. 2004).

Fig. 3.

The process of signal transduction in TCSs

nsdA and nsdB

The pleiotropic negative regulatory gene nsdA can be found in many Streptomyces spp. The nsdA genes from various Streptomyces spp. share 77–100% similarity with each other, and nsdA homologous genes are also present in many Streptomyces strains (Yu et al. 2006). NsdA, which contains a TPR structural domain, plays a negative role in sporulation, morphological differentiation and antibiotic synthesis. The overproduction of actinorhodin (ACT), calcium-dependent antibiotic (CDA) and methylenomycin was detected in a nsdA mutant, and the deletion of the nsdA in Streptomyces lividans also resulted in the expression of silent ACT biosynthetic genes. These results indicated that NsdA may silence the ACT biosynthetic gene cluster by repressing the expression of CSRs (Yu et al. 2006). There is also a TPR-like structural domain in the nsdB gene product whose disruption gives rise to ACT production, but NsdB does not affect morphological differentiation. The deletion of nsdB, which is a negative regulator of CDA, resulted in an increase in CDA production. nsdB has been shown to control antibiotic biosynthesis along with nsdA but has no influence on the expression of nsdA at the RNA level (Yu et al. 2006).

afsR2 and afsB

The protein encoded by afsR2 is 63 amino acids long (Lee et al. 2000), and AfsR2 is a positive regulator of ACT and undecylprodigiosin (RED). Lee et al. (2000) cloned a regulatory gene from S. avermitilis that was homologous to afsR2 from S. lividans and S. coelicolor. The integration of multiple copies of afsR2 into the wild-type strain resulted in approximately twofold overproduction of avermectin. ACT and RED can be regulated by pleiotropic regulatory genes in addition to synthetic gene clusters. afsB encodes a DNA-binding protein and is a pleiotropic regulator that is essential for ACT biosynthesis. An increase in copies of afsB significantly improved the production of ACT and RED. The introduction of afsB into S. lividans triggered the transcription of ACT biosynthetic genes that were otherwise silent (Horinouchi et al. 1989). afsB provides positive regulation by stimulating its target genes and can trigger RED biosynthesis in S. lividans.

cprA and cprB

In S. coelicolor, the gene products of cprA and cprB are highly similar to each other. Onaka et al. (1998) found that the deletion of cprA led to a sharp reduction of ACT and RED. The introduction of cprA into the parental strain resulted in increased antibiotic production. Those results demonstrated that cprA positively regulated the biosynthesis of ACT and RED (Onaka et al. 1998). The disruption of cprB led to a precocious overproduction of ACT, but no change was detected in the production of RED. These results revealed that cprB only negatively regulated the biosynthesis of ACT (Onaka et al. 1998). Another study showed that cprA and cprB activated antibiotic biosynthesis in S. coelicolor via the GBL quorum-sensing pathway (Bhukya et al. 2014).

adpA and arpA

adpA, which is a pleiotropic regulator, encodes a 405-amino-acid protein containing a HTH DNA-binding motif in its central region. AdpA showed high sequence similarity to the transcriptional regulators of the AraC/XylS family (Gallegos et al. 1997). To determine the function of adpA in S. griseus, a disruption strain was generated using in-frame deletion. No streptomycin was detected in the disrupted strain, but streptomycin production was recovered when adpA was introduced, showing that adpA exerted a positive effect on streptomycin biosynthesis (Higo et al. 2012; Ohnishi et al. 1999). ArpA contains a region resembling the HTH motif that is present in many transcriptional families. ArpA is an A-factor receptor protein that negatively controls the production of streptomycin in S. griseus. ArpA behaved as a repressor-type regulator for streptomycin production (Onaka et al. 1995). adpA, arpA and A-factor form a widespread regulatory cascade in Streptomyces, termed the A-factor-adpA-arpA regulatory cascade. There is a model for the A-factor regulatory cascade that leads to streptomycin biosynthesis (Ohnishi et al. 1999): A-factor gradually accumulates, and when the concentration of A-factor reaches a certain point, it binds ArpA and causes ArpA to dissociate from the promoter, thus leading to the transcription and translation of adpA. AdpA then activates the transcription of strR. Therefore, the induction of StrR positively regulates the transcription of most of the streptomycin biosynthetic genes (Retzlaff and Distler 1995).

afsQ1/afsQ2 and cutR/cutS

afsQ1/afsQ2 is representative of the TCSs, with afsQ1 encoding an aspartic acid RR and AfsQ2 belonging to the sensing kinases. AfsQ2, located in the membrane, is autophosphorylated at His-294 upon sensing an environmental signal, and the signal is then transferred to the Asp-52 residue of the AfsQl protein in the cytoplasm (Ishizuka et al. 1992). afsQ1/afsQ2 plays a pleiotropic role in the secondary metabolism of antibiotics. cutR/cutS is the second TCS found in Streptomyces that represses secondary metabolism, and cutR/cutS also negatively regulates antibiotic production. The time taken to produce ACT is 20 h shorter in the cutR/cutS mutant than in the parental strain. The inactivation of cutR/cutS triggers an increase in ACT production, and the introduction of cutR reverses this change (Chang et al. 1996).

afsR-p/afsS

Parajuli et al. (2005) isolated the afsR-p gene from S. peucetius ATCC 27952, and it had greater than 50% sequence similarity to afsR from S. coelicolor. The overproduction of doxorubicin, γ-actinorhodin, clavulanic acid and streptomycin (slight), respectively, was detected in strains of S. peucetius, S. lividans, S. clavuligerus and S. griseus strains that carried the afsR-p gene (Parajuli et al. 2005). This demonstrated that AfsR-p may activate various CSRs in antibiotic biosynthetic gene clusters in different ways, leading to the speculation that phosphorylated AfsR-p binds to the promoter region of afsS and that the latter then triggers other regulators to induce the production of certain secondary metabolites (Parajuli et al. 2005).

orfX/orf41 and phoR/phoP

Disruption of the orfX gene resulted in a significant but incomplete loss of the production of avermectin in S. avermitilis (Hwang et al. 2003). The increase in avermectin may result from the role of orfX itself or from the collaboration of orf41 with orfX. Recently, a new TCS, phoR-phoP, was found in S. lividans and S. coelicolor, and PhoP was indeed a member of the OmpR family. The PhoR-PhoP system may activate the formation of ACT and RED via a specific repressor protein with phosphate-controlled promoters, acting via a cascade mechanism (Sola-Landa et al. 2003).

valP/valQ and rapR/rapS

valP and valQ, which can regulate validamycin in S. hygroscopicus 5008, may encode a TCS regulatory protein consisting of a HK and a Sigma B PP2C-like phosphatase (Bai et al. 2006). RapR and RapS in Streptomyces rapamycinicus ATCC 29253 share high sequence identities with the RRs and HKs, respectively, of TCSs. Gene expression analysis demonstrated that most of the rapamycin biosynthetic genes were negatively controlled by rapS (probably in collaboration with rapR) and rapY (a CSR for the biosynthesis of rapamycin) (Yoo et al. 2015). In addition, RapS represses the expression of rapY gene and RapR/S is a repressor of rapamycin biosynthesis.

rapA1/A2, abrA1/A2 and abrC1/C2/C3

The rapA1 in S. coelicolor encodes a protein that belongs to the OmpR family, while the sequence of RapA2 shows characteristics that are typical of HKs (Lu et al. 2007). Lu et al. (2007) found that RapA1/A2 is an activator of ACT and a yellow cryptic polyketide (yCPK). The effect exerted by rapA1/A2 on the biosynthesis of these antibiotics may also depend on two CSRs, actII-ORF4 and kasO. The disruption of rapA1/A2 and subsequent experiments with the disrupted strain indicated that rapA1/A2 was a positive TCS regulator of ACT [which is encoded by a type II polyketide synthase (PKS)] and yCPK [which is encoded by a type I PKS in S. coelicolor (Lu et al. 2007)]. abrA1/A2 is also a TCS regulator that negatively regulates ACT, RED and CDA in S. coelicolor. However, abrC1/C2/C3 is composed of two HKs and one RR that positively regulate the abovementioned antibiotics. AbrC1 and AbrC2 are HKs, while AbrC3 is a RR (Yepes et al. 2011).

ohkA

OhkA was reported to be an orphan HK in S. coelicolor that repressed the biosynthesis of five known secondary metabolites: ACT, RED, CDA, yCPK and albaflavenone (Lu and Jiang 2013). The deletion of ohkA in S. coelicolor caused a drastic increase in the biosynthesis of antibiotics, especially of ACT and CDA (Räty et al. 2002). OhkA negatively regulates secondary metabolites by repressing CSRs in S. coelicolor.

Other regulatory genes

In addition to CSRs and pleiotropic regulators, there are many other regulatory genes that deserve attention. These include barZ in Streptomyces virginiae, which regulates virginiamycin (Pulsawat et al. 2007); ppk from S. lividans (Ghorbel et al. 2006 ); hyg1 and hyg3 (Palaniappan et al. 2006) from S. hygroscopicus NRRL 2388; and some regulatory genes whose functions remain unknown, such as claR, which is related to cephamycin C and clavulanic acid. Regulatory genes of this type are shown in Table 3.

Table 3.

Other regulatory genes

Regulatory gene(s)	Gene(s) from	Product(s) of regulation	Function	Notes	References
hyg1 and hyg3	S. hygroscopicus	Hygromycin A		Hyg1 and Hyg3 show sequence similarities to AfsR and StrR, respectively	Palaniappan et al. (2006)
ppk	S. lincolnensis	ACT	−	Positively controlled by phoR/phoP	Ghorbel et al. (2006)
barZ	S. virginiae	Virginiamycin			Bate et al. (1999 and Pulsawat et al. (2007)
farR2	S. lavendulae FRI-5	Nucleoside antibiotics and indigoidine		FarR2 is a GBL receptor	Kitani et al. (2008)
claR	S. clavuligerus	Cephamycin C and clavulanic acid	Cephamycin C: − Clavulanic acid: +	ClaR contains two HTH motifs and is homologous with the LysR family	Pérez-Redondo et al. (1998)
stgR	S. coelicolor	ACT and RED	−	StgR belongs to the LysR family of transcriptional regulators and regulates antibiotics by indirectly repressing redD and actII-ORF4	Mao et al. (2013)
lndYR	S. globisporus	Landomycin	+	Encodes a GntR-like regulator of the YtrA subfamily	Ostash et al. (2011)

+ Represents positive regulation, − represents negative regulation

Discussion

The regulation of secondary metabolite biosynthetic gene clusters in Streptomyces spp. is currently receiving substantial attention. Many transcription units are apparently regulated by several metabolite regulatory genes via transcriptional activation or repression, and biosynthetic gene clusters often collaborate with transcriptional regulators. Furthermore, it is reported that many Streptomyces strains can produce antibiotics. Analyses of the genomic sequences of many Streptomyces spp., such as S. coelicolor and S. avermitilis, have also been completed. These analyses provide a basis for post-genomic projects involving Streptomyces and will effectively promote studies of the structure, function and expression regulation of biosynthetic genes. In addition, many regulatory genes can also be expressed in other strains and can influence antibiotic production by heterologous expression combined with other methods. For example, the integration of aveR, orfX, or afsR in the chromosomes of S. hygroscopicus promoted rapamycin production by approximately 3.8-fold, 1.2-fold or slightly, respectively (Huang et al. 2011). Currently, we can effectively influence antibiotic production by manipulating regulatory genes involved in pathway-specific regulation or pleiotropic regulation in Streptomyces. For example, as introduced in this paper, the aveR mutants in S. avermitilis lost the ability to synthesize avermectin, whereas the overexpression of this gene increased the yield (Ikeda et al. 2003). The overproduction of ACT, CDA and methylenomycin was detected in the nsdA mutant of S. coelicolor (Yu et al. 2006). Hence, studying the regulatory mechanisms of secondary metabolite biosynthesis, inactivating the transcriptional repressors and overexpressing the transcriptional activators in natural producing strains may allow the optimization of antibiotic production. Further, it will provide a crucial theoretical basis for improving antibiotic production and using regulators to activate silent gene clusters, thereby leading to the discovery of new drugs.

Compliance with ethical standards

Funding

This study was funded by the Open Research Program of Key Laboratory of Synthetic Biology, the Chinese Academy of Sciences (SYN201612) and the Key Program of Sichuan Science and Technology Project (2017GZ0430).

Conflict of interest

All of the authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.