Development of a strictly regulated xylose-induced expression system in Streptomyces

Abstract

BackgroundGenetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces.ResultsTo determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of d-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by d-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions.ConclusionIn this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0991-y) contains supplementary material, which is available to authorized users.

The ROK‐family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis

“…XylA (xylose isomerase) catalyses isomerization of xylose to xylulose, and XylB (xylulose kinase) mediates phosphorylation of xylulose to xylulose‐5‐phosphate. XylR acts as a repressor of xylA and xylB (Swiatek et al ., 2013; Noguchi et al ., 2018). Gene numbers for xylABR in S. avermitilis are sav_718 2 ( xylA ), sav_7181 ( xylB1 ), and sav_7180 ( xylR ).…”

“…Concerning the control of xylose uptake, Rok7B7 acts as a direct repressor of xylFGH but not of another xyl gene locus, xylABR , consistent with the observation that rok7B7 deletion in S. coelicolor had no effect on xylABR expression (Swiatek et al ., 2013). XylR repressed xylA and xylB (Swiatek et al ., 2013; Noguchi et al ., 2018), and addition of xylose derepressed xylA expression in S. avermitilis (Noguchi et al ., 2018), but the relationship between XylR and xylFGH was not previously investigated. In the present study, the expression of the xylFGH operon completely escaped glucose repression in ∆rok7B7, but xylose still induced transcription of these genes, suggesting that xylFGH is controlled by other regulator(s).…”

Xylose-Inducible Promoter Tools for Pseudomonas Species and Their Use in Implicating a Role for the Type II Secretion System Protein XcpQ in the Inhibition of Corneal Epithelial Wound Closure

“…Xylose-inducible plasmid systems have been developed for a number of other Gram-negative bacteria, such as Caulobacter crescentus (34), and for Gram-positive species, including Bacillus subtilis, Clostridioides difficile, Lactococcus lactis, Staphylococcus xylosus, and Streptomyces lividans (35)(36)(37)(38)(39). The xylose-responsive transcription factors for these systems include members of the AraC family, such as XutR, used in this study, LacI, MarR, and the ROK family.…”

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

“…Constitutive promoters ermE* promoter Mutation at the promoter of the erythromycin resistance gene of Streptomyces erythraeus [31] SF14P promoter Genome of Streptomyces ghanaensis phage I19 [34] kasOP promoter Promoter of SARP family regulator in Stretpomyces coelicolor A3 [33] gapdh promoter Promoter of glyceraldehyde-3-phosphate dehydrogenase in Streptomyces griseus [35] rpsL promoter Promoter of 30S ribosomal protein S12 in Streptomyces griseus [35] 195 native or synthetic promoters High-throughput screening in S. venezueale [58] 32 native promoters Transcriptome data-based selection in S. albus [38] 166 native promoters Transcriptome data-based selection in S. coelicolor [39] 2 native promoters Multi-omics data-based selection in S. coelicolor [59] Inducible promoters tipA promoter Thiostrepton-induced promoter [40] nitA promoter ε-caprolactam-induced promoter [45] xylA promoter Xylose-induced promoter [46] tcp830 Tetracycline-induced promoter [43] PA3-rolO Resorcinol-induced promoter [44] P21-cmt Cumate-induced promoter [44] Terminators Fd Bidirectional transcription termination originated from E. coli phage fd [48] TD1 Bidirectional transcription termination originated from Bacillus subtilis phage Φ29 [49] RBS AAAGGAGG Typical RBS sequence of S. coelicolor [134] 192 native or synthetic RBSs High-throughput screening in S. venezueale [58] 4 native RBSs Multi-omics data-based selection in S. coelicolor [59] Reporter genes luxAB cassette n-Decanal as substrate; absorbance at 490 nm wavelength [61] amy gene Soluble starch with 3,5-dinitrosalycilic acid (DNS) as substrate; absorbance at 540 nm wavelength [64] xylE gene Catecol as substrate; absorbance at 375 nm wavelength [62] gusA gene p-Nitrophenyl-β-D-glucuronide as substrate; absorbance at 415 nm wavelength [63] eGFP Green fluorescent protein; excitation wavelength 470-490 nm and emission wavelength 515 nm [65] sfGFP Green fluorescent protein; excitation wavelength 488 nm and emission wavelength 500~550 nm [58] mRFP Red fluorescent protein; excitation wavelength 584 nm and emission wavelength 607 nm [67] mCherry Red fluorescent ...…”

“…The nitA promoter, which originates from the nitrilase promoter of Rhodococcus rhodochrous, is induced by a complex of ε-caprolactam and the transcription regulator NitR [45]. The recently developed xylA promoter is a strictly regulated xylose-induced expression promoter [46]. Additionally, several glycerol-inducible systems have been developed for Streptomyces; however, these systems have not been utilized because glycerol treatment may alter intrinsic cellular metabolism [47].…”