Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces

Abstract

There is a great demand for precisely quantitating the expression of genes of interest in synthetic and systems biotechnology as new and fascinating insights into the genetics of streptomycetes have come to light. Here, we developed, for the first time to our knowledge, a quantitative method based on flow cytometry and a superfolder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. Single cells of filamentous bacteria were obtained by releasing the protoplasts from the mycelium, and the dead cells could be distinguished from the viable ones by propidium iodide (PI) staining. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. Furthermore, an insulator (RiboJ) was recruited to eliminate the interference between promoters and RBSs and improve the modularity of regulatory elements. Seven synthetic promoters with gradient strength were successfully applied in a proof-of-principle approach to activate and overproduce the cryptic lycopene in a predictable manner in Streptomyces avermitilis. Our work therefore presents a quantitative strategy and universal synthetic modular regulatory elements, which will facilitate the functional optimization of gene clusters and the drug discovery process in Streptomyces.

Keywords: flow cytometry; modular regulatory elements; natural product; single-cell resolution; synthetic biology.

Fig. 1.

(A) Workflow of single-cell quantification for gene expression in Streptomyces by flow cytometry. Effect of different buffers on the viability of protoplasts, released from the mycelium of S. venezuelae, indicated by scatter plot (B) and histogram (C), with the population of viable cells being gated. RFP, red fluorescent protein.

Fig. 2.

Distinguishing viable individuals from dead ones by PI staining. Images of the PI-stained mycelium (A) and protoplasts (B) of S. venezuelae harboring kasOp*-driven sfGFP were taken by a confocal laser-scanning microscope. (C and D) The dead individuals were separated from the viable ones by PI staining. A weak promoter (ermEp*) (C) and a strong promoter (kasOp*) (D) were both demonstrated to show the advantage of PI staining.

Fig. 3.

Correlation of GFP expression with mRNA abundance, measured by quantitative RT-PCR. Open circle, gapdhp (SG); solid circle, rpsLp (SA); open square, rpsLp (RE); solid square, ermEp*; solid triangle, gapdhp (KR); solid upside-down triangle, rpsLp (TP); open triangle, rpsLp (CF); open upside-down triangle, kasOp*. Error bars, data are presented as mean ± SD obtained from at least three experiments performed on different days.

Fig. 4.

Relative strength of native and synthetic promoters and RBSs evaluated in S. venezuelae ISP5230. (A) Relative strength of 15 native or engineered promoters (gray) and 44 sequenced synthetic promoters (white) with kasOp* (*) as the reference. The widely used promoter ermEp* is shown in red. (B) The relative strength of 15 native RBSs (gray) and 41 sequenced synthetic RBSs (white) with the RBS of capsid protein from phage ϕC31 (*) as the reference. Error bars, data are presented as mean ± SD obtained from at least three experiments performed on different days.

Fig. 5.

Performance of the RiboJ insulator in Streptomyces to eliminate interferences between the promoter and RBS. (A and B) A scheme of the combined seven promoters and nine RBSs to form a full combinatorial library of expression control elements without RiboJ (A) or with RiboJ (B). Heat maps show GFP fluorescence for all combinations of promoter (P, columns) and RBS (R, row) elements driving the expression of GFP reporter gene for the with-RiboJ (F) and without-RiboJ (E) cases. Each value was obtained by flow cytometry with three experimental duplications. Analysis of variances by full factorial ANOVA was performed for the without-RiboJ (C) and with-RiboJ library data (D). Promoter: GSV, gapdhp (SV); GSA, gapdhp (SA); GSG, gapdhp (SG); RTP, rpsLp (TP); GCF, rpsLp (CF); REL, gapdhp (EL); KSC, kasOp*. RBS: TER, terminase (ϕC31); TAP, tape measure protein (ϕC31); KSC, kasO (SC); TAI, tail protein (ϕC31); NUK, nucleotide kinase (ϕC31); HEL, helicase (ϕC31); CAP, capsid protein (ϕC31); GSG, GAPDH (SG); RCF, 30s ribosomal protein S12 (CF).

Fig. 6.

Lycopene production in S. avermitilis under the control of different promoters with the presence of RiboJ. Promoter strength is shown by the relative strength to the kasOp* promoter. Solid circle, SP12; open circle, SP18; solid square, SP23; open square, SP26; open triangle, kasOp*; open diamond, SP43; open upside-down triangle, SP44. Data are expressed as mean ± SD of the results of three parallel studies. Promoter sequences are listed in SI Appendix, Table S3.