Development of an Unnatural Amino Acid Incorporation System in the Actinobacterial Natural Product Producer Streptomyces venezuelae ATCC 15439

Abstract

Many Actinobacteria, most notably Streptomyces, produce structurally diverse bioactive natural products, including ribosomally synthesized peptides, by multistep enzymatic pathways. The use of site-specific genetic incorporation of unnatural amino acids to investigate and manipulate the functions of natural product biosynthetic enzymes, enzyme complexes, and ribosomally derived peptides in these organisms would have important implications for drug discovery and development efforts. Here, we have designed, constructed, and optimized unnatural amino acid systems capable of incorporating p-iodo-l-phenylalanine and p-azido-l-phenylalanine site-specifically into proteins in the model natural product producer Streptomyces venezuelae ATCC 15439. We observed notable differences in the fidelity and efficiency of these systems between S. venezuelae and previously used hosts. Our findings serve as a foundation for using an expanded genetic code in Streptomyces to address questions related to natural product biosynthesis and mechanism of action that are relevant to drug discovery and development.

Keywords: Streptomyces; green fluorescent protein; natural products; p-azido-l-phenylalanine; p-iodo-l-phenylalanine; unnatural amino acids.

Figure 1

a) Schematic overview of amber suppression-based unnatural amino acid (UAA) incorporation; b) structures of UAAs p-benzoyl-l-phenylalanine (pBpa), p-azido-l-phenylalanine (pAzPhe), and p-iodo L-phenylalanine (pIPhe) used in this study.

Figure 2

a) Schematics of pSUA2, pSUA3, pSUA4, pSUA5, opt-pSUA2, and opt-pSUA2 (no His) vector inserts containing elements of the amber suppression reporter system; b) Anti-His Western blots of Ni-NTA affinity purified eGFP proteins obtained from S. venezuelae harboring opt-pSUA2 (lane 1), pSUA3 (lane 2), pSUA4 (lane 3), pSUA5 (lane 4), and opt-pSUA2 lacking the C-terminal His tag on MjTyrRS (lane 5). The molecular weights of MjTyrRS and eGFP are 35 kDa and 27 kDa, respectively.

Figure 3

a) Schematics of the pSUAl-pBpaRS, pSUAl-pAzPheRS, pSUAl-pIPheRS, opt-pSUA2-sfGFP, and pSUA5-sfGFP vector inserts; b) Anti-His Western blot of Ni-NTA affinity pwified sfGFP proteins obtained from S. venezuelae harboring pSUAl-pBpaRS grown in the presence (lane 1) and the absence (lane 2) of 1 mM pBpa, pSUAl-pAzPheRS grown in the presence (lane 3) and the absence (lane 4) of 1 mM pAzPhe, pSUAl-pIPheRS grown in the presence (lane 5) and the absence (lane 6) of 1 mM plPhe, opt-pSUA2-sfGFP (lane 7), and pSUA5-sfGFP (lane 8). The blot was imaged for 165 s. c) Amber suppression efficiencies of the same eight protein samples used for Western blot analysis (Figure 3b), measured by fluorescence quantification, and normalized to wild-type sfGFP signal. GFP excitation was carried out at 485 nm, bandwidth 9 nm; and GFP emission was detected at 520 run, bandwidth 15 run.

Figure 4

a) SDS-PAGE of purified sfGFP proteins obtained from S. venezuelae harboring pSUAI pAzPheRS grown in the presence of I mM pAzPhe (lane I), pSUAl-pAzPheRS grown in the presence of 5 mM pAzPhe (lane 2), pSUAI-plPheRS grown in the presence of I mM plPhe (lane 3), and opt-pSUA2-sfGFP (lane 4). b) Streptavidin Western blot of purified sfGFP proteins obtained from S. venezuelae harboring pSUAl-pAzPheRS grown in the presence (lane 1) and the absence (lane 2) of 1 mM pAzPhe, incubated with 2 µM DBCO-PEG₄-biotin at 25°C for 12 h, and re-purified by Ni-NTA affinity chromatography to remove excess DBCO-PEG4-biotin. The blot was imaged for 5 s.

Figure 5

Deconvoluted ESI-MS data of GFP proteins purified from S. venezuelae. The aaRS, GFP-TAG, UAA, concentration of UAA, and growth media used for each protein expression experiment is shown to the right of each spectrum. The percentage of UAA incorporation, as estimated by the ratios of peak heights, is also shown to the right of each spectrum where appropriate. Peak I corresponds to GFP in which phenylalanine has been incorporated (Spectra H, I), Peaks II and V to GFP with tyrosine or p amino-l-phenylalanine incorporated (Spectra A, C-K), Peak III and VI to GFP with pAzPhe incorporated (Spectra C-G), and Peak N to GFP with pIPhe incorporated (Spectrum B). Peaks 1-N correspond to proteins with the N-terminal methionine removed, and Peaks V and VI to proteins with the N-terminal methionine retained (Spectrum F only). See Table S2 for observed and expected masses for each peak in each spectrum, and Figures S2-S12 for raw and deconvoluted ESI-MS spectra.

Figure 6

Excitation (upper) and emission (lower) spectra of GFP proteins purified from S. venezue/ae harboring opt-pSUA2-sfUFP (solid line) and pSUA1-pBpaRS-Y66TAG (dashed line). The excitation spectrwn was obtained by excitation over the 410-510 nm range and detection of emission at 540 nm; and the emission spectrum was obtained by excitation at 446 nm and detection of emission over the 480-580 nm range. To aid in comparison of peak shapes, both spectra were normalized, with the intensity of each dashed line spectrum adjusted to 95% of the intensity of the solid line spectrum at the excitation and emission maxima.