A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Summary

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

Abstract

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 μL) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 μM) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

Introduction

Cell-free transcription-translation (TX-TL) systems provide an ideal prototyping platform for synthetic biology to implement rapid design-build-test-learn cycles, the conceptual engineering framework for synthetic biology¹. In addition, there is growing interest in TX-TL systems for high-value recombinant protein production in an open-reaction environment², for example, to incorporate non-standard amino acids in antibody-drug conjugates³. Specifically, TX-TL requires a cell extract, plasmid or linear DNA, and an energy solution to catalyze protein synthesis in batch or semicontinuous reactions. Wh....

Protocol

NOTE: See Table 1 and Table 2 for recipes for GYM medium and agar plate and S30A and S30B wash buffers.

1. Preparation of solutions and general guidance

1. Keep all solutions, cells (post-growth), and cell extracts on ice after preparation, unless an exception is stated.
2. Store stocks for 1 M Mg-glutamate, 4 M K-glutamate, 40% (w/v) PEG 6000, 1 g/mL polyvinylsulfonic acid at room temperature, and all other stocks at -80 °C. Minimize the number of freeze-thaw cycles to avoid chemical degradation.
3. For the preparation of energy solution stocks (see Table 3....

Results

This detailed protocol is provided as an example to help the user establish a Streptomyces TX-TL system based on the S. venezuelae ATCC 10712 model strain (Figure 1). The user may seek to study other Streptomyces strains; however, the growth/harvesting stages of other strains with longer doubling times or distinct growth preferences will need to be custom-optimized to achieve peak results. For the representative result, the mScarlet-I fluorescent protein from the p.......

Discussion

In this manuscript, a high-yield S. venezuelae TX-TL protocol has been described with detailed steps that are straightforward to conduct for both experienced and new users of TX-TL systems. Several features from existing Streptomyces⁴⁵ and E. coli TX-TL⁴¹ protocols have been removed to establish a minimal, yet high-yield protocol for S. venezuelae TX-TL^5,26. The workflow recommend.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2.5 L UltraYield Flask	Thomson	931136-B
3-PGA (>93%)	Sigma	P8877
384 Well Black/Clear Bottom Plate	ThermoFisher	10692202
Ammonium chloride (98%)	Fluorochem	44722
ATP, CTP, UTP, GTP (100 mM solution, >99%)	ThermoFisher	R0481
Carbenicillin (contact supplier for purity)	Melford	C46000-25.0
D-(+)-glucose (contact supplier for purity)	Melford	G32040
DFHBI (≥98% - HPLC)	Sigma	SML1627
DTT (contact supplier for purity)	Melford	MB1015
FlAsH-EDT2 (contact supplier for purity)	Santa Cruz Biotech	sc-363644
Glucose-6-phosphate (>98%)	Sigma	G7879
HEPES Free Acid (contact supplier for purity)	Melford	B2001
L-glutamic acid hemimagnesium salt tetrahydrate (>98%)	Sigma	49605
Magnesium chloride (98%)	Fluorochem	494356
Malt extract	Sigma	70167-500G
PEG-6000	Sigma	807491
Pierce 96-well Microdialysis Plate, 10K MWCO	ThermoFisher	88260
Poly(vinyl sulfate) potassium salt	Sigma	271969
Potassium glutamate (>99%)	Sigma	G1149
RTS amino acid sampler	5 Prime	2401530
Sodium chloride (99%)	Fluorochem	94554
Supelclean LC-18 SPE C-18 SPE column (1 g)	Sigma	505471
Yeast Extract	Melford	Y1333
Equipment
Platereader	BMG	Omega