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

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

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.

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. While Escherichia coli TX-TL is the dominant cell-free system, a number of emerging non-model TX-TL systems have attracted attention for different applications^4,5,6,7,8. Key advantages of TX-TL include flexible scalability (nanoliter to liter scale)^9,10, strong reproducibility, and automated workflows^8,11,12. In particular, automation of TX-TL permits the accelerated characterization of genetic parts and regulatory elements^8,12,13.

In terms of reaction setup, TX-TL requires both primary and secondary energy sources, as well as amino acids, cofactors, additives, and a template DNA sequence. Nucleotide triphosphates (NTPs) provide the primary energy source to drive initial mRNA (ATP, GTP, CTP, and UTP) and protein synthesis (only ATP and GTP). To increase TX-TL yields, NTPs are regenerated through the catabolism of a secondary energy source, such as maltose¹⁴, maltodextrin¹⁵, glucose¹⁴, 3-phosphoglycerate (3-PGA)¹⁶, phosphoenolpyruvate¹⁷, and L-glutamate¹⁸. This inherent metabolic activity is surprisingly versatile, yet poorly studied, especially in emerging TX-TL systems. Each energy source has distinct properties and advantages in terms of ATP yield, chemical stability, and cost, which is an important consideration for scaled-up TX-TL reactions. So far, current protocols for E. coli TX-TL have reached up to 4.0 mg/mL (~157 µM) for the model green fluorescent protein (GFP), using a blend of 3-PGA (30 mM), maltodextrin (60 mM), and D-ribose (30 mM) as the secondary energy source¹⁹.

Recently, there has been a rising interest in studying secondary metabolite biosynthetic pathways in TX-TL systems^20,21,22. Specifically, Actinobacteria are a major source of secondary metabolites, including antibiotics and agricultural chemicals^23,24. Their genomes are enriched with so-called biosynthetic gene clusters (BGCs), which encode enzymatic pathways for secondary metabolite biosynthesis. For the study of Actinobacteria genetic parts and biosynthetic pathways, a range of Streptomyces-based TX-TL systems have recently been developed^5,6,25,26. These specialized Streptomyces TX-TL systems are potentially beneficial for the following reasons: [1] provision of a native protein folding environment for enzymes from Streptomyces spp.²⁶; [2] access to an optimal tRNA pool for high G+C (%) gene expression; [3] active primary metabolism, which potentially can be hijacked for the supply of biosynthetic precursors; and [4] provision of enzymes, precursors, or cofactors from secondary metabolism present in the native cell extract. Hence, a high-yield S.venezuelae TX-TL toolkit has recently been established to harness these unique capabilities⁵.

Streptomyces venezuelae is an emerging host for synthetic biology with a rich history in industrial biotechnology^5,27,28,29 and as a model system for studying cell division and genetic regulation in Actinobacteria^30,31,32. The main type strain, S. venezuelae ATCC 10712, has a relatively large genome of 8.22 Mb with 72.5% G+C content (%) (Accession number: CP029197), which encodes 7377 coding sequences, 21 rRNAs, 67 tRNAs, and 30 biosynthetic gene clusters²⁷. In synthetic biology, S. venezuelae ATCC 10712 is an attractive chassis for the heterologous expression of biosynthetic pathways. Unlike most other Streptomyces stains, it provides several key advantages, including a rapid doubling time (~40 min), an extensive range of genetic and experimental tools^5,28, lack of mycelial clumping, and sporulation in liquid media^28,33. Several studies have also demonstrated the use of S. venezuelae for heterologous production of a diverse array of secondary metabolites, including polyketides, ribosomal and nonribosomal peptides^{34,35,36,37,38}. These combined features make this strain an attractive microbial host for synthetic biology and metabolic engineering applications. While S. venezuelae is not the dominant Streptomyces model for heterologous gene expression, with further developments, it is primed for broader use within natural product discovery.

This manuscript presents a detailed protocol (Figure 1) for a high-yield S. venezuelae TX-TL system, which has been updated from the original previously-published protocol²⁶. In this work, the energy solution and reaction conditions have been optimized to increase protein yield up to 260 μg/mL for the mScarlet-I reporter protein in a 4 h, 10 μL batch reaction, using a standard plasmid, pTU1-A-SP44-mScarlet-I. This plasmid has been specifically designed to enable various methods of detecting protein expression. The protocol is also streamlined, while the energy system has been optimized to reduce the complexity and cost of setting up cell-free reactions without compromising the yield. Along with the optimized TX-TL system, a library of genetic parts has been developed for fine-tuning gene expression and as fluorescent tools for monitoring TX-TL in real time, thereby creating a versatile platform for prototyping gene expression and natural product biosynthetic pathways from Streptomyces spp. and related Actinobacteria.

In this work, the recommended standard plasmid (pTU1-A-SP44-mScarlet-I) can be used to establish the S. venezuelae TX-TL workflow in a new laboratory and is available on AddGene (see Supplemental Table S1). pTU1-A-SP44-mScarlet-I provides the user with the flexibility to study other open-reading frames (ORFs). The mScarlet-I ORF is codon-optimized for S. venezuelae gene expression. The SP44 promoter is a strong constitutive promoter that is highly active in both E. coli and Streptomyces spp.³⁹. The plasmid has two unique restriction enzyme sites (NdeI, BamHI) to allow the sub-cloning of new ORFs in-frame with a joint C-terminal FLAG-tag and fluorescein arsenical hairpin (FlAsH) binder tag system. Alternatively, both tags can be removed with the inclusion of a stop codon after sub-cloning a new gene. With this base vector, the high-yield expression of a range of proteins has been demonstrated, namely proteins from the oxytetracycline biosynthesis pathway and an uncharacterized nonribosomal peptide synthetase (NRPS) from Streptomyces rimosus (Figure 2). In terms of mRNA detection, the pTU1-A-SP44-mScarlet-I standard plasmid contains a dBroccoli aptamer (in the 3'-untranslated region) for detection with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) probe. For increased flexibility, a toolset of EcoFlex⁴⁰-compatible MoClo parts has also been made available on AddGene, including an EcoFlex-compatible Streptomyces shuttle vector (pSF1C-A-RFP/pSF2C-A-RFP) and a range of pTU1-A-SP44 variant plasmids expressing superfolder green fluorescence protein (sfGFP), mScarlet-I, mVenus-I, and β-glucuronidase (GUS). In particular, the pSF1C-A plasmid is derived from pAV-gapdh²⁸ and is cured of BsaI/BsmBI sites for MoClo assembly. pSF1C-A-RFP/pSF2C-A-RFP is equivalent to pTU1-A-RFP/pTU2-A-RFP from EcoFlex⁴⁰ but contains additional functionality for conjugation and chromosomal integration in Streptomyces spp. using the phiC31 integrase system²⁸.

The first stage of the protocol involves the growth of the S. venezuelae ATCC 10712 or a closely related strain, cell harvest at mid-exponential phase, cell wash steps, and equilibration in S30A and S30B buffers. This stage requires three days, and the time for cell growth can be used to prepare the remaining components as described below. The harvested cells are then lysed by sonication, clarified, and undergo a run-off reaction. At this final stage of preparation, the cell extracts can be prepared for long-term storage at -80 °C to minimize loss of activity. For the assembly of TX-TL reactions using this protocol, a Streptomyces Master Mix (SMM) is presented, with the option of a Minimal Energy Solution format (MES) that gives comparable yields. Further, it is recommended to streak a fresh culture of S. venezuelae ATCC 10712 from a -80 °C glycerol stock onto a GYM agar plate and incubate at 28 °C for at least 48-72 h until single colonies are visible. Only fresh cultures should be used for the following steps.

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) such as 3-PGA (requires pH adjustment), follow the guidance provided in the E. coli TX-TL protocol⁴¹.
   NOTE: All components are fully soluble in ddH₂O and stored as aliquots in the -80 °C freezer.
4. Defrost individual stocks or energy solutions (described later) on ice. Heat the amino acids stock at 42 °C with vortexing for ~15-30 min to solubilize all amino acids.
5. As some amino acids (L-Cys, L-Tyr, L-Leu) precipitate on ice, while minimizing rest time, leave this solution at room temperature and use a vortex to dissolve.
6. Add the calculated volumes (Table 3) of stock solutions and water and mix well using a vortex.
7. Aliquot the energy solution as 20-100 µL aliquots per tube, or as desired, on ice and store at -80 °C until further use.

2. Preparation of S. venezuelae ATCC 10712 cells

1. Day 1-Media/buffer preparation and overnight pre-culture
   1. Prepare 1 L of sterile GYM liquid medium in a 2 L baffled flask, as described in Table 1. See the Table of Materials for equipment/chemical/reagent sources.
   2. Prepare 1 x 50 mL of sterile GYM liquid medium in a 250 mL Erlenmeyer flask, as described in Table 1.
   3. Prepare 100 mL of 1 M HEPES-KOH pH 7.5, 100 mL of 1 M MgCl₂, and 500 mL of 4 M NH₄Cl solutions to make 1 L of S30A and 1 L of S30B wash buffers. See Table 2 for the recipes.
   4. Prepare the overnight pre-culture. Pre-warm the sterile 50 mL of GYM liquid medium in a 250 mL Erlenmeyer flask to 28 °C for 30 min.
   5. Inoculate a single colony of S. venezuelae ATCC 10712 (or related strain) from a GYM agar plate into prewarmed 50 mL of GYM liquid medium and incubate at 28 °C, 200 rpm for 16 h (overnight pre-culture).
2. Day 2-Prepare daytime pre-culture and main growth culture.
   1. Pre-warm 50 mL of sterile GYM liquid medium in a 250 mL Erlenmeyer flask at 28 °C for 30 min.
   2. Transfer 1 mL of overnight pre-culture into pre-warmed 50 mL of GYM liquid medium and incubate at 28 °C, 200 rpm for 8 h (daytime pre-culture).
   3. After this growth period, check the OD₆₀₀ in a spectrophotometer using a 1:10 dilution with sterile GYM medium in a 1 mL (1 cm path length) plastic cuvette.
      NOTE: The OD₆₀₀ should have reached at least 3-4. If there is poor growth, it is advisable to repeat steps 2.2.1-2.2.2.
   4. Sub-culture 0.25 mL of daytime pre-culture into 1 L of liquid GYM medium in 2 L baffled flasks.
   5. Shake overnight at 28 °C, 200 rpm for 14 h.
3. Day 3-Harvest cells
   1. After the previous incubation period (14 h), record the OD₆₀₀ of the main culture. Dilute the overnight culture 1:10 with fresh GYM medium for OD₆₀₀ measurement.
      NOTE: The OD₆₀₀ should have reached 3.0-4.0 at this stage.
   2. If OD₆₀₀<3.0, increase the shaking speed to 250-300 rpm and grow until an OD₆₀₀ of 3.0 is reached. Grow for no longer than an additional 2 h (16 h in total).
   3. If OD₆₀₀>3.0, transfer the cultures to centrifugation containers and rapidly cool on wet ice for 30 min.
   4. While waiting for the cell culture to cool on ice, prepare 4 mL of fresh 1 M dithiothreitol (DTT), S30A and S30B buffers, as described in Table 1, and keep them on ice. See the Table of Materials for chemical/reagent source.
   5. Pre-weigh an empty 50 mL centrifuge tube and pre-chill at -20 °C.
   6. Add 2 mL of 1 M DTT to 1 L of S30A buffer on ice and mix well.
      NOTE: Add DTT to the S30A and S30B wash buffers only before using them.
   7. Centrifuge cells at 6,000 × g, 4 °C, 10 min, and carefully discard the supernatant in a quick and single motion.
      NOTE: If the pellet is disturbed, maximize cell retention with residual GYM medium and continue the protocol.
   8. Add 500 mL of S30A buffer and resuspend the cells by shaking the centrifugation bottles vigorously until the cell clumps are homogeneously dispersed.
   9. Centrifuge the cells at 6,000 × g, 4 °C, 6 min, and carefully discard the supernatant.
      NOTE: Although the cell pellet will be firmer at this point, some cells will remain in suspension (see Figure 1). Treat as described in 2.3.7 and retain as many cells as possible.
   10. Repeat steps 2.3.8-2.3.9.
   11. Add 2 mL of 1 M DTT to 1 L of S30B buffer on ice and mix well. Add 500 mL of S30B buffer to the cells. Repeat step 2.3.9.
   12. Resuspend the cell pellet in 10 mL of S30B buffer and transfer to the pre-weighed, pre-chilled 50 mL centrifuge tube. If required, transfer the residual cells with an additional 5-10 mL of S30B buffer. Fill to 50 mL with S30B.
   13. Centrifuge cells at 6,000 × g, 4 °C, 10 min, and carefully discard the supernatant.
   14. Repeat step 2.3.13.
   15. Carefully aspirate the remaining S30B supernatant with a 100-200 µL pipette.
   16. Weigh the wet cell pellet.
       NOTE: Typical wet cell pellet weight for 1 L of overnight GYM culture (OD₆₀₀ = 3.0) is ~4.5 g.
   17. For every 1 g of wet cells, add 0.9 mL of S30B buffer. Resuspend the cells using either a Pasteur pipette or vortex.
   18. Centrifuge briefly (~10 s) up to 500 × g to sediment the cells.
       NOTE: The protocol can be paused at this point, and cells can be frozen on either liquid nitrogen or dry ice and stored at -80°C. For safety, wear appropriate personal protective equipment (PPE) when handling liquid nitrogen, including face shields and gloves.

3. Cell lysis by sonication to obtain the crude cell extract

NOTE: At this stage, the user can choose to disrupt the cells by sonication either in 1 mL fractions (option 1) or as a larger cell suspension (5 mL) in a 50 mL tube (option 2). Both options have been detailed below to ensure reproducibility, as the final volume of the cell suspension can change due to the loss of cells during previous harvesting and wash steps. A new user should attempt option 2.1 first to establish the protocol.

1. Cell Lysis by sonicating in 1 mL fractions
   1. Using a 1 mL pipette tip (cut off the end of the tip to increase the bore size), transfer 1 mL of the cell suspension into 2 mL microcentrifuge tubes.
      NOTE: If the cells are frozen, rapidly thaw the 50 mL tube containing the pellet in lukewarm water prior to cell lysis. Transfer the tube to wet ice as soon as the pellet has begun to defrost, and chill for 10 min.
   2. Place each microcentrifuge tube in a beaker of ice water, using a plastic tube rack to hold the tube for sonication.
      NOTE: Due to the sensitivity of the cell extract to overheating, it is critical to ensure that the tubes do not warm up to prevent protein precipitation and reduced enzymatic activity.
   3. Use a sonicator probe with a 3 mm diameter tip and clean it with 70% (v/v) ethanol and double-distilled water (ddH₂O). Lower the sonicator tip into the cell suspension until it is ~1 cm below the liquid surface.
   4. Input the following settings into the sonicator: 20 kHz frequency, 65% amplitude, 10 s pulse ON time, 10 s pulses OFF time, 1 min total sonication time.
   5. Run the sonication protocol. Move the tube up/down and sideways during the first two resting cycles to ensure the cells are evenly sonicated. Record the energy input.
      NOTE: For safety, wear appropriate hearing protection during sonication. The viscosity will decrease as cells are disrupted, and the pale cream wet cell pellet should turn into a homogenous brown fluid. The recommended energy input is 240 J per mL of wet cells. If the cells are only partially lysed, the suspension will still appear cream-colored with viscous clumps of cells, particularly on the sides of the tube.
   6. Invert the tube 2-3 times and repeat the sonication for an additional one or two 10 s cycles, mixing frequently until the cells are fully disrupted.
2. Cell Lysis by sonicating a 5 mL cell suspension
   1. If the cells are frozen, rapidly thaw the 50 mL tube containing the pellet in lukewarm water with shaking before cell lysis. Transfer the tube to wet ice as soon as the pellet has begun to defrost, and chill for 10 min.
   2. Briefly spin the tube at 500 x g to sediment the cells.
   3. Place the 50 mL tube in a beaker of ice water for sonication.
      NOTE: Due to the sensitivity of the cell extract to overheating, it is critical to ensure that the tubes do not warm up to prevent protein precipitation and reduced enzymatic activity.
   4. Use a sonicator probe with a 6 mm diameter tip and clean it with 70% (v/v) ethanol and ddH₂O (see the visual schematic of 6 mm probe in Figure 1). Lower the sonicator tip into the cell suspension (~5 mL) until it is ~1 cm below the liquid surface.
   5. Input the following settings into the sonicator: 20 kHz frequency, 65% amplitude, 10 s pulse ON time, 10 s pulses OFF time, 1 min total sonication time per mL of wet cells (5 min in total).
   6. Run the sonication protocol. Move the tube up/down and sideways during the first two resting cycles to ensure the cells are evenly sonicated.
      NOTE: For safety, wear appropriate hearing protection during sonication. The viscosity will decrease as the cells are disrupted, and the pale cream wet cell pellet should turn into a homogenous brown fluid. Record the energy input. An optimal energy input of 240 J per mL of wet cells (~1200 J in total from 5 min sonication) is recommended.
   7. If some cells remain intact, follow the guidance from step 3.1.5.
   8. Transfer the cell extracts into 2 mL microcentrifuge tubes.

4. Cell extract clarification and run-off reaction

1. Centrifuge the lysed cells at 16,000 × g for 10 min at 4 °C to remove the cell debris. Transfer the supernatant into 1.5 mL microcentrifuge tubes as 1 mL aliquots.
2. Perform the run-off reaction for the cell extracts. Incubate the 1.5 mL tubes containing the cell extracts at 30 °C for 60 min on a heat block or incubator without shaking.
3. Centrifuge the cell extracts at 16,000 × g for 10 min at 4 °C. Pool the supernatants into a 15 mL centrifuge tube. Mix the supernatant by inverting the tube five times until homogenous, then keep it on ice. Invert gently to avoid the formation of air bubbles.
4. Dilute 10 µL of the cell extract 100-fold with S30B buffer and measure the total protein concentration using a Bradford assay with three technical repeats (see Supplemental Material S2 for Bradford assay guidance).
5. If the protein concentration is 20-25 mg/mL, transfer the cell extracts as 100 µL aliquots into new 1.5 mL tubes, flash-freeze in liquid nitrogen, and store at -80 °C.
   NOTE: For safety, wear appropriate PPE when handling liquid nitrogen, including face shields and gloves.
6. If the protein concentration is <20 mg/mL, repeat the crude extract preparation steps to ensure high-quality cell extract and TX-TL yields are comparable to the previously published work⁵.

5. Preparation of plasmid DNA template

1. Purify the pTU1-A-SP44-mScarlet-I plasmid (pUC19 origin) from a freshly transformed E. coli plasmid strain (DH10β, JM109) grown in 50 mL of LB culture (with 100 mg/mL carbenicillin) using an appropriate plasmid DNA purification kit as per manufacturer's instructions.
2. Elute the plasmid in 2 x 300 µL of nuclease-free water and combine the fractions.
3. Add 0.1 volumes (66 μL) of 3 M sodium acetate (pH 5.2).
4. Add 0.7 volumes (462 μL) of isopropanol.
5. Incubate the DNA at -20 °C for 30 min.
6. Centrifuge at 16,000 × g for 30 min at 4 °C and discard the supernatant.
7. Add 2 mL of 70% (v/v) ethanol to the DNA pellet.
8. Invert the tube 3-4 times to resuspend the plasmid DNA pellet.
9. Centrifuge at 16,000 × g for 5 min at 4 °C and discard the supernatant.
10. Repeat steps 5.7-5.9 and remove all visible liquid.
11. Air-dry the DNA pellet for 10-30 min or dry for 5 min with a vacuum centrifuge.
12. Resuspend the dried pellet with 600 µL of nuclease-free ddH₂O.
13. Measure the DNA concentration and purity using a spectrophotometer.
14. Prepare 50-100 µL aliquots and store at -20 °C.
    ​NOTE: A high DNA concentration in the range of 500-1000 ng/µL is recommended due to the tight volume constraints of cell-free reactions. Dilute the plasmid DNA stock to 80 nM; 168 ng/µL pTU1-A-SP44-mScarlet-I plasmid is equivalent to 80 nM.

6. Preparation of the Streptomyces Master Mix (SMM) solution

1. Amino acid solution
   1. Use the amino acid sampler kit to avoid manual errors and reduce preparation time, following the manufacturer's instructions provided online.
   2. Dilute the 20x amino acid stock solution using ddH₂O to a final concentration of 6 mM (5 mM L-Leu).
   3. Further dilute to 2.4 mM (2 mM L-Leu) within the 2.4x SMM solution (see Table 3).
      ​NOTE: The final concentration in the TX-TL reaction is 1 mM 19x amino acids and 0.83 mM L-Leu.
2. Energy solution and additives
   1. Prepare the other components in the 2.4x SMM solution by following the recipe described in Table 3.
   2. Alternatively, prepare a 2.4x Minimal Energy Solution (MES), following the recipe described in Table 3.

7. Setting up a standard S. venezuelae TX-TL reaction

1. Thaw the cell extract, SMM (or MES) solution, and plasmid DNA on ice. Pre-chill a 384-well plate at -20 °C.
2. Set up TX-TL reactions where 25% of the volume is plasmid DNA, 33.33% is cell extract, and 41.67% is SMM solution; keep them on ice to avoid start time bias.
   NOTE: A standard TX-TL template has been provided (Table 4) to calculate the volume of reagents needed based on the number of reactions. The standard volume for a 33 µL reaction is as follows: 11 µL of cell extract, 13.75 µL of SMM, and 8.25 µL of plasmid DNA.
3. Gently vortex the mixture for ~5 s at a low-speed setting to ensure the solution is homogenous. Avoid foaming/bubble formation.
4. Transfer 10 µL aliquots into three wells of a 384-well plate as a technical triplicate without introducing air bubbles. Seal the plate with a transparent cover and spin at 400 × g for 5 s.
5. Incubate the reaction at 28 °C either in an incubator (for end-point readings) or a plate-reader without shaking.
   NOTE: Reactions typically require 3-4 h to reach completion. See Supplemental Material S2 for guidance on a plate reader and mScarlet-I standard measurements.

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 pTU1-A-SP44-mScarlet-I standard plasmid (Figure 2 and Figure 3) was optimized to provide high-yield expression in S. venezuelae TX-TL with a range of detection methods (SDS-PAGE, fluorescence). In addition, this standard plasmid was modified to demonstrate the synthesis of a range of secondary metabolite enzymes from S. rimosus (Figure 2)⁵. Finally, a potential workflow for scaled-up natural product biosynthesis is shown as a schematic for using a model pathway from the early-stages of heme biosynthesis. The workflow is potentially adaptable to other secondary metabolite biosynthetic pathways. As a guideline, this protocol should provide a minimum yield of 2.8 μM for sfGFP and 3.5 μM for mScarlet-I/mVenus from the expression plasmids provided on AddGene. These figures allow for typical batch variation (up to 28%) observed in previous data⁵, although yields greater than 10 μM mScarlet-I have been achieved with optimal batches (unpublished data).

Measuring S. venezuelae TX-TL of the mScarlet-I gene using five distinct methods
The expression of the pTU1-A-SP44-mScarlet-I standard plasmid is shown, with the measurement of mScarlet-I expression using five different methods: 1. real-time fluorescence measurement of mRNA using the dBroccoli aptamer, 2. real-time fluorescence measurement of immature mScarlet-I protein using the FlAsH tag system, 3. real-time fluorescence measurement of mature mScarlet-I protein, 4. in-gel fluorescence staining of mScarlet-I using FlAsH tag, and 5. Coomassie blue staining of total cell-free proteins. For this data, the reactions were set up in 2 mL microcentrifuge tubes as 33 µL reactions (for end-point samples) or as a 10 μL technical triplicate in 384-well plates in a plate reader. A triple-tagged (N-terminal His₆, C-terminal Flag and C-terminal FlAsH) mScarlet-I protein was separately purified to create a calibration standard for measurements, using the pET15b-mScarlet-I plasmid, which is described further in Supplemental Material S2. The data for these experiments are shown in Figure 3. Further details of the in-gel fluorescence staining method are available in Supplemental Material S3.

S. venezuelae TX-TL of early-stage heme biosynthesis
To serve as a model natural product biosynthetic pathway, the 'one-pot' biosynthesis of uroporphyrinogen III (uro'gen III) was performed using the pTU1-A-SP44-hemC-hemD/cysG^A-hemB expression plasmid⁵. This model biosynthetic pathway was chosen as uro'gen III is highly oxygen-sensitive and rapidly oxidizes (loss of six electrons) to uroporphyrin III, which displays strong red fluorescence. This enables easy detection of the reaction in real time using fluorescence measurements and/or HPLC-MS (Figure 4), as previously described⁵. In addition, these reactions were studied using either a batch or semicontinuous method. A semicontinuous reaction is a strategy, which uses a micro-dialysis device^42,43 that provides additional energy (NTPs, secondary energy source) and amino acids to prolong the reaction time and increase protein synthesis yields. Here, the semicontinuous method is used to scale up the heme model reaction and separate the TX-TL proteins from the reaction product to facilitate the purification and analysis by HPLC-MS. Further details of methods are available in Supplemental Material S4 or for data, see previous work⁵. Semicontinuous cell-free reactions are also described in earlier work^42,43. The example schematic workflow demonstrated here (Figure 4) is potentially adaptable to other natural product biosynthetic pathways.

Figure 1: Overview of the Streptomyces venezuelae TX-TL protocol. A protocol summary is illustrated, including a recommended time frame of three days. The protocol is broken down into distinct stages of cell growth, cell harvest, cell wash, cell lysis by sonication, clarification, run-off reaction, master mix (SMM) preparation, plasmid DNA preparation, and the TX-TL reaction assembly. The full protocol is described in detail within the text, along with guidance and practical tips. Abbreviations: SMM = Streptomyces Master Mix; TX-TL = transcription-translation. Please click here to view a larger version of this figure.

Figure 2: High-yield protein synthesis from high G+C (%) genes. (A) Synthesis of sfGFP, mVenus-I, and mScarlet-I fluorescent proteins. (B) Synthesis of biosynthetic enzymes from Streptomyces rimosus. Abbreviation: EV = Empty Vector; NRPS = nonribosomal peptide synthetase. The figure is modified from ⁵. Please see the protocol and supplemental files for reaction setup and methodology. Please click here to view a larger version of this figure.

Figure 3: Measurement of TX-TL five-ways with the pTU1-A-SP44-mScarlet-I plasmid. (A) Plasmid design including the following features: SP44 is a strong constitutive promoter active in Streptomyces spp. and E. coli; pET-RBS is derived from the pET expression plasmids and is highly active in both Streptomyces spp. and E. coli^5,40; Streptomyces codon-optimized mScarlet-I gene, which encodes a red-fluorescent protein derivative⁴⁴; C-terminal FLAG-tag for affinity chromatography purification or western blotting detection; C-terminal FlAsH tag for fluorescent labeling for in-gel staining or real-time measurement of nascent protein synthesis; dBroccoli aptamer for real-time mRNA measurement using the DFHBI probe; Bba_B0015 transcription terminator, which are highly efficient in S. venezuelae ATCC 10712⁵; ampicillin resistance marker; and pUC19 origin of replication. (B) Real-time mRNA expression, detected with the dBroccoli aptamer and the DFHBI probe (excitation 483-14 nm, emission 530-30 nm). (C) Real-time nascent protein synthesis detection with FlAsH-EDT₂ fluorescent probe (excitation 500-10 nm, emission 535-10 nm). (D) Real-time fluorescence measurement of mScarlet-I synthesis (excitation 565-10 nm, emission 600-10 nm). (E) In-gel staining with the FlAsH-EDT₂ fluorescent probe. (F) Coomassie blue staining of total TX-TL proteins with purified His₆-mScarlet-I standard for comparison. Reactions were run under the conditions described in the protocol with 40 nM of plasmid DNA template. All fluorescence data are represented as RFU, and error bars (standard deviation of three technical repeats) are represented within a grey shaded area. Abbreviations: TX-TL = transcription-translation; FlAsH = fluorescein arsenical hairpin; DFHBI = 3,5-difluoro-4-hydroxybenzylidene imidazolinone; RFU = relative fluorescence units. Please click here to view a larger version of this figure.

Figure 4: Schematic workflow for the S. venezuelae TX-TL semicontinuous reaction. An example workflow for natural product TX-TL, using the early-stage heme biosynthetic operon and downstream analysis by HPLC-MS. Reactions and analysis are detailed in the supplemental material. The figure is modified from ⁵. Abbreviations: SMM = Streptomyces Master Mix; TX-TL = transcription-translation; ALA = 5-aminolevulinic acid; SPE = solid phase extraction; ESI-MS = electron spray ionization-mass spectrometry; HPLC-MS = high-performance liquid chromatography-mass spectrometry. Please click here to view a larger version of this figure.

Table 1: Recipe for GYM bacterial growth medium and GYM agar plate. Please click here to download this Table.

Table 2: Reagents for preparing S30A and S30B wash buffers. This information was adapted from Kieser et al.⁴⁵ Abbreviation: DTT = dithiothreitol. Please click here to download this Table.

Table 3: Recipe for making the S. venezuelae MES and SMM solutions. Abbreviations: MES = Minimal Energy Solution; SMM = Streptomyces Master Mix; NTP = nucleoside triphosphate; PEG 6000 = polyethylene glycol 6000; 3-PGA = 3-phosphoglycerate; G6P = glucose-6-phosphate; PVSA = polyvinylsulfonic acid. Please click here to download this Table.

Table 4: Recipe for S. venezuelae TX-TL reaction. Abbreviations: MES = Minimal Energy Solution; SMM = Streptomyces Master Mix; TX-TL = transcription-translation. Please click here to download this Table.

Supplemental Table S1: Plasmids for S. venezuelae TX-TL workflow. Abbreviation: TX-TL = transcription-translation. Please click here to download this Table.

Supplemental Material S2: mScarlet-I calibration standard preparation and plate reader measurements. Please click here to download this File.

Supplemental Material S3: FlAsH-tag methods. Abbreviation: FlAsH = fluorescein arsenical hairpin. Please click here to download this File.

Supplemental Material S4: Semicontinuous reaction, purification, and HPLC-MS. Please click here to download this File.

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 recommended here is to ensure that S. venezuelae is growing rapidly in the chosen rich medium, to be able to inoculate the final culture in the evening. This allows cell harvest at peak growth the following morning and permits the user to harvest and prepare the active cell extract on the same day. By following this streamlined protocol, it is expected that a single researcher can complete the protocol conveniently in a three-day framework. A complementary plasmid toolkit has also been provided for the S. venezuelae TX-TL system, including a strong expression plasmid system (pTU1-A-SP44-mScarlet-I), which provides broad functionality for mRNA/protein analysis. This standard plasmid is powered by the constitutive SP44 promoter that is highly active in a range of Streptomyces spp. and in E. coli³⁹. To demonstrate the initial potential of the S. venezuelae TX-TL toolkit, the representative results show the high-yield synthesis of a range of fluorescent proteins, secondary metabolite enzymes, and the biosynthesis of a model natural product pathway (from heme biosynthesis).

Overall, the protocol contains a detailed description of the S. venezuelae TX-TL system, as well as practical tips for preparing the three essential components of the TX-TL reaction: (1) cell extract, (2) Streptomyces Master Mix (SMM) solution, and (3) plasmid DNA. This protocol does not require specialized equipment and only requires routine microbiology and biochemistry skills; hence, it is accessible to most laboratories. The protocol is suited for small-scale (10-100 µL) and larger-scale reactions (~2.5 mL), although some optimization of reaction size/aeration may influence protein yield. The recommended reaction volume is 33 μL in a 2 mL tube or 10 μL in a 384-well plate. The crude extract takes five days to prepare by a single person starting from a glycerol stock. Each liter (L) of culture yields at least 5 mL of cell extract (equivalent to ~1500 x 10 µL TX-TL reactions)-this is a conservative estimate and accounts for sample loss during wash steps and cell extract clarification. Each stage of the protocol is independent and can be optimized by the user to meet their needs. A major limitation for all cell-free systems is batch variation^46,47. Generic factors include pipetting error, user experience, media batch variation, and equipment differences. We specifically introduce a master mix to minimize pipetting error and provide detailed instructions that cover media and equipment use. To date, the protocol is reproducible by a range of users in at least five UK research groups. However, it is unknown what role biological variation contributes to cell-free batch variability. Alongside global gene expression regulation differences, genome plasticity in Streptomyces spp. is widely reported and is a potential contributor⁴⁸. To investigate batch variation, it is recommended to grow up to four separate 1 L cultures derived from four single colonies grown overnight. Previously, up to 28% variation (in terms of standard deviation) was observed between four biological batches (4 L per batch provided ~20 mL of cell extract)⁵. Based on these data, a reasonable minimal target for a new user is 2.8 μM for sfGFP and 3.5 μM mScarlet-I/mVenus-I using the plasmids that are available on AddGene-these targets are 30% lower than the average observed in previous data. If downstream HPLC-MS analysis is desired, the PEG 6000 can be removed from the master mixes, although a decrease in the overall TX-TL yield can be expected by up to 50%.

In terms of the potential of specialized Streptomyces cell-free systems^5,6, there is a growing desire to develop new wet-laboratory tools for bioprospecting applications such as natural products. The Streptomyces genus is steeped in the history of natural product discovery, including antibiotics, herbicides, and pharmaceutical drugs⁴⁹. The increasing knowledge gained from whole-genome sequencing projects and the latest bioinformatic tools^50,51,52 has revealed an unprecedented level of natural products encoded by BGCs within microbial genomes⁵³. Unlocking this genetic information-which is anticipated to hold new drugs/chemicals and enzymes useful to biotechnology-will require the development of new synthetic biology strategies, including novel expression systems and a range of metabolic engineering tools⁵⁴. Specialized Streptomyces-based TX-TL systems are advantageous to study genes and regulatory elements from Actinobacteria and related genomes for the following reasons: [1] availability of a native protein folding environment²⁶, [2] access to an optimal tRNA pool for high G+C (%) gene expression, and [3] an active primary metabolism for the potential supply of biosynthetic precursors. In addition, a key advantage of cell-free systems is the high-throughput characterization of genetic parts and gene expression, using next-generation sequencing¹³ and acoustic liquid handling robotics^8,11,12. In summary, the S. venezuelae TX-TL toolkit⁵ provides a complementary tool in the field of synthetic biology for natural products. The S. venezuelae TX-TL toolkit will support the further development of S. venezuelae as a model system and provide a method to engineer novel synthetic biology parts/tools and explore secondary metabolite biosynthetic pathways and enzymes.

The authors declare that they have no competing financial interests.

The authors would like to acknowledge the following research support: EPSRC [EP/K038648/1] for SJM as a PDRA with PSF; Wellcome Trust sponsored ISSF fellowship for SJM with PSF at Imperial College London; Royal Society research grant [RGS\R1\191186]; Wellcome Trust SEED award [217528/Z/19/Z] for SJM at the University of Kent; and Global Challenges Research Fund (GCRF) Ph.D. scholarship for KC at the University of Kent.