Part:BBa_K4011003

Fre-SttH

Fre-SttH is a fusion protein used to halogenate the sixth carbon of tryptophan (Trp) in the dye production of 6, 6’dibromoindigo (tyrian purple) and 6, 6’dichloroindigo (tyrian red), with the help of another fusion protein: TnaA-FMO. Fre-SttH is composed of two separate domains: Fre and SttH, fused together with a rigid linker. This is a part in a part collection where we enable the production of indigo, tyrian purple, and related dyes from tryptophan in E. coli.

The part collection includes: Parts expressing Fre-SttH to convert Trp to 6-X-Trp. BBa_K4011003 and BBa_K4011012 . Parts expressing fusion protein TnaA-FMO to convert 6-X-Trp into indigoid dyes. BBa_K4011004 BBa_K4011005 BBa_K4011013 BBa_K4011014 BBa_K4011015 and BBa_K4011019 .

Our part collection can be used to help and inspire future teams to design and perfect different indigoid dye production pathways in E. coli, adding to the collection.

Usage and Biology

Fre-SttH is a fusion protein used to halogenate the sixth carbon of the tryptophan in the dye production. Fre-SttH is composed of two separate domains: Fre is from E.coli and SttH is from Streptomyces toxytricini. They are fused by a rigid linker with the protein sequence EAAAKEAAAK. SttH is a trp-6-haloganese that requires FADH2 as a cofactor to convert trp and halogen ions into 6-X-trp, and is highly insoluble in E.coli. Therefore, Fre, a highly-soluble flavin reductase which reduces FAD to FADH2 from E.coli, is fused with SttH as a N-terminal soluble tag, enabling the protein to become soluble and eliminating the need for costly FADH2 cofactors to be added. We express Fre-SttH in ptac system and measured its enzymatic activity using HPLC (Lee et al, 2021).

Characterization

SDS-PAGE

We expressed Fre-SttH under T7 promoter in E. coli BL21(DE3) (Fig. 1A). SDS-PAGE of the sample showed decent Fre-SttH expression but suboptimal water solubility, since the supernatant sample has less intense target band compared to the whole cell sample (Fig. 1B).

For producing tyrian purple, Fre-SttH and TnaA has to be in two different strains, so we used a ΔTnaA E. coli strain, courtesy of Sha Zhou. However, our ΔTnaA E. coli was supplied as E. coli DH5α, which was incompatible with the T7 promoter.

Because of the need for a ΔTnaA E. coli strain, we decided to switch from the T7 system to the E. coli DH5α compatible ptac system. We constructed two ptac plasmids, ptac-Fre-SttH and ptac-histag-Fre-SttH, and transformed them into E. coli DH5α ΔTnaA (Fig. 1C).

We then induced expression of both proteins and performed SDS-PAGE. Results show that histag-Fre-SttH expression and solubility were poor, but Fre-SttH had extremely high expression and solubility (Fig. 1D). Thus, ptac-Fre-SttH in E. coli DH5α ΔTnaA was used for all further experiments.

Figure 1: Construction and expression of Fre-SttH proteins. A) Schematic representing E. coli BL21 (DE3) transformed with histag-Fre-SttH plasmid. B) SDS-PAGE analysis of histag-Fre-SttH expressed by E. coli BL21 (DE3) represented by A. C) Schematic representing E. coli DH5a ΔTnaA transformed with histag-Fre-SttH and Fre-SttH plasmids. D) SDS-PAGE analysis of Fre-SttH and histag-Fre-SttH expressed by E. coli DH5a ΔTnaA represented by C.

HPLC

In order to model the enzymatic dynamics of Fre-SttH, we induced Fre-SttH expression and added the substrates. We then took samples of the culture in 6-hour-intervals for 24 hours for HPLC, and calculated the relative concentrations of trp and 6-X-trp (Fig. 2C & D). To learn about the detailed experimental setup, visit our experiment and measurement pages.

For sample added with trp and NaBr, the concentration of trp decreased from approximately 1.5 mM to 0.5mM, while the concentration of 6-Br-Trp increased from approximately 0mM to 1.0mM (Fig. 2A). We obtained similar result from sample with trp and NaCl, which the trp concentration went from approximately 1.7mM to 0.4mM and 6-Cl-Trp concentration increased from 0 to 1.3mM (Fig. 2B). For both samples, the yield of trp to 6-X-trp conversion by Fre-SttH was near 100%. As trp still remains after 24h in both cultures, a fermentation time of 36 or 48h might yield a higher concentration of 6-X-Trp, should it be desired. From this, we acquired several enzymatic constants for Michaelis-Menten equation, which was vital to our modeling team.

Figure 2: Quantifying the production of 6-X-Trp in Fre-SttH. A) Concentration-time graph of Trp and 6-Br-Trp in ptac-Fre-SttH in E. coli DH5a ΔTnaA with 1.5mM Trp and NaBr. B) Concentration-time graph of Trp and 6-Cl-Trp in a culture of ptac-Fre-SttH in E. coli DH5a ΔTnaA with 1.7mM Trp and NaCl. C) HPLC results, from top to bottom, of Trp, 6-Br-Trp, 040 + NaBr culture at 0, 6, 12, 18, and 24h. D) HPLC results, from top to bottom, of Trp, 6-Cl-Trp, 040 + NaCl culture at 0, 6, 12, 18, and 24h.

The effectiveness of Fre-SttH in different strains of TnaA-FMO

TnaA and FMO are two vital but separate enzymes for converting trp/6-Br-trp to our indigo and tyrian purple dye. We designed and engineered three strains of E. coli DH5α ΔTnaA: ptac-TnaA-rbs-FMO (RBS; in this strain TnaA and FMO are expressed as separate proteins), ptac-TnaA-rigid linker-FMO (RL), and ptac-TnaA-Flexible linker-FMO (FL) to test the halogenation effectiveness of Fre-SttH

Figure 3: Measuring dyes production of different TnaA-FMO strains from Trp, 6-Cl-Trp, and 6-Br-Trp. A) Pictures of TnaA-RL-FMO, TnaA-FL-FMO, and TnaA-RBS-FMO with Trp, 6-Cl-Trp, or 6-Br-Trp added. B) Comparison of production titers of TnaA-RL-FMO, TnaA-FL-FMO, and TnaA-RBS-FMO with Trp, 6-Cl-Trp, or 6-Br-Trp added, pictured in A. Error bars denote two standard deviations from the mean.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 576
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 948
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 424