Membrane Rudder

- Violacein & Deoxyviolacein synthetic pathway

Background

Dynamically and artificially regulating the direction of biochemical pathway in vivo has remained a challenge for scientists. We are now to achieve this goal through controlling the aggregation state of different enzymes in branched biological reactions based on Membrane Scaffold. We named this universal device Membrane Rudder. Theoretically this device could sense a large amount of signals. We chose blue light and rationally designed RNA molecule D0 as controlling signal. Branched violacein & deoxyviolacein synthetic pathway is recruited to verify the feasibility of Membrane Rudder.

There are three branches in violacein & deoxyviolacein synthetic pathway. We could identify the different end-products through high-performance liquid chromatography (HPLC). The change of products proportion will demonstrate the efficiency of direction alteration.

Violacein & Deoxyviolacein Biosynthetic Pathway

The violacein & deoxyviolacein biosynthetic pathway includes 5 key enzymes which work in sequence. VioA, a flavoenzyme and VioB, a heme protein, work together to oxidize and dimerize tryptophen into IPA imine dimer. Then VioE induces an indole rearrangement, producing prodeoxyviolacein acid, also known as PVA.

Fig.1 :Details of violacein & deoxyviolacein biosynthetic pathway. The left pathway painted in purple indicates the synthetic pathway of purple pigment deoxyviolacein.The right pathway painted in violet indicates the synthetic pathway of purple pigment violacein. The green pathway indicates the nonenzymatic production of green pigment deoxychromoviridans.

The main intermediate, PVA now is produced by the sequential catalyzation of VioA, VioB, VioE, then branches are about to appear.

There exists one intrinsic E.coli enzyme that automatically contribute to an additional side reaction, further modifying PVA into one green pigment called deoxychromoviridans.

The last two enzymes, VioC and VioD are flavin-dependent oxygenases. VioC alone transforms PVA into a purple pigment called deoxyviolacein, the second product, while it could also act cooperatively with VioD. The 3rd branch works like this: VioD hydroxylates 5-position indole ring, then the other 2-position indole ring is processed by VioC to create the oxindole. It is in this way could violacein be produced.

Design of Experiment

We tried to create Membrane Rudder controlled by light signal and RNA signal to switch direction of biochemical reaction dynamically and artificially in E.coli.

Membrane Rudder Sensing Light Signal

Light is an intriguing signal to regulate E.coli activity because it is easy to obtain, highly tunable and nontoxic. A metabolite-coupled light-switchable system could be quite fascinating.

We constructed our device as demonstrated in Fig.2. In light-sensing Membrane Rudder, VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. VVD, a photoreceptor of Neurospora crassa, can form dimer in the presence of blue light and disassociate as light is off. When blue light is present, VioD with VVD-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when blue light is off, VioD with VVD coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.

Fig.2 :Construction details of light-sensing Membrane Rudder. VVD acts as a blue light sensor.

For VioB and VioE only function normally in dimereic state, free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E to ensure the normal function of the whole system.

Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of light-sensing Membrane Rudder are incubated under blue light. The other group of bacteria expressing full set of light-sensing Membrane Rudder are incubated in the dark.

The BioBrick Part VVD-MA5-vioC and VVD-MA6-vioD is [http://partsregistry.org/Part:BBa_K771205 Part:BBa_K771205] and [http://partsregistry.org/Part:BBa_K771206 Part:BBa_K771206], respectively. For MA2-VioA, MA3-VioB, MA4-VioE, the corresponding part is [http://partsregistry.org/Part:BBa_K771201 Part:BBa_K771201], [http://partsregistry.org/Part:BBa_K771202 Part:BBa_K771202], [http://partsregistry.org/Part:BBa_K771203 Part:BBa_K771203], respectively.

Membrane Rudder Sensing RNA Signal

So far, Membrane Accelerator and Membrane Rudder are both post-translation controlling device to regulate metabolic flux of the host cell. To connect this relatively isolated post-translation control system to genetic circuits, we employed RNA signal, which is present in cytoplasm. When certain RNA molecule with two aptamer domains is present in cells, their cognate aptamer binding proteins can thus aggregate together.

By now, if we want to assemble certain proteins through external signal, we need to find corresponding signal-induced dimer or oligomer. But if we place RNA D0 (with PP7 and MS2 aptamer domains) under various promoters regulated by different signals, approaches to induce dimerization would be expanded sharply. Thus, Membrane Rudder could sense much more signals.

We constructed our device as demonstrated in Fig.3. In RNA-sensing Membrane Rudder, VioA, VioB, VioE and VioC with interacting Membrane Anchors constitutively aggregate. RNA D0, can aggregate with RNA aptamer binding protein MS2 and PP7. So when RNA D0 is present, VioD with MS2-coupled Membrane Anchor would aggregate with assembly of VioA, B, C and E, making violacein the dominant final product. But when RNA D0 is absent, VioD with MS2-coupled Membrane Anchor will disassociate with VioA, B, C and E. Thus the biosynthetic pathway for deoxyviolacein is switched on.

Fig.3 :Construction details of RNA-sensing Membrane Rudder. RNA aptamer binding protein MS2 and PP7 acts as an RNA sensor.

Free VioB and free VioE were coexpressed with membrane anchored VioA, B, C, D and E to ensure the normal function of the whole system.

Bacteria in experimental group were induced at a L-Arabinose concentration of 0.1%. One group of bacteria expressing full set of light-sensing Membrane Rudder are incubated under blue light. The other group of bacteria expressing full set of light-sensing Membrane Rudder are incubated in the dark.

Results and Discussion

Membrane Rudder Sensing Light Signal

Overview

By attaching Enzymes VioA, B, C, D and E to Membrane Anchors that could sense blue signal, we managed to change the proportion of final products by switching on or off blue light signal. When blue light is on,

Moreover, a significant decrease of side products such as deoxychomoviridans was detected when membrane complex system was present.

Direction Alteration

The switch of the reaction can be realized by the extracellular signals. Through the light induction, the reaction producing deoxyviolacein by VioC alone is inhibited due to the lack of interaction with PVA. On the other hand, as long as light is restrained from the bacteria, however, the above reaction is initiated leading to the production of deoxyviolacein.

Fig.3 :HPLC result (right) and the ratio(left) of deoxyviolacein in sample with light induction and without light induction. Fig. B represents the deoxyviolacein HPLC peak results from sample with no light induction. Fig. C represents the deoxyviolacein HPLC peak results from sample with light induction. Results indicate that the amount of the deoxyviolacein from the sample without light induction is tremendously larger than that from the sample with light induction. The red part in the Fig.A represents the portion of deoxyviolacein and the blue part represents violacein

We ran an HPLC (SHIMADZU LC-20AP,C18 reversed column) test with the purple samples extracted from the bacterium culture.Then we ran an mass spectrometry (Thermo Ultra GC-ISQ) test and verified the corresponding molecule role of every main peak. The HPLC results show that the peak of deoxyviolacein appears at about 9 minutes after the injection. We find out that under the induction of light, production of deoxyviolacein was inhibited (to almost 0%) while most products were violacein. Without the light induction, however, the pathway leading to the deoxyviolacein was initiated and thus deoxyviolacein was produced(about 8%). Such results confirmed our membrane rudder system and indicated that by controlling the extracellular signal, such as light, we are able to manipulate the branched chain reactions, thus producing the target products we desire.

Decrease in Side Products

Membrane complex system would help to reduce the amount of side-products to some extent. Under the case that the enzymes involved in the side reaction are situated in the cytoplasm, they are less competent than the core enzymes attached on the membrane to mediate the subsequent reactions due to the spatial obstacle.

Fig.4 :The HPLC peak area of deoxychromoviridans from sample with light induction and free vioABCDE control group.

From the HPLC results, we detected a distinct decrease of side-product, deoxychomoviridans. Judging from the peak area, we find out that the amount of deoxychromoviridans from rbs-vio control group is nearly eight times that from light-induced group. Such experiment results coincided with our anticipation and demonstrated that the membrane complex system would enhance the usage of substrates and thus reduce the amount of intermediate products and other subsequent side-products.

Now Trying

Multiple choices for signal control over reaction is the next target for membrane rudder, so we constructed a modified set of membrane assemblies where a molecule named RNA D0 offered another way to regulate direction.

As illustrated in the Construction section, we fused MS2 and PP7 aptamer domain to membrane assembly 1 and membrane assembly 2 correspondingly, and they are the cognate binding domains of a RNA molecule we found named RNA D0. If the RNA D0 gene is set behind a genetic circuit, its transcription can be regulated by the circuit and on the other hand, will lead to aggregation of membrane assembly 1 and 2.

If VVD and RNA D0 are put in the system together,they could aid in realizing free switch between 2 or 3 branches of reaction. More overwhemingly, theRNA D0 modified system establish a connection between genetic circuit (in other words,traditional control mode on transcription level) and membrane rudder(our novel dynamic control system on protein level).

We have constructed the PP7 and MS2 standardized BioBrick part, [http://partsregistry.org/Part:BBa_K771111 Part:BBa_K771111] and [http://partsregistry.org/Part:BBa_K771112 Part:BBa_K771112], respectively.

All necessary parts have been constructed and submitted to the registry while relating confirmatory experiments are being conducted.

Reference

1. Balibar, C. J. and C. T. Walsh (2006). "In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum." Biochemistry 45(51): 15444-57.

2. Hoshino, T. "Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core." Appl Microbiol Biotechnol 91(6): 1463-75.

3. Shrode, L. B., Z. A. Lewis, et al. (2001). "vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation." Fungal Genet Biol 32(3): 169-81.