Not content with simple fermentation to produce our target compounds, we sought to address the gaps in current biomanufacturing and incorporated various features into Coup Dy'état. Our system was designed with three main principles in mind: commercial viability, eco-friendliness, and emerging technology adoption (Figure 1).

In our project, we set out to demonstrate the possibility of converting waste to dyes in E. coli via dynamic and stress regulation, achieved using computer-assisted optogenetic control. The illustration below encapsulates how all the features are integrated together to complete the bioproduction process.

The various parts of our system are designed to be modular; they can be incorporated into another system and be expected to work. With the advent of more biomanufacturing technologies, additional features can be assimilated seamlessly. Coup Dy'état is thus a technological platform where microbial production of any chemicals can be optimized.

With Coup Dy'état, we hope to set the standard for biomanufacturing.

We are certain that our approach is much less harmful to the environment than the production of synthetic dyes. But what if we compared our project to traditional methods of obtaining natural dyes? While synthetic biology opens up new vistas for the bioproduction of heterologous compounds, we can only consider ourselves a superior alternative to plant cultivation and extraction if the process of acquiring the sugar feedstock for fermentation consumes significantly less land, water, and other resources ^[1].

But that’s not all. From sucrose derived from sugarcane in Brazil to glucose extracted from corn in the USA, the demand for sugar sources in biomanufacturing competes with demands for animal feeds and human consumption ^[2]. Additionally, sugarcane farming takes a substantial toll on the environment, fuelling deforestation, soil degradation, water pollution, and biodiversity loss ^[3].

Our team realized that we needed to ensure that our bioproduction method is itself eco-friendly and sustainable. We thus turned to lignocellulosic waste. It is the underutilized, non-edible portion of plants found in empty fruit bunches, the major biomass byproduct of the palm oil industry ^[4]. This is incinerated in abundant quantities in plantations all over Indonesia, the largest palm oil producer in the world, contributing to air pollution and haze ^[4] which plague her neighbours, including Singapore.

All these burning issues can be alleviated if we can feed lignocellulosic waste to our bacteria. However, there is a major hurdle. Xylose and glucose are the major constituents of lignocellulose, and economically feasible bioconversion requires one to use both sugars at the same time. Unfortunately, this is hindered by carbon catabolite repression (CCR), a regulatory mechanism where expression of genes necessary for the utilization of secondary carbon sources is suppressed by the presence of a preferred substrate, which is often glucose (Figure 2a and b) ^[5].

Recently, CCR has been shown to be released by performing two substitution mutations in the xylose operon regulatory protein (XylR) gene in E. coli, which creates a strain that is able to co-utilize xylose and glucose efficiently. This mutant, XylR*, has a higher affinity to its DNA binding site, and thus activates its promoter independently of the co-activator, the cAMP receptor protein (CRP) (Figure 2c). As a result, expression of xylose metabolic and transport genes occurs even in the presence of glucose ^[5]!

This had huge implications for our project as converting waste into dyes is now viable. We thus constructed a plasmid which allows overexpression of XylR* upon induction by IPTG (Figure 3), and demonstrated its effectiveness in enhancing xylose utilization in E. coli BL21 Star (DE3) by conducting growth experiments.

We deviated from our initial plan of engineering a xylose-utilizing strain like what was accomplished in the study, it would be easier to transform this construct into our dye-producing bacteria, allowing xylose to serve as the feedstock. This also enables potential optimization of the expression levels of XylR* and balancing of metabolic flux, which may vary depending on the biosynthetic pathway or fermentation conditions.

Along the targeted pathway, we took special interest in a key intermediate compound, naringenin. Naringenin, a flavanone, is extremely valuable due to its antioxidant, anti-inflammatory, antiviral, and anti-tumour properties ^[6]. More intriguingly, naringenin is also the intermediate platform compound for the biosynthesis of other flavonoids, many of which are potential candidates for textile dyes (Figure 4).

While commercial naringenin can be added as the substrate during the biomanufacturing process, we find it desirable to achieve de novo biosynthesis given its strategic location in the biosynthetic pathway, which provides us with the basis for the synthesis of many other high-value flavonoids. Besides reducing costs, this would eliminate the need to introduce expensive chemical precursors ^[7], which is consistent with our team’s vision of involving less chemicals in the biomanufacturing process.

Serendipitously, there was a recent breakthrough involving the production of naringenin from xylose via a synergistic co-culture system comprising E. coli and S. cerevisiae ^[6]. Subsequent literature review also revealed the advantages of co-culture biosynthesis, with each independent strain devoted to the functional expression of different stages of the biosynthetic pathway, allowing for reduced metabolic burden, flexibility of employing suitable hosts, and adjustable biosynthetic strength ^[8]. Inspired by these revelations, we decided to adopt a co-culture strategy as well, especially considering the length of the biosynthetic pathway and how we intended to use xylose in our feedstock.

Therefore, we sought to produce naringenin from tyrosine in a single E. coli strain, which could then be co-cultured with cells responsible for the synthesis of our final compound, thus completing the pathway. This process involves catalysis by four enzymes - phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), Oryza sativa polyketide synthase (OsPKS), and malonyl-CoA synthetase (MCS) ^[9] put together in a single plasmid (Figure 5a).

Currently, our de novo construct carries three of the enzymes necessary for naringenin production. While PAL is placed under a constitutive promoter, OsPKS and MCS are strategically put under the control of a P_lac promoter instead (Figure 5b). This enables overexpression of the two more crucial enzymes involved in the latter stages of the pathway, thus helping to drive the reaction forward.

By supplementing the medium with coumaric and malonic acid as substrates, we demonstrated the production of significant amounts of naringenin in E. coli BL21 Star (DE3) cells carrying the de novo construct. The production of naringenin was verified using High Performance Liquid Chromatography (HPLC), while the expression of the enzymes was confirmed via SDS-PAGE.

With the eventual complete construct, de novo biosynthesis of naringenin can be achieved with just the introduction of this plasmid. This design complements the use of xylose as feedstock to achieve a much more environmentally-friendly bioproduction of naringenin, and ultimately, dyes.

The choice of target compound was given careful consideration as its production is the raison d’être of Coup Dy’état, and the design of all the features in our system would revolve around its production. Our desire to renounce synthetic dyes prompted us to look for natural alternatives, which would be the first product of our system.

We explored the production of various flavonoids, all derived from the intermediate platform compound, naringenin. Chrysanthemin (pink), callistephin (orange), and theaflavin (reddish-brown) were our original candidates as many enzymes are shared across their biosynthetic pathways. A range of colours could then theoretically be produced from a mixture of these compounds, whose relative concentrations could be varied by controlling the expression of key enzymes (Figure 6). However, the promiscuity of the enzymes and inherent leakiness of the pathway necessitate extremely tight control of gene expression which was technically challenging to accomplish.

The major turning point occurred when we interviewed designers and synthetic dye companies and learned that such colours were not commercially viable. Primary colours are preferred, as they could be mixed to produce the desired hue. Following this, we opted to focus our efforts on the synthesis of luteolin, a yellow flavone located downstream of naringenin too (Figure 6).

Notwithstanding the warmth and optimism that it evokes, the colour yellow has a murky history. The vivid Indian Yellow dye was derived from the urine of cows which had been fed only mango leaves and were thus kept in a perpetual state of near-starvation, yellow to be dubbed the “cruellest” colour ^[10]. Presently, the synthetic yellow compound PCB-11, which possesses a plethora of toxic effects, is found ubiquitously in textiles and other consumer products ^[11]. Luteolin, which is perhaps the oldest European dye, enjoys a much better reputation. It is deemed to possess the best light fastness amongst natural yellow dyes, and is still used to dye silk, wool, cotton, and leather ^[12]. A bright future for yellow seemed to be in the cards with luteolin.

Once we confirmed that we would be producing luteolin, we expressed the two enzymes required to produce it from naringenin in E. coli: flavone synthase (FNS) from Petroselinum crispum and flavonoid 3’-hydroxylase (F3’H) from Arabidopsis thaliana (Figure 6), as heterologous expression of these enzymes, albeit in Streptomyces albus, was successful in producing small amounts of luteolin ^[13]. E. coli BL21 Star (DE3) was selected to be our chassis as it is an RNase knockout strain, which enhances protein expression and dye production. It was also shown to be a suitable host for flavonoid synthesis ^[15]. The expression of the enzymes FNS and F3’H are optogenetically controlled (see: Blue Light Repressible System).

Through multiple rounds of biosynthesis, we were able to successfully demonstrate the production of luteolin via HPLC. The levels of mRNA and protein expression were also investigated using qPCR and SDS-PAGE respectively.

Where induction of gene expression is necessary, chemical inducers are staples owing to their capability to trigger high expression levels ^[16]. However, the drawbacks are numerous: potential cellular toxicity, non-reversible induction, delayed diffusion, and high costs ^{[17] [18]}. All these problems are exacerbated in large-scale biomanufacturing (Figure 7).

Enter optogenetics. The allure of being able to manipulate cellular behaviours using light is causing optogenetics to make waves in the field of synthetic biology. Light-based induction offers many advantages: non-toxicity, reversibility, precise spatiotemporal control, tunability, and availability ^{[19] [20]} (Figure 7). Hence, optogenetics offers a compelling alternative to traditional chemical induction, particularly in biomanufacturing, a view that has been corroborated by several experts in the field.

We adopted the EL222 blue light system, thus renouncing the use of chemical inducers. Originating from the marine bacterium Erythrobacter litoralis HTCC2594, EL222 is a photosensitive DNA binding protein, with a N-terminal light-oxygen-voltage (LOV) domain and a C-terminal helix-turn-helix (HTH) DNA binding domain. Irradiation by blue light of wavelength 450nm exposes the hitherto sequestered HTH, facilitating dimerization and subsequent DNA binding ^[21].

In Coup Dy'état, the repressible system was employed to facilitate dynamic gene regulation (see: Cell-Machine Interface). With energy conservation in mind, blue light is shone during the shorter growth phase, and switched off during the lengthier production phase to derepress the expression of enzymes. This is achieved by placing the DNA binding site of EL222 between the -35 and -10 hexamers of the consensus promoter in E. coli, creating the blue light repressible promoter P_BLrep ^[21]. As a result, EL222 acts as a repressor, blocking the binding of RNA polymerase and repress gene expression in the presence of blue light. In the dark, RNA polymerase can now bind, and gene expression takes place (Figure 8).

To elucidate the strength, sensitivity, and leakiness of P_BLrep, and its inducible counterpart, P_BLind, we performed many rounds of characterization experiments. This was essential in providing us with insights for its subsequent integration into bioproduction to drive target gene expression. The characterization involves the use of mRFP as a reporter placed under the control of the promoters. However, the output was not representative of the ON/OFF status of the promoter as a result of the accumulation of mRFP, which has a long half-life. To resolve this, we introduced degradation tags of varying strengths so that the mRFP produced would be targeted for quicker proteasomal degradation (Figure 9). This improves on our previous model for promoter characterization.

Through our characterization experiments, we also found P_BLrep to be better suited for our system due to its relatively lower leakiness, further justifying our choice of a repressible system. Hence, we replaced the chemically-inducible promoters, which were originally used to control the expression of our biosynthetic genes F3’H and FNS, with P_BLrep (Figure 10). To verify that our blue light repressible system works as intended, we demonstrated that cells that were exposed to blue light throughout the course of biosynthesis had a significantly lower yield.

Our project thus built upon the landmark study on optogenetically-regulated bioproduction in yeast ^[22] and demonstrated that the use of this technology with bacteria shows promise. We envision this to be the future of biomanufacturing.

Stress, and the cell’s response to it, is a universal phenomenon. In large industrial bioreactors, stress is exacerbated owing to the large variation in the local environment as compared to small bench-top fermenters or shake-flask cultures. This is the result of a longer global mixing time, causing significant heterogeneity in the medium. Besides hydrodynamic shear stress, cells are also subjected to stress induced by fluctuations in the level of oxygen, pH, substrate, and other parameters ^[23]. As Dr Nic Lindley said, “take a sample point in a bioreactor and you get an image, take a look of the whole bioreactor and you get a film.”

All these compound the difficulty in translating the biological potential of the cell as demonstrated in the laboratory into actual production. One type of stress, however, applies to bench-top experiments in the laboratory and large-scale processes in the industry, and across the bioproduction of any compounds. This opens up the possibility of investigating and regulating it in bioproduction.

This particular stress is that induced by the expression of recombinant proteins. The depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, hampering the growth and expected performance of engineered cells in an unpredictable manner ^[24]. Stress regulation was accomplished by decreasing the expression of a synthetic construct in response to increasing burden. Regulated cells outperformed controls in protein yield ^[25], a discovery with significant implications for the future of biomanufacturing.

Therefore, we strove to improve our yield by incorporating a stress reporter module into our system, which studies the level of stress induced by our constructs and facilitates stress regulation. The module consists of the mRFP gene placed under the control of a burden-responsive promoter P_htpG1 (Figure 11a). This particular promoter showed high promise as a biosensor for burden, as it displayed the best ON/OFF characteristics compared to other promoters that were similarly upregulated upon the induction of synthetic constructs ^[25].

Using this part, we were able to quantify the level of stress induced by GFP production by measuring the fluorescence level, thus demonstrating proof of concept (Figure 11b). Our constructs were subsequently tested and shown to impose a substantial metabolic burden (Figure 11c). Characterization of the P_htpG1 promoter will hopefully pave the way for stress regulation and the ensuing optimization of the bioproduction process.

Biomanufacturing involves the introduction of synthetic constructs often with the aim of maximizing flux towards heterologous pathways, placing tremendous burden on cells, attenuating their growth and curtailing their production (see: Stress Reporter). Cells’ natural propensity to grow and multiply is thus directly in conflict with synthetic biologists’ desire to maximize their production.

However, prioritization of both growth and production could conceivably be accomplished using dynamic control. This separates fermentation into two phases: a growth phase where cells are cultivated to high densities, followed by a production phase where expression of non-native pathways takes place ^[52]. Dynamic control can be achieved through these means - chemical induction, autoregulation, and computer-assisted feedback control. As compared to the other two, computer-assisted feedback control is tunable, versatile, modular - it is easier to incorporate additional modules e.g. nutrient- and substrate-sensing module s^[53], can perform more complex logic, and places less metabolic burden on the cell. These advantages that comes with giving control over to the user will only become more significant as our system scales up.

Computer-assisted feedback control enjoys a particularly excellent synergy with optogenetics, as light sources are easily programmed to turn on/off. However, using light to control transcription remains a challenge in itself. Even state-of-the art methods are unable to simultaneously achieve precision and repeatability, nor can they withstand changing operating culture conditions ^[54]. Furthermore, based on our literature review, custom optogenetic tools have only been designed for well plates, which are small-volume, high-throughput systems. These tools are inadequate for our project as our objective is to scale up to a large-volume, low-throughput bioreactor.

Our first design focus was then to create an in silico automated feedback control system, a theoretically effective and feasible strategy for overcoming current challenges in light-controlled gene expression ^[55] as compared to typical open-loop feedback control systems for gene expression ^[56]. Our second design focus was to accommodate larger-volume containers for scaled-up cell cultivation. We thus produced solutions based on these design considerations.

Hardware

We designed two complementary hardware systems. The first fulfills users’ needs to characterize their optogenetic circuit in scaled-up systems. The second enables optogenetic biomanufacturing. See our Hardware page for more on how we actualized our final designs.

PDF-LA!

Plate-Dish-Flask Light Apparatus (PDF-LA!) is a suite of three fully programmable devices compatible with standard laboratory equipment. Users design their own optogenetic circuit, and have the choice of characterizing their cells in 12-well plates, petri dishes, or Erlenmeyer flasks. The containers, placed in the corresponding devices, will be illuminated using LEDs. These LEDs can be programmed for varying intensities and durations.

The first design was a device for the 12-well plate, P-LA!. We designed P-LA! to improve on another design which had been loaned to us by a different university, and hence gain experience of creating custom tools for optogenetics research.

P-LA! is better than the previous design because it can accommodate 12-well plates from different manufacturers, whereas the other design could only fit a specific 12-well plate model. P-LA! also uses significantly less material. Fun fact: despite its name, P-LA! was printed with ABS filament rather than PLA filament! All our 3D-printed designs use ABS filament as it was made readily available to us by our university, and its material properties were deemed sufficient for our purposes.

Incremental design improvements based on user feedback

After optimizing our device for the 12-well plate, it was time to scale up and create devices for petri dishes and Erlenmeyer flasks. These two items of standard laboratory equipment were chosen based on feedback from research fellows in NUS. To provide users a more integrated solution, we produced a single device which can be used for both, DF-LA!.

As such a device had not been designed previously, there was no existing structure to improve or draw inspiration from, and our team started from scratch. This resulted in many design iterations (Figure 13).

The first iteration is for a single petri dish, which can be illuminated from top and bottom. Users wanted a system with the additional option to test two petri dishes at once, and the second iteration was conceived.

The second iteration was designed such that the components to illuminate a single petri dish from the bottom could be stacked with another set of the same components to create a structure that could expose the petri dish to light from the top and bottom. This symmetry is advantageous as the functionality of a single unit doubles, and was therefore retained and developed in subsequent design iterations.

After that, we attempted to design a separate device for the Erlenmeyer flask, but realized that it would be much simpler and more elegant to adapt the second iteration to also accommodate the flask. The third iteration thus has an extra ring in between the devices for the petri dish. This acts as a holder for the flask. Also, the “teeth and slots” feature used to join the components together has moved from the center of the walls of the cylinder to the outer edge of the cylinder. We intended to shake the flask, and were thus concerned that the walls of the slot and the teeth would be too thin and might break due to shear stresses. We thus modified this feature for the third iteration.

Major changes from the third iteration to the fourth iteration are the increase in the height of the flask adapter so that the flask could also be illuminated from both top and bottom, the increase in the number of LEDs available to illuminate the flask or dish, and modifying the shape of the electronics container so that the whole structure could be cylindrical. The reason for the last change was fivefold - firstly, to reduce the material expended during 3D-printing, secondly, so the device would occupy a smaller area, thirdly, for the user’s comfort during transport as the inconsistency in shape made the structure unwieldy to carry, fourthly, to make the design aesthetically pleasing, and last but definitely not least, the design now becomes infinitely more modular as users can now continuously stack combinations of D-LA!, the petri dish module, and F-LA!, the erlenmeyer flask module, into a single column. As the components are lightweight, structural failure is not a concern, and the number of units a user can stack is only limited by the microcontroller. Many petri dishes and flasks can be illuminated simultaneously while still occupying the same area as a single testing unit. For more on the increased functionalities of our final iteration of DF-LA!, please visit our Hardware page.

Users reported satisfaction with the final design iteration. Please visit our Hardware page for more on how we conducted user testing and learned from user feedback.

Light Wait

Light Wait is a 500 ml working volume bioreactor system, with features enabling optogenetic biomanufacturing, and computer-assisted automated feedback control. Light Wait comprises 4 components - a fermentation chamber, a peristaltic pump, a 2-in-1 OD and fluorescence sensor, and an Arduino Uno microcontroller board (Figure 14). The microcontroller is not shown for visual clarity. We omitted it as it was not something we designed ourselves but an off-the-shelf component. For a user guide of how the entire system operates, and our design of the system as a whole, please visit our Hardware page. This page will focus on the design considerations of the individual components, which are summarized as follows.

Peristaltic Pump

The peristaltic pump was adapted from the design by the 2015 Aachen iGEM team, since their design was also for continuous pumping in a bioreactor. Three modifications were made.

The first modification replaces their pentagonal spring rotor base with a circular rotor base (Figure 15).

This was so that we could countersink flat-head bolts into the rotor structure, compared to having raised bolts such as pan-head bolts (Figure 18).

Design improvements

When we recreated Aachen 2015’s pump, we discovered that no matter how carefully we mounted the rotor onto the shaft of the stepper motor, one screw head would always drag along the pump plates, implying that the rotor was tilted at an angle.This caused the pump to have insufficient torque to move the media through the silicone tubes.

The screw attaching the rotor to the stepper motor shaft is a flat end screw, and the rotor should not be tilted at an angle. Troubleshooting further would yield diminishing marginal returns, as other possible sources of error such as inaccuracies in 3D-printing would not be within our power to resolve. Moreover, the angle was very slight and otherwise undetectable by visual inspection. An obvious solution would be to decrease the height of the central rotor column, and then shift the entire rotor assembly upwards, but this would not be ideal. As the load moves further along the shaft, the angular deflection of the shaft increases. While the increase in deflection would definitely be negligible in this case, such a solution would be bad design practice. Hence, we elected to countersink the bolts because this approach allows us to easily visually inspect the rotor to ensure clearance between it and the faceplate of the stepper motor.

However, a flat-head bolt of equivalent size could not be countersunk directly into the Aachen design as there was too little material remaining to hold the bolt in place. We then simplified the rotor base, and thus produced what you see in Figure 17.

Our second modification was to add material for the fastening screw. Even the 2017 Aachen team acknowledged that a drawback of the 2015 team’s design was the instability of the bolt and nut combination fastening the rotor to the stepper motor shaft (Figure 19) ^[57].

The third modification we made was to reposition the inlet and outlet for the silicone tubing.

Aachen 2015’s design stacked several Plexiglass layers on the stepper motor’s faceplate, forming a housing to hold a silicone tube inside a circular path. We noticed that Aachen 2015’s silicone tubes had a smaller outer and inner diameter than the tubes we planned to use, as the working volume of their bioreactor is less than ours. After analyzing their design, we realized that we only needed to modify one layer

Our first design for the

Experimental Validation

To test our hardware systems and prove their utility and functionality, we came up with and executed experimental plans.

summary of experiment plans: Jiacheng

Please visit our Hardware page for our detailed experimental plans and results.