Full-bore Biotech

Adam Duckett speaks to the University of Warwick researchers who are recoding microbes into competitive chemical factories

IN A LAB at the University of Warwick, PhD student Sepideh Bourenjanshirazi is comparing glass beakers filled with purple liquid – the perfect visual cliché of a laboratory researcher at work. Look a little closer though and we’ll discover that Bourenjanshirazi and her supervisors are pushing towards something far from the commonplace.

Bourenjanshirazi is engineering bacteria to function as more efficient chemical factories, hijacking their DNA to fully open their “metabolic valves”.

“We are looking to increase their productivity using cheap substrates,” she says.

If successful, the work could help biotechnology better compete with more economical petrochemical production of commodity chemicals. It could also enable microbial factories to produce previously undiscovered chemicals that presently only exist in trace amounts or as intermediates for fractions of a second.

Her project is barely a month old, and at this early stage Bourenjanshirazi is engineering the cells to produce the purple pigment violacein which provides a simple visual indicator of how the research is progressing.

This experimental work builds on modelling research published by Bourenjanshirazi’s supervisor Alexander Darlington, who is assistant professor in control and engineering biology at Warwick.

Darlington says: “We’re trying to take computational design tools from control theory and overlay them into the engineering biology design cycle to help experimentalists build systems that perform optimally.”

In January, he co-published research in Nature Communications (doi.org/g82p72) modelling a microbial factory’s production pathways.

“Bacteria are soups of protein in a fat coat,” he says. “Those proteins control how the external sugar substrate [added as a feedstock to a bioreactor] gets converted into either cell growth or a product you’re interested in.”

Darlington predicts he can almost double chemical production by selectively switching off growth once cells have reached a certain size. This flies in the face of conventional practice which currently involves switching on production instead.

The journey to genetic engineering

A sense of dissatisfaction helped bring Darlington to where he is today. He studied genetics as an undergraduate, then shifted his focus during his master’s to understanding how those individual genes work together to make a cell function.

“How does the emergent behaviour of a cell come out of all of those individual interacting components…and if we can understand the system, can we hack it and make it do useful things?”

During his PhD he looked at how genes interact to constrain a cell’s synthesis of chemicals and developed theoretical design tools for understanding the trade-offs – how turning on and off certain genes impacts performance.

“And then during my postdoc, I got a little…frustrated is the wrong word but I read all of these cool science papers about fine control of genes, making products and so on, but none of it was happening in the real world. It was all happening in academic labs. We’re publishing nice papers but I asked myself how can we actually design circuits like gene regulatory networks to control metabolism as part of a transition to a sustainable chemicals industry?”

So, he applied for a Royal Academy of Engineering research fellowship that married his background in biosynthetic constraints and his interest in metabolic engineering. In 2021, the academy supported Darlington in building a research group working at the interface of engineering and biology.

Flick the growth switch, not production

“Biologists or biotechnologists very often experiment first. They’ll build things based on intuition rather than a design. We’ve taken a model-guided design approach to study all of the different potential genetic circuits that control production and growth.”

In the latest paper, published with bioengineers including Ahmad Mannan at Imperial College London, the team analysed the genetic circuits involved in growth and chemical production, creating a multi-scale model that Darlington says captures “the whole fermenter level performance that one would expect from turning genes on and off”.

“We tested around 500 different control mechanisms, and found two that were new to research, which offers a clear pathway toward more efficient bio-based synthesis of chemicals.”

He expects this will enable the sustainable manufacture of everything from drugs to plastics.

“The way that most systems are engineered is that you activate synthesis during cell growth.”

Manufacturers add a chemical to the bioreactor that prompts the cells to start producing a desired product.

You could take the control a step further by giving manufacturers two genetic switches – one that turns on production and another that turns off cell growth. But Darlington’s research suggests the benefit of doing so might be minimal and comes with the cost of having to engineer twice as many genes. He believes manufacturers will be better served by having cells that grow and synthesise chemicals from the off, and then once they’re fattened up there’s only a single switch to flick – one that turns off growth.

“Most people at the moment are just controlling synthesis. Our modelling suggests that controlling growth, over controlling synthesis, is more important for maximising productivity.”

Hacking bottlenecks

“From the point of view of your biochemical engineering readers, I would say that fundamentally the interesting part of this [research paper] is that we might be regulating the wrong thing. They’ll be spending all their time thinking about regulating production and we would suggest they should regulate growth.”

And this is where Bourenjanshirazi comes in. In January, she joined as the team’s first experimentalist. She is tasked with engineering these genetic controls into living cells, taking the research a step closer to industrial application.

But how does she get those new controls into the cells?

The genetic modification starts with the team placing an online order for the genes they need. They upload their gene sequence and a contract manufacturer syntheses it onto a circular piece of DNA called a plasmid, which is then mailed to Warwick. In the lab, Bourenjanshirazi adds the plasmid sample to a solution containing the microbes she wants to modify – in this case E.coli – and then heat- or electro-shocks them, which prompts the cells to absorb the little rings of code. As well as smuggling in the new genetic controls for production and growth, the plasmid also sneaks in code for antibiotic resistance. Later, she’ll add in antibiotics and voila any cells without the plasmid are killed, leaving a pure colony of modified E.coli.

The testing can then begin. Presently, Bourenjanshirazi is using a BioLector microbioreactor to perform high throughput screening of the modified E.coli. Dozens of separate millilitre samples are added to the machine and different conditions are tested to see how the bacteria perform.

“We are using different carbon sources to see how protein production and growth rate would change and what’s the interplay between these two,” Bourenjanshirazi says. They also test how the bacteria behave in the harsher conditions they might experience in industry.

“The results tell us what the limiting step is and what we could do next to overcome them.”

For example, they might experiment with using different media – this is the soup of nutrients and growth factors in which the bacteria live and hopefully thrive. Or they might decide to tinker again with the genetic code to prevent production of an unwanted intermediate or modify the so-called metabolic flux that occurs within the cells. This flux is the rate at which the cascade of chemical reactions occurs as the cell converts chemical A to chemical B, and then B to C and so on. The rates at each step are influenced by enzymes – catalysts in protein form. If the team concludes that production of a particular enzyme is too low and is holding back the cascade, they can re-engineer the bacteria to produce more of it, in a bid to overcome this rate-limiting step.

“The more enzyme you make, it’s like opening a valve that will speed up the reaction,” Darlington says.

The bioreactor includes a filter module that allows Bourenjanshirazi to evaluate the cultures in real-time including their biomass and fluorescence without removing samples from the machine. To take advantage, the team has modified the DNA responsible for producing the specific protein they are focused on by splicing in genes from jellyfish. This bolts on an extra sequence of protein that glows under UV light, allowing the team to conveniently monitor how much of their target protein is being produced.

Purple patch

We return to the purple samples that Bourenjanshirazi has produced. One is really vibrant, while the second is much more dilute. The weaker one was grown using a poor-quality carbon source and no supplements.

“We’re wondering whether our dynamic growth switch could let us make this second one more efficient so it produces more chemical from a low quality media that’s cheaper for industry,” Darlington says.

“The work that Sepideh is doing is going to benchmark our model to enable us to make sure it is quantitatively predictive of how these strains perform.”

At the end of Bourenjanshirazi’s PhD, Darlington hopes the team will have strains of E.coli that have been modified to produce a range of chemicals including vitamins and food supplements.

“The outcome of the next three years will be the validation of the design principles from the paper and the generation of strains which show superior productivity and yield. This will be a proof of concept that the methods work – that the approach works and is worth applying in the industrial setting.”

We’re on a molecule hunt

The second supervisor in the project is Christophe Corre, a professor in Warwick’s life sciences school, whose research includes hunting for as yet unknown high-value chemicals and getting microbes to efficiently manufacture them. He is working with a soil bacteria called Streptomyces.

Corre is engineering bacteria to enhance their production of sunscreen molecules. By selectively inhibiting each protein in the strain one by one, he is analysing its effect on molecule production, transport across the cell membrane, and secretion into the bioreactor.

“They are actually famous in the sense that most antibiotics we use clinically originate from this type of bacteria. And we know that they have the ability to produce millions of molecules which nobody has ever seen.”

By studying their DNA, he can identify the chemicals they are capable of producing. However, they do not produce these chemicals under laboratory conditions.

“They may produce them in nature in response to specific signals when they actually need this compound. But in the lab, it’s all another matter.”

The team’s research could provide a system for the team to produce samples of novel chemicals that can then be screened by industry to see whether they might be useful as drugs, food supplements or insecticides.

Improving industrial production

Corre says the strains of Streptomyces used today by industry in fermenters to manufacture insecticides have been developed somewhat ineffectively using natural selection. He expects the work Darlington and Bourenjanshirazi are doing could offer a more rational way of developing highly productive strains.

Darlington has addressed the impact of the work for those in bioengineering, but what does it mean for chemical and process engineers working in more traditional sectors?

“So, at the moment, biochemical engineering is really good for pharmaceuticals because there’s no other way of doing it. You know, there’s no other way of making biologics other than by bio. But in terms of producing commodity chemicals, then really, petrochemicals are still winning. They’ve been winning for a long time because we’re very good at using them. But if we want to transition to a circular economy where we stop using petroleum products and start using plastic waste or spare biomass from farms, then we need bio-based manufacturing.

“I hope that in five years’ time…we develop robust strains that can be used in industry.

“This is another piece of ammunition to try and improve the cost competitiveness of bio-based chemical production.”