In any bioreactor, over time, only a certain percentage of cells actually produce what they have been engineered to express, says Enduro Genetics CEO Christian Munch. The rest are essentially freeloaders, consuming valuable feedstocks without making anything useful, which can make the unit economics of the whole enterprise pretty challenging.
“Sometimes only 15-20% of the cells are responsible for producing your target product. And after 60-80 generations, the cells may completely lose the ability to produce,” notes Munch, a former Novozymes executive very familiar with the economics of industrial fermentation. “There are various factors that impact declining production at scale, but for each generation, there are mutants that say, if ‘I stop making target protein X, I can grow faster,’ and they will ultimately take over the bioreactor.”
Which is no surprise if you think about it, says Munch, who caught up with AgFunderNews at the recent SynBioBeta conference. Microbial cells do not naturally produce dairy proteins, vaccines, human milk oligosaccharides or other bioproducts that cells have been genetically engineered to express, so there’s “no evolutionary competitive advantage” to making them.
He adds: “And even if all your cells are producing the target, some are really high producers and others are not, so you can still get a really inefficient process.”
More productive bioproduction over a longer period
Enduro, a Copenhagen-based startup spun out of the Technical University of Denmark by Dr. Peter Rugbjerg (CSO) and Dr. Christian Munck, is addressing this fundamental problem in biomanufacturing (declining production at scale, and poor yields) with technology “that sounds almost too good to be true” when you first come across it, says Munch.
In a nutshell, Enduro “effectively tricks cells into thinking that they have to produce the target substance in order to survive,” by linking the expression of essential genes, which are critical for the survival of an organism, and high-production of the target substance in a cell, adds Munch.
“Our solution [‘Enduro Sense’] is a genetic biosensor that couples with essential genes in the cell and that means that only cells that are high-producers of the target substance can grow or proliferate. The essential genes are upregulated when the cell produces the target substance and downregulated when they don’t.”
The net result is that high productive cells dominate the bioreactor, enabling firms to sustain bioproduction over longer time scales, claims Munch, who says Enduro’s genetic biosensors have been tested in E.Coli and Bacillus strains of bacteria to baker’s yeast and filamentous fungi, producing everything from milk proteins and mevalonic acid to antibodies used in drug development.
“A successful implementation can lead to 30%+ improvements in titer and yields and the ability to run fermentations five times longer as you’re effectively shifting the culture towards the highest producing cell variants. Enduro Sense also works completely independently of the product pathway and works with cells that do intracellular expression [where the target product is expressed inside cell] or extracellular expression [where the target product is secreted from the cell into the media].”
By making existing biomanufacturing capacity more productive, clients can also potentially avoid capex it had planned to spend on additional or larger bioreactors, he claims.
Reverse evolution?
He adds: “We’re kind of putting everything in reverse. Right now there is a selective evolutionary pressure against high-producing cells because it places a burden on them. With our tech, we’re reversing that so high-production confers an evolutionary benefit.”
In one study conducted by Enduro cofounder Dr. Rugbjerg and others at the Technical University of Denmark, researchers controlled the expression of one nonconditionally essential gene operon with a mevalonic acid biosensor. The resulting production organism retained high-yield mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, by which time the regular strain wasn’t producing anything, says Munch.
Implementing Enduro Sense
Implementing Enduro Sense into a production strain is done one time at the master cell bank level and requires no changes to the media (no antibiotics, no additives) or production process, he says. “We don’t introduce any foreign DNA and we don’t tamper with the production pathway.”
First, Enduro conducts an analysis of fermentation samples at different stages of a fermentation using RNA-seq (a technique that reveals the presence and quantity of RNA molecules in a biological sample) and Enduro’s proprietary algorithm. Next, it designs sensor-essential gene constructs from its algorithm and its library. After that, either Enduro or the client can generate multiple variant candidates with Enduro Sense, screen and select the best ones based on phenotypic performance in shake flasks, and then validate them in a bioreactor.
Some clients are happy to send Enduro their strain, “and we do everything in-house,” says Munch. For others concerned about IP, he says, “We can send the constructs, the sequences that they need to implement this as a data file, so they can synthesize the DNA and do the transformation themselves with our guidance, and we can help them interpret the results. We’ve done implementations with clients where their strain never left their premises.”
Too good to be true?
According to Munch, who has been CEO for a year, “It sounded too good to be true when I first came across it, because I saw immediately that if it works, it’s foundational game-changing technology for an industry I worked in for 14 years [at Novozymes]. It’s using biology to solve a biologic problem.
“When I joined, Enduro was in stealth mode. We reached out to a bunch of companies, who said yes, the problem you claim to be solving is real, and if this works, it’s almost too good to be true, what’s the catch? Will it work with my [production] host? So we started to do projects with them and sort out IP and so on, and now we have agreements with a number of leading industry players that have fully recognized that if this works, this is a game changer.”
Enduro has “foundational IP that covers both product and method claims related to exercising this type of technology,” he adds.
Enduro secured seed funding from NOON Ventures in 2022 and has also secured grant funding from the European Innovation Council (EIC). It is now looking to raise a series A round, says Munch.
“What investors can see is that we are solving problems that affect everyone in biomanufacturing, whether you’re making vitamins or antibiotics. We’re a tools company with a licensing and royalty model, not a product company.”
Further reading:
