by Natalia Bateman, September 2018

Talking to Ben Long is like suddenly landing on an episode of Star Trek: he speaks about bombarding chloroplasts with foreign DNA, using miniature shot guns to penetrate plant cells and about designing icosahedral compartments filled with carbon dioxide.

Credit: Stuart Hay, ANU

I am just waiting for him to teleport any time now, as he explains the core of his research obsession for the last 15 years: the tiny glasshouses, or carboxysomes, located inside blue-green algae.

Ben is a keen cyclist and a plant scientist. With the first, he learnt to appreciate the dry Canberra landscapes and roads. The second, is a career that has given him a ticket to explore the inside of plant leaves, a place as alien as Mars, for people who, like me, haven’t had the opportunity to walk inside this world.

Ben’s interest in science started when he was in high school, thanks to a biology teacher who made his students to hand-in their reports as scientific articles. “I chose biology because I felt less threatened by it than by other subjects. Biology is also always applicable and easily related to everyday life. You can just look around you and see biology in action every day,” he says.

He started studying biology in La Trobe University with an emphasis in botanical biochemistry. It was there, in a plant physiology lab, where Ben had his first contact with the organisms that have been at the centre of his research career: cyanobacteria. They are better known for their blooms that can be toxic to humans and cause public closures of pools, lakes and rivers. They are also known as blue-green algae, a confusing nickname, as they are not algae at all.

“My honours project was in microbial ecology, working on the production of toxins by blue-green algae and the environmental effects that affect how it occurs. I continued through this path with my PhD, working out the underlying questions of the physiology and biochemistry of cyclic peptide toxin production. These peptides are incredibly toxic to humans and I wanted to know why cyanobacteria produce them,” he says.

“This led me to embark in a postdoc at the University of Surrey in the UK, where they were looking at the processes that control cyclic peptide antibiotic production in bacterial systems and how they can be manipulated. It was a logical progression from my PhD studies and gave me a new perspective on the broad solutions that evolution allows microbes to live in diverse environments”

But cyanobacteria were calling Ben again back home. He returned to Australia, this time to ANU in Canberra, and began working with aspects of blue-green algae that he didn’t even know existed.

“While I was focussed previously on toxin production by cyanobacteria, I’d given very little thought to how they have evolved highly efficient CO2 concentrating mechanisms to help them photosynthesise. This special compartment is called the carboxysome.”


Mimicking the secrets of cyanobacteria

At ANU, Ben began to work on carboxysomes, the bacterial micro compartments that resemble a 20-sided die shape glass house inside the cell. In cyanobacteria, carboxysomes house Rubisco, the enzyme in charge of fixing CO2 during photosynthesis. They are one of the reasons why cyanobacteria are so efficient at capturing this gas.  Carboxysomes allow bicarbonate in, so it can be converted into CO2 around Rubisco.

Ben with his team. From left to right Nghiem Nguyen, Eng Kee Au, Wei Yih Hee, Eiri Heyno, Professor Dean Price, Professor Susanne von Caemmerer and Dr Ben Long. Credit: Lannon Harley, ANU

“Rubisco is notoriously inefficient at doing its job. It is a slow enzyme and it finds it difficult to differentiate between CO2 and O2. Having Rubisco encapsulated inside carboxysomes, like cyanobacteria do, means that the short-sighted Rubiscos have mostly CO2 around them, which makes photosynthesis much more efficient,” he says.

Ben’s mission at ANU, was to explore an innovative solution: to make photosynthesis more efficient by putting carboxysomes into crop plants such as wheat and rice. Until then, this was considered a wildly speculative, fantastical project, belonging more to science fiction than science.

“Dean Price, Murray Badger and Susanne von Caemmerer thought about this idea of improving plant performance by inserting carboxysomes in plants chloroplasts. This is how our “Moon shot” project began. We now have funding from the Gates foundation to do it, but, as NASA did during the mercury missions, we are just getting off the ground. We know where the moon is – although in our case it looks like a very productive crop plant – and we are designing the machine to get there.”

Inserting carboxysomes is like inserting turbo-charged engines inside the cell. After a long process, the team has now managed to produce minimal carboxysomes in tobacco plants with Rubisco inside them. It is the first time that someone has been able to put Rubisco inside a compartment inside a plant cell.

The team’s next challenge is to put working bicarbonate pumps inside the cell, which will make Rubisco work faster.

The CO2 concentrating mechanism inside cyanobacterial cells is like a turbo-charged form of photosynthesis. Most crop plants use a relatively inefficient carbon fixation engine called C3 photosynthesis. It’s slow and misfires often and can only overcome these limitations by investing in more of the slow Rubisco enzyme, using large amounts of water and nitrogen fertilizer; a little like an old 2-stroke engine with poor fuel efficiency.

The cyanobacterial system, however, is more like a fuel-injected turbo engine. It’s fast and efficient and could use less water and nitrogen fertilizer than C3 plants currently use. This means that by inserting the carboxysome system into crop plants, scientists have the potential to dramatically increase the photosynthetic efficiency of crop plants and consequently, their yield.


Engineering carboxysomes

Building a carboxysome inside a plant cell is a complex engineering task.

“It is a bit like inserting a fish gill into a whale,” Ben says, doubting a bit about using the analogy.

And just like explaining how to do a heart transplant over the phone, Ben tries to summarise the complex process of creating carboxysomes inside a cell. It has taken his team more than 5 years to get to this point.

A carboxysome graphic representation in the front and an image of the carboxysomes inserted in tobacco (background)

“In order to achieve our goal, we need to introduce genes for a carboxysome into the chloroplast of a plant cell. Chloroplasts are the green factories inside plant cells, responsible for carrying out photosynthesis.”

“To do this, we build the strands of DNA we want and stick them to tungsten beads. These beads become ammunition in a miniature shotgun that we use to blast the DNA into leaf tissue from the plant we want to transform. The beads penetrate the plant cells and the chloroplast. If we’re lucky, we end up with little plants that contain the genes we want with the cyanobacterial Rubisco and parts of the carboxysome.”

The next step is to check the plants to see if the carboxysomes are there with the enzyme Rubisco inside them. While Ben’s team had tobacco plants sitting in the growth cabinet ready to be tested, there was every reason to expect failure.

“We knew that the proteins and genes were there, but the idea that structural carboxysomes could be built with just four proteins, and the high risk of this project from the beginning, meant that we didn’t rush to see if we’d succeeded. In fact, our plants sat in the cabinet for a month or two before we tooled-up and decided it was time to see what they had produced”.

Ben applied a technique for purifying carboxysomes from cyanobacteria to test the team’s successfully transformed tobacco plants.

Excitingly, this yielded a creamy band of pure protein, evidence that not only the carboxysomes were inside the tobacco plant chloroplasts but also the cyanobacterial Rubisco was inside them along with the proteins that they were expecting to be present.

The final piece of evidence needed was to actually see the structures inside the cell using an electron microscope.

“In our team, Wil does all the electron microscopy. I remember him sending me photos of the screen as he was looking at the electron microscope. To see these small structures, just like those you’d find in cyanobacterial cells, but inside plant chloroplasts, was unbelievable. When we realized we had done it we were absolutely thrilled.”

“Now that we have the genetic construct that will be the base for our bionic engine, we need other 5 or 6 genes to make sure they work correctly. Our next step is to get the functionality right”.

“We are currently trying to produce the same thing in the bacterium E. coli, because the downside of using tobacco plants is that it takes them year or two to produce seeds and three months to get those plants to mature and extract the carboxysomes. In contrast, we can do the same thing in E coli and literally grow them overnight. This cuts experimental time to a couple of days instead of months.”

Read Ben’s latest publication in Nature Communications here

Cycling, taking risks and planned serendipity

When Ben is not at the lab bench, he is cycling around the Canberra landscape.

“I am a keen cyclist and Canberra is the perfect place to move on two wheels. Like the goal of my research, cycling is a highly efficient way to reach your destination and a great alternative to the costly and energy-demanding car. Also, in science, like in cycling, you begin your ride expecting to go from point A to B, but quite often you finish in point C instead, which is often more fun, interesting and gives you a perspective on the landscape you may not have otherwise noticed”.

“Getting to this point in our research has been a long process but now finally we know that it can work, and that we are going in the right direction. We took risks but we got it right. We used evidence and built our decisions on how to build the carboxysome on the solid foundations provided by other scientists in our field. We had logical reasons for choosing the genes we did, based on a really interesting work by a couple of research groups in the US. So, we had some ideas of which proteins should form a minimum carboxysome structure, but we weren’t entirely sure it would work. I call this process planned serendipity.

“We began this project with a specific goal and an idea of how to go about achieving it.

“However, just like riding a bike, we were confronted by some obstacles and had to consider detours.”

Ben says that one of the things he has learned during his experience as a researcher is that there is a fine line between doings things the way you first envisioned, and taking risky diversions.

“Luckily, I have had the freedom to break the rules a little and to take these risks. I try to do the same within my research team. I encourage students to break their own rules occasionally and take the road less travelled. The road may not take you where you expected, but you’ll always see something new.”