Biocatalysis: the next molecular biology revolution

The biocatalysis group at CSIRO Molecular Science, led by Prof Michael Zachariou, has established an impressive pipeline of research and is working towards developing a more sustainable and renewable chemical industry in Australia.

From identifying new ways of accessing potential chemical building blocks from plant biomass, to developing novel bioprocessing technologies and their use in pharmaceutical applications, this team's researchers have made leaps in progress since the group was put together three years ago.

"Our aims are to create new biologically based chemical industries," Zachariou explains. "The drivers behind that are that petrochemicals are very polluting, they are becoming scarce, they are becoming costly and they are not environmentally friendly."

The chemical industry is an old industry. But it is changing -- moving towards deriving many of its current synthetic chemicals using biological means, Zachariou says. Citing companies like Dupont, Cargill Dow and BASF, Zachariou says these companies have made this their mission, with the aim of creating a more sustainable chemical industry.

And this is also what Zachariou's team is doing -- investigating the use of biological sources, such as trees and grains, to make chemicals, and exploring ways in which low-value plant biomass can be converted to high-value chemicals using biocatalysts. Hand in hand with this they are discovering new biocatalyst agents, using these to produce novel compounds as well as developing novel bioprocessing technologies.

Biocatalysts comprise microbes and their enzymes. Such biological systems, including engineered microbes, can be employed to produce particular chemicals that can then be used to produce plastics or herbicides or other chemicals of interest.

"We are trying to use biomass as a feed stock to generate chemicals, new chemicals," said Zachariou.

At the same time Zachariou says that developing and using this type of process generates a different type of value system for the rural sector, because their sources of biomass, such as grain crops or trees, could be used to generate chemicals or plastics or biofuels.

Accessing the biomass

Zachariou's team is looking at finding ways of effectively breaking down plants, such as trees and grains, so that carbon sources can be released for microbes or enzymes to use to make chemicals.

One component of the biomass they are looking at is lignin, which, along with cellulose, forms the cell walls of plants. This tough substance that is said to give strength and rigidity to plants can constitute up to 35 per cent of the world's plant biomass, and is very rich in polyphenolic compounds -- structures very similar to petrochemical compounds that are used as starting materials in the production of plastics.

Lignin is a rich source of potentially useful chemicals, and Zachariou cited a Norwegian study that found it contained 600 different compounds, "some at fairly low levels and some at fairly high levels, up to 10 and 15 per cent, or 20 and 30 per cent depending on the type of wood that was used."

Yet because lignin is so difficult to break down, or depolymerise, it is primarily burned and used as an energy source.

"If you could actually break lignin down effectively and efficiently, then you could use 35 per cent of the world's plant biomass to make your chemicals that were previously being made using synthetic means," says Zachariou.

His team is looking at combining chemical and biological methods to try and find an effective process for breaking down lignin. Zachariou said chemical methods currently in use employ quite toxic substances and enzymatic methods -- for example enzymes such as laccases and peroxidases produced by microbes -- would provide a more efficient and manageable process.

"The chemical methods are quite extreme," said Zachariou. "Things like nitrobenzene oxidation are used, which are very extreme conditions, so you can't really forsee that someone could scale that up effectively. If we can learn from the chemical methods how they work and what their weak points are, and learn from the many biological methods that are out there as well, hopefully we can combine the two together and use the strengths of both to make a much more scalable, more efficient process."

Cellulose

Zachariou's team is also looking at ways of efficiently breaking down the cellulose component of plants. The aim is to find effective cellulases to break down this insoluble complex carbohydrate and release it as glucose.

"There is a lot of interest in the US right now to take plant based matter and utilise its energy to make ethanol, a fuel, for obvious reasons," says Zachariou. "For example, the US National Renewable Energy Laboratory (NREL) is taking biomass and trying to develop processes to make fuel ethanol from cellulose."

According to Zachariou, 40 per cent of the cost of breaking down cellulose into glucose is in the enzymes. Thus, finding more effective and less costly enzymes to do this is an aspect of this work.

Eucalyptus oil

Another substance Zachariou's team is looking at is cineole, which is found in eucalyptus oil. Cineole is a bicyclic monoterpene and is quite a strong antiseptic -- it is currently used in lysterine and Vick's vapour rub, but these are fairly low volume uses and what the researchers are trying to do is find high volume uses for it.

They have taken cineole, fed it to microbes and isolated those that can grow effectively on this antiseptic.

"They [the microbes] end up hydroxylating the cineole in a single step," Zachariou says. "And that is something that synthetic chemists have extreme difficulty in doing, well it's never been done in a single step. They can do it eventually in multiple steps, with poor yields."

Adding a hydroxyl group to a compound makes it more functional, and this is what happens when cineole is hydroxylated. As Zachariou explains, "You are giving it a functional tag and you can then decorate it with other compounds, without that hydroxyl group cineole is not as reactive."

The researchers have generated 50 to 60 novel compounds using cineole as a base, and they are currently screening these for a range of high-volume uses such as application as degreasing agents, surfactants, herbicides, insecticides, anti-fungal agents, and anti-protozoan agents. Zachariou predicts that they will have a good indication of how effective these new compounds are by the end of the year.

Funded by the Rural Industries Research and Development Corporation (RIRDC), this work links into the Western Australian government's initiative to address dryland salinity, which is encouraging farmers to plant eucalyptus trees to help reduce the water table, thus relieve salinity problems. The potential of producing cineole adds value to the eucalyptus trees, providing another incentive, or making it more likely, that farmers would plant eucalyptus trees.

Discovering new microbes

In order to discover new microbes and their enzymes that can depolymerise plant material, the researchers have developed a tool they call Evolver technology, which does just this.

"We just launched this technology at the Enviro 04 conference in Sydney and we've been quite successful because the beauty of this technology is that we can discover these new biocatalysts or microbes within an average of about three to four weeks timeframe," says Zachariou. "Over the last year we've isolated 60 or 70 bugs, probably 10 to 20 per cent of them don't appear to exist on any DNA database so we consider them at the moment as new types of microbes."

As well as enabling the researchers to isolate microbes quickly, the other side to this technology is the unique microbial source that it uses to isolate potential new biocatalysers. Zachariou cannot say what this source is, but says it is uniquely Australian, highly populated, dynamic and saves them a lot of time and effort.

"This technology allows us to generate the environment within the technology itself, without leaving the lab," Zachariou explains.

In the business of discovering novel enzymes or microbes, companies have been known to go to extremes to obtain permits and licenses from governments or local communities to biomine specific environmental sites. US company Diversa, for example, wanted to isolate a lipase so went to the depths of the ocean to find whale carcasses and isolate microbes from within the fatty tissues of the whale in the hope that they contained lipases. But the technology Zachariou's team has developed negates the need to do this.

The Evolver technology also requires no assay development. Zachariou says the process involves starting with the microbial source, adding a carbon source, such as cineole, then the technology does its thing and they end up with about four or five species of microbe which they then purify out through monoculturing.

Australian made enzymes

Zachariou's team is also looking at producing novel technologies to carry out, and improve the cost efficiency, of biocatalytic processes. He says there are some companies in Australia that import significant amounts of enzyme, in the thousands of tonnes per month, from overseas companies.

The enzymes are used to breakdown components in agricultural feeds -- such as amylase for breaking down starch, or phytase to release phosphorous.

"What we are looking at is supplying these Australian companies with a different approach for making enzymes so that they wouldn't be reliant on overseas suppliers, they could actually make the enzymes themselves fresh every time they need them at significantly reduced costs," says Zachariou.

They have demonstrated proof of concept for this work and expect things to be well under way towards the end of this year.

Zachariou's team is also working on how to improve the processes for isolating these proteins and enzymes by looking at novel purification tags and different ways of isolating and purifying protein therapeutics.

"A lot of the tags that are out there are amino acid based sequences, we're trying to move away from amino acid based sequences and go towards more chemical based sequences for use as purification tags," Zachariou says.

Protein tags are used so that genetically engineered proteins can be retrieved from the host cells, such as yeast or bacteria, in which they are made. By getting the host cell to clone in six histidines, for example, to the target protein, the protein can then be purified via an affinity column from the rest of the proteins in the cell.

Instead of getting the host to clone in six histidines to the target protein, Zachariou's team is working to getting the host to put on a chemical moiety which would then be used as the purification tag.

This concept has potential application in the pharmaceutical industry because it gets around the problems amino acid tags have with illiciting an immune response.

"There are currently about 100 protein therapeutics on the market, none of them have been purified using tags or have any purification tags on them because there is a possibility that the tag itself can become an immunogen and elicit an immune response in the patient," explains Zachariou.

"The other issue is even if they accept that it could be immunogenic what can we do about it? One option is to cleave the tag off, but it is not a clean cleave, you are still left with one or two amino acids that are not part of the natural protein."

This impurity creates problems with regulatory bodies because the molecule is not the same as the natural protein, thus proof that it has the same effect as the natural molecule is required. In addition, cleaving off the tag from the protein requires a protease, which would need to be made under the same requirements as the target molecule and, since proteases are known to be promiscuous, would have to be extremely specific.

Attaching a chemical moiety as a tag for affinity purifying protein therapeutics would get around these problems because the chemical could be cut off without affecting the integrity of the target molecule since the cleaving entity would not attack amino acids. This process would also produce a more pure batch of the natural form of the protein.

Training

Zachariou says his team has a connection to the pharmaceutical industry through Swinburne University of Technology. "The connection there is training," he says. "It's all about training."

CSIRO is collaborating with Swinburne to introduce a postgraduate course in good manufacturing practice (GMP) in the second semester this year -- a certificate, diploma and masters will be offered. GMP is integral to the manufacturing of products in the pharmaceutical industry, products must be compliant with the Australian Therapeutic Goods Administration (TGA) and the US Food and Drug Administration (FDA) regulatory requirements for use in humans.

This is an issue for all pharmaceutical and biotech industries because they have to train their staff to do this, or send them to specialist training courses in GMP.

"A lot of the issues associated in manufacturing some of these products is the process development," says Zachariou. "Australia is very good at the discovery end of things, the research side of things, but does not have adequate experience in either the process development of these great products they have discovered and has limited experience in the manufacturing component such as the GMP."

Although he thinks some people would disagree with him on this, Zachariou says the fact that there are only about 100 protein therapeutic compounds approved for sale as pharmaceutical compounds in the world suggests that not many people have had the experience of having developed a product that is successful and carrying it through to the manufacturing process.

Thus, Swinburne and CSIRO are taking up this niche area and will train people in process development or manufacturing practices. And according to Zachariou there will be a demand for this type of graduate in the future.

"This area of biocatalysis has been defined by the OECD as the third scientific revolution, after the agricultural and pharmaceutical revolutions, it is believed that industrial biocatalysis is really the next revolution to come through science and through biotech."