Paints, varnishes or cleaning products: most contemporary chemical products are made from crude oil and natural gas. Researchers in the EU, including some at the University of Stuttgart, want to change this situation by coaxing the soil bacteria Pseudomonas putida to produce commodity chemicals from sugar. To achieve this, they are using tools from the field of synthetic biology.
Humans have already been using microorganisms for thousands of years to make beer, wine, sourdough and cheese. One of the first industrial mass-production applications began in 1916 with the fermentation of starch or sugar by bacteria from the Clostridium acetobutylicum genus to produce the solvents Acetone and Butanol. The biochemist and later President of Israel, Chaim Weizmann, had previously isolated the bacteria and optimised the biotechnological process. However, following the Second World War, the burgeoning petrochemical industry made it more cost-effective to synthesise chemicals from crude oil rather than have bacteria do the work. Now, faced with rapidly depleting crude oil reserves, interest in bacterial factories is once again increasing.
Professor Ralf Takors, Director of the Institute of Biochemical Engineering (IBVT) at the University of Stuttgart, is less interested in Acetone and more, among other things, in microbiologically produced Butanols and their gaseous descendants, Butene and Butadiene. Initially, Butanol was primarily needed for the production of artificial rubber. Today, it is widely used as a solvent in the paints and varnishes industry and as an additive in cleaning products. It also serves as a precursor material for the aromatic substances used in perfume and is under discussion as a potential biofuel component. The Butanol derivatives are used, for example, as intermediates for plastics and tyre rubber. Thus far, they have been produced exclusively by chemical means.
Like Weizmann over a century ago, Takors and his team also use sugar as a basis for the microbial fermentation process, but use other bacterial species, which have tended to be rather neglected by bioengineers. Floating in the murky culture fluid in a three-litre glass fermenter at the Institute of Biochemical Engineering are bacteria of the genus Pseudomonas putida, or more precisely of the KT2440 strain, which are being pumped in a circular motion via an extensive system of hoses. In another 200-litre bioreactor, Takors’ researchers are testing the biotechnical mass production of biobutanol and its derivatives.
“Omnivores” with Great Potential
“This strain has several benefits”, says the bioprocesses engineer, “it can tolerate both the accumulation of organic solvents, such as Butanol, and oxidative stress, which can occur during the cultivation process in gigantic bioreactors”. Takors’ laboratory “pets” originated in the soil where they live on plant roots, feeding on almost anything they can fi nd. They can even decompose toxic substances or evacuate them from the cell thanks to inbuilt “export pumps”, which is why they are often found on polluted sites.
In addition, they also produce the cellular reducing agents NADH and NADPH. They serve – for enzymes, for example – as cofactors that counteract oxidative stress. On the other hand, bioengineers can exploit this surplus reducing agent to transform sugar into significantly more chemically reduced products than has hitherto been possible from a bioengineering perspective. It is this natural robustness that will potentially enable Pseudomonas putida to become the new stars of bioengineering.
Until recently, bioengineers were usually thwarted by the low tolerance levels of their chosen bacterial strains to organic solvents. Even Weizmann only achieved a meagre harvest with his bacteria. “In high concentrations”, Takors explains, “the bacterial strains that have been used to date consume a lot of sugar and energy just to deal with these harsh conditions, which reduces their efficiency”. However, high production yields are necessary to keep the production costs down, as the existing oil-based products already sell for ridiculously low prices of less than one euro per kilogram. Because, unlike Weizmann's bacteria, Pseudomonas putida does not naturally produce biobutanol and its derivatives, it has to be “empowered” to do so by means of synthetic biology.
Researchers are currently collaborating in the Eu project “EmPowerPutida” to develop ways of genetically reprogramming the metabolism of Pseudomonas putida such that it will produce these products in addition to a particular herbicide. “We want to use this example to demonstrate that it functions successfully, which will increase the strain's acceptance for industrial applications”, says Takors. He is collaborating in the project together with his colleague Bernhard Hauer, Director of the Institute of Biochemistry and Technical Biochemistry (IBTB), and other partners from Germany, the United Kingdom, the Netherlands, Portugal, Switzerland and Spain.
Conversion to Bacterial Factories
“The biggest challenge was to coax the omnivorous Pseudomonas putida into not consuming the products it makes itself”, says Takors. To this end, the partners fi rst reduced the bacterium's genome to the basic functions it needs to survive. Bernhard Hauer's team then equipped the bacterium with the genetic material needed to create novel metabolic pathways that culminate in the desired product. “We also hypothesise based on enzymes from other bacteria, look at their structure and refine the enzymes with the aid of computer-based and evolutionary-learning methods such that they will, for example, accept other substrates”, Hauer explains.
To further increase yields, the partners ensure that the bacterial cell opens all available channels for the absorption of sugar and primarily uses it for subsequent production activities rather than to fuel its own growth. Takors’ research group designed novel, patentable regulatory circuits and gene switches intended to ensure that the bacteria continue to produce the desired products even under adverse conditions. As Takors explains: “the bacteria mustn't shut down their metabolic processes when conditions for growth are poor, but should instead maximize production”.
The EU is providing over 5 million euro of funding for the four-year project via its Horizon 2020 framework program. By the end of the project next year, the researchers want to further optimize both the production strains and the production process in large fermenters to achieve even greater yields. “All we could have done ourselves would have been to develop the enzymes” says biochemist Hauer. “The nice thing about the EU project is that, with the aid of the other partners, we were able to assemble everything to produce a viable, functioning bacterial strain”. This was one of just three projects submitted in response to the original call for applications in relation to Synthetic Biology that received funding, of which two involved Pseudomonas putida. As Takors emphasizes: “this demonstrates the importance of these bacteria”.