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We depend heavily upon dwindling petroleum and other fossil reserves not only for fuel, but also for an array of valuable chemicals. It is estimated that about 20 % of crude petroleum is used for products other than transportation and industrial fuels. Petrochemicals are for example used as raw materials in the production of pharmaceuticals and polymers that we use day-to-day.
Renewable resources are an attractive alternative to petroleum, but for them to be used, innovation is required to turn microorganisms into economically viable and powerful ‘miniature factories’. “People have been talking about expanding the potential of nature – and in particular of microbes – for a long time,” says Vítor Martins dos Santos from Wageningen University in the Netherlands, coordinator of the EU-supported project EMPOWERPUTIDA.
To this end, classical engineering principles have been introduced to cell engineering. “The EMPOWERPUTIDA team is building a bacterial ‘chassis’ for biocatalysis – the use of bacterial enzymes linked to each other in a network to perform chemical reactions that cannot be performed cost-effectively using traditional methods,” explains Martins dos Santos.
A bacterial ‘chassis’ of choice
No organism can fill every biosynthesis need because each microbe has its strengths and limitations. For instance, Escherichia coli is a well-established platform for protein production. Pseudomonas putida, a Gram-negative, saprotrophic (it feeds on dead matter) bacterium, is very robust under harsh industrial conditions and resistant to chemical stresses.
Strains of P. putida are found in soil and water habitats where there is oxygen. They flourish by metabolising a wide range of compounds, including toxic chemicals such as those found in oil spills, paint and other industrial wastes. Their metabolic flexibility stems from an unusual wealth of genetic determinants and the ability to regulate the expression of many different metabolic pathways.
Scientists take P. putida and turn it into something valuable: “The desirable traits of P. putida are enhanced, others are replaced, and those that are inconvenient are removed, making it a versatile ‘chassis’ capable of generating scores of chemicals with exceptional efficiency. The bacterial chassis can then become a bioreactor that will self-replicate indefinitely at user’s will.
“This part of the project fits into the realm of synthetic biology, where researchers employ new ways of systems design. The EMPOWERPUTDA team takes advantage of mathematical models and computer-aided design to deliver precisely engineered, tailor-made whole-cell factories,” explains Martins dos Santos.
Promoting a bio-based economy
Determining which traits are essential for P. putida – and should therefore stay – and which can go, has proven challenging. However, programming the bacterium as a catalyst in a bioreactor promises a significant increase in biofuel yield and production of a broad range of valuable chemicals. Most encouraging, the redesign of the bacterial genome will be more efficient than the traditional ‘trial-and-error’ approaches for metabolic engineering of bacteria.
EMPOWERPUTIDA scientists are not only engaged in a race to the bottom. For synthetic biology applications, the goal is to streamline and optimise rather than minimise P. putida’s genome. The bacterial genome normally contains thousands of elements that are helpful for real-world survival but have little value for the production of biofuels and other chemicals of interest.
Cellular blueprints created using data from various studies allow researchers to reconstruct sophisticated ‘virtual’ cells computationally. Once the basics of cellular life have been simulated with high fidelity, the insights gained could guide experimental construction of synthetic cells with novel and optimised functions.
“This will be a leap in biotechnology, similar to Henry Ford's creation of the assembly line that revolutionised manufacturing,” concluded Martins dos Santos.
“As was the case with the assembly line, which had tremendous impacts well beyond car manufacturing, all industries will benefit from such whole-cell factories utilising renewable resources and generating little or no waste.”