The approach essentially reprogrammes an enzyme in baker’s yeast to produce the more valuable short-chain fatty acids (SCFAs).
“The presented technology is applicable far beyond baker’s yeast,” the research paper stated. “It can be plugged into essentially all currently available fatty-acid overproducing microorganisms.
The technique differs from similar processes in that it is a minimally invasive protein engineering approach that leaves the molecular mechanisms of the enzyme in question - Fatty Acid Synthase (FAS) - unchanged.
This enzyme is normally responsible for catalysing long-chain fatty acid formation - the kind that have proved beneficial in fats and oils, including olive oil, soybean oil, fish, nuts, avocado and meat.
To produce SCFAs in the quantities that are commercially viable currently involves a complex extraction process using certain plants such as coconut.
This new process may initially have the potential to produce high-value constituents for cosmetics, active pharmaceutical ingredients, antimicrobial substances, aromas or soap.
However, the principles shared in the research paper means there’s no reason why SCFAs specifically used for packaging food applications among others, can be produced in the required quantities.
Highlights of technique
Research groups led by Professor Martin Grininger and Professor Eckhard Boles at Goethe University Frankfurt began by identifying specific sites located on the three-dimensional structure of the FASs.
The objective was to understand how the FAS counted cycles in order to determine how long the fatty acid chain should be.
Remarkably, they identified a set of only five mutations that are capable of significantly controlling FA chain-length regulation.
“We found a type of ruler which measures the length of the fatty acid”, said Dr Grininger.
“We modified this ruler in such a way that the fatty acid synthase measures incorrectly and releases shorter chains. All this took place first of all on the computer and in the test tube.”
Next, these modified fatty acid synthases were placed in yeast Saccharomyces cerevisiae, where they began secreting short-chain fatty acids in large quantities.
“This development is just the start,” the researchers said. “We want now, through similar modifications on other large enzyme complexes, that is, polyketide synthases, to synthesize other new types of molecule for industries which are not readily available otherwise.”
MPT domain
The challenge of producing SCFAs from S. cerevisiae is a complicated one. Yeast FAS has a complex and highly organised structure.
Added to the researcher’s intention to target the MPT domain of the FAS enzyme, while maintaining FAS structure and function, further complicated matters.
“Among the mutations employed in FAS engineering, the MPT mutation is, however, clearly a costly one,” the study admitted.
“As a call for a first improvement of the presented system, FAS engineering needs to be optimised towards increased affinities of MPT for short-chain acyl-CoAs to increase acyl export rates without decreasing malonyl import.”
S. cerevisiae does not naturally produce fatty acids in large quantities. However, its robustness, pH tolerance, simple nutrient requirements and long history in industry make it a suitable candidate biocatalyst for such purposes.
In addition, the yeast FAS, while very efficient in producing fatty acids, is not optimal for the production of SCFAs.
In S. cerevisiae, amounts of 400 milligrams per litre (mg /l−1) free FAs, and more recently of 2.2 grams per litre (g/ l−1) and even up to 10.4 g l−1 in glucose have been achieved. However, they were mainly long FAs, from C12 to C18 in saturated and monounsaturated form).
More recent techniques used the enzyme thioesterase in the production of SCFAs in S. cerevisiae in a more invasive approach that resulted in amounts of up to 111 mg l−1.
Source: Nature Communications
Published online ahead of print: doi:10.1038/ncomms14650
“Engineering fungal de novo fatty acid synthesis for short chain fatty acid production.”
Authors: Jan Gajewski, Renata Pavlovic, Manuel Fischer, Eckhard Boles & Martin Grininger