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Rewiring bacteria for large scale biofuel and bioplastic production

Changing the fatty acid metabolism of bacteria could pave the way toward a more sustained environment

By Benjamin Boettner

Organisms that died many millions of years ago are the source of today’s natural petroleum resources. As phytoplankton and zooplankton, they sank to the bottom of ancient oceans and formed sediments under ground, and were exposed to extreme heat and pressure. This process turned the molecules that built all of their outer and inner membranes, known as fatty acids, into the main hydrocarbon component of petroleum.

However, to become useful for the production of fuels, plastics and detergents, the petroleum-derived hydrocarbons must be chemically ‘cracked’ into shorter chains of medium length with 8 to 12 hydrocarbon molecules and the right set of properties. With increasing length of the hydrocarbon chains, their consistency changes from gas to liquid to wax.

This image shows Wyss Institute researchers Marika Ziesack and Jeffrey Way evaluating E. coli bacteria that express engineered thioesterase enzymes to produce medium-length fatty acids. Credit: Wyss Institute at Harvard University

To help avoid further exhausting fossil fuels and the environmental burden posed by refining and cracking petroleum, metabolic engineers are working towards ‘designer fatty acids’ with just the right chain lengths that can be generated in large bacterial cultures, by leveraging the microbes’ own fatty acid synthesis machineries.

“For the production of biofuels and bioplastics, we aim for medium chain lengths that make fatty acids liquid, and give them the desired boiling point, low freezing temperatures and viscosity to be the major components of gasoline and diesel fuels,” said Jeffrey Way, Ph.D., Senior Staff Scientist at the Wyss Institute for Biologically Inspired Engineering. “But thus far we have been missing the right type of enzyme that could efficiently produce medium-length fatty acid chains in bacterial systems.”

In a new protein engineering study published in Applied and Environmental Microbiology, led by Way and Wyss Institute Core Faculty member Pamela Silver, Wyss Institute researchers now provide the basis for creating that missing enzyme link.

Fatty acids are synthesized in the cytoplasm of E. coli bacteria in several steps: a first hydrocarbon unit is attached to a protein know as acyl carrier protein (ACP) and then sequentially elongated with additional units. Finally, the completed fatty acid chain with a length of 16 or 18 carbons is transferred to glycerol via acyl transferases and incorporated into the membrane. Since this biosynthesis machinery does not produce free fatty acids of medium chain length, the team explored a thioesterase (TE) enzyme from the flowering plant Cuphea palustris, which was known to be able to generate medium-length fatty acid chains.

In fact, the C. palustris TE comes in two flavors that are highly similar in the amino acid sequences that determine their size, three dimensional structure and functions, one that generates long fatty acids with 14 hydrocarbon units and is highly productive, and one that is able to generate the desired fatty acids with 8 hydrocarbon units albeit with a comparatively low productivity. However, how these differences between the two enzymes came about has remained mysterious.

“We hypothesized that by swapping amino acids between the two TE enzymes and expressing them in E. coli, we could identify chimeric enzymes capable of producing medium chain fatty acids with high productivity and new chain lengths,” said first author Marika Ziesack, a Graduate Student co-mentored by Silver and Way.

The Wyss Institute team modeled the structures of two closely related thioesterase enzymes from the flowering plant Cuphea palustris to be able to zoom into and identify regions important for medium-length fatty acid specificity. Credit: Wyss Institute at Harvard University

To pinpoint candidate amino acids or divergent sequences of amino acids, the team modeled 3-dimensional structures of the two TE variants using molecular structures from other TE enzymes that had been described at great detail as a basis. “The two TE enzyme models enabled us to map out interesting regions that the two enzymes differed in. As a result, we importantly engineered a new enzyme that now could generate medium-chain fatty acids with high efficiency, and that in addition was also highly stable, which is important for biotechnological processes,” said Ziesack.

This advance was based on unexpected insights into how the two enzymes’ 3D structures were related to the mechanisms they deployed. It was known that TEs capture the ACP-attached fatty acid chain in an elongated interior pocket and cleave it at the pocket’s entrance. “In addition to structural features that determine the size of the pocket in the two TEs, and the rate of extrusion after cleavage and their overall productivity, we found, that a patch of amino acids on the surface of the TEs is more positively charged in the variant with medium-chain specificity and helps it to bind ACP stronger. In turn, this induces ACP to pull at the fatty acid chain and assists the TE in cleaving off shorter fatty acid chains,” said Way.

“With this study, we fill an important piece into our bigger effort to rewire different stages of fatty acid metabolism of bacteria towards the production of designer molecules for biofuels and bioplastics that could be produced at industrial scale. Being able to do this, in the longer run, could tremendously help save natural resources and sustain our environment,” said Silver, who is also the Elliot T. and Onie H. Adams Professor of Biochemistry and Systems Biology at Harvard Medical School (HMS).

The study was also authored by other former and present members of the Wyss Institute including Nathan Rollins, Aashna Shah, Brendon Dusel and Gordon Webster; and funded by the U.S. Department of Energy and Harvard’s Wyss Institute for Biologically Inspired Engineering.

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