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Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria

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Abstract

Photosynthetically derived fuels, such as those produced by microalgae, are touted as a future renewable energy source and a means for achieving energy independence. Realization of these claims, however, will require fuel production rates beyond the native capabilities of these microorganisms. The development of a metabolic engineering toolkit for microalgae will be key for reaching the production rates necessary for fuel production. This work advances the toolkit for cyanobacterial fuels by exploring the use of eukaryotic algal gene sources for free fatty acid biosynthesis rather than the traditional bacterial and plant sources. Many species of eukaryotic algae naturally accumulate high levels of triacylglycerol, a compound requiring three fatty acid side chains. Triacylglycerol accumulation implies that eukaryotic algae have naturally efficient enzymes for free fatty acid production, representing an unexplored resource for metabolic engineering targets. In this work, the model cyanobacterium, Synechococcus elongatus PCC7942, was engineered for free fatty acid production by targeting three main rate-limiting steps: (1) fatty acid release, catalyzed by a thioesterase, (2) fixation of carbon by ribulose-1,5-bisphosphate carboxylase/oxygenase, and (3) the first committed step in fatty acid biosynthesis, acetyl-CoA carboxylase. The recombinant acyl-ACP thioesterase and acetyl-CoA carboxylase were derived from the model green alga, Chlamydomonas reinhardtii CC-503. By targeting these proposed rate-determining steps, free fatty acid production was improved on a cell weight basis; however, the overall concentration of excreted free fatty acid did not increase. Recombinant gene expression was optimized by using native promoters, and while expression improved, the free fatty acid yield did not likewise increase. From physiological measurements, it was determined that free fatty acid production in S. elongatus PCC7942 is ultimately limited by the negative physiological effects associated with free fatty acid synthesis rather than bottlenecks within the metabolic pathway. This work demonstrates the successful expression of algal genes in a cyanobacterial host, but further improvement in free fatty acid yields will only be possible when the negative effects of free fatty acid production are mitigated.

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Acknowledgments

This work was supported by the Harry S. Truman Fellowship in National Security Science and Engineering and the Laboratory Directed Research and Development program. Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000. The author is grateful to Dr. James Laio (University of California, Los Angeles) for providing pSA126 and to the Kansas Lipidomics Research Center for performing GC/MS analysis on the extracted FFAs. Instrument acquisition and method development at the Kansas Lipidomics Research Center was supported by NSF grants MCB 0455318 and DBI 0521587, K-INBRE (NIH Grant P20 RR16475 from the INBRE program of the National Center for Research Resources), and NSF EPSCoR grant EPS-0236913 with matching support from the State of Kansas through Kansas Technology Enterprise Corporation and Kansas State University.

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The author has no conflict of interest to declare.

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  1. Department of Bioenergy and Defense Technologies, Sandia National Laboratories, P.O. Box 5800, MS 1413, Albuquerque, NM, 87185-1413, USA

    Anne M. Ruffing

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Correspondence to Anne M. Ruffing.

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Ruffing, A.M. Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria. J Appl Phycol 25, 1495–1507 (2013). https://doi.org/10.1007/s10811-013-9993-7

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  • DOI: https://doi.org/10.1007/s10811-013-9993-7

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