Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-l-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l−1) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

[1]  V. J. Chapman Seaweeds and their uses , 1950 .

[2]  Martin Kumar Patel,et al.  Medium and Long-term Opportunities and Risks of the Biotechnological Production of Bulk Chemicals from Renewable Resources , 2006 .

[3]  Isabelle Migneault,et al.  Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. , 2004, BioTechniques.

[4]  A. Burgard,et al.  Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. , 2011, Nature chemical biology.

[5]  Jeong Wook Lee,et al.  Systems metabolic engineering of microorganisms for natural and non-natural chemicals. , 2012, Nature chemical biology.

[6]  David G Hendrickson,et al.  Differential analysis of gene regulation at transcript resolution with RNA-seq , 2012, Nature Biotechnology.

[7]  W. M. Stewart,et al.  The Contribution of Commercial Fertilizer Nutrients to Food Production , 2005 .

[8]  S. Withers,et al.  Glycoside cleavage by a new mechanism in unsaturated glucuronyl hydrolases. , 2011, Journal of the American Chemical Society.

[9]  N. Friedman,et al.  Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data , 2011, Nature Biotechnology.

[10]  T. Norton,et al.  Seaweeds and their Uses. , 1981 .

[11]  S. Horn,et al.  Ethanol production from seaweed extract , 2000, Journal of Industrial Microbiology and Biotechnology.

[12]  Y. Yoshikuni,et al.  Implementation of stable and complex biological systems through recombinase-assisted genome engineering , 2013, Nature Communications.

[13]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[14]  C. Valdes Can Brazil Meet the World's Growing Need for Ethanol? , 2011 .

[15]  C. Boulton,et al.  Growth and metabolism of mannitol by strains of Saccharomyces cerevisiae. , 1987, Journal of general microbiology.

[16]  C. Nakamura,et al.  Metabolic engineering for the microbial production of 1,3-propanediol. , 2003, Current opinion in biotechnology.

[17]  Susanne B. Jones,et al.  Macroalgae as a Biomass Feedstock: A Preliminary Analysis , 2010 .

[18]  J. Keasling,et al.  Microbial engineering for the production of advanced biofuels , 2012, Nature.

[19]  Bruce E. Dale,et al.  Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST) , 2009, Proceedings of the National Academy of Sciences.

[20]  Satu Hilditch Identification of the fungal catabolic D-galacturonate pathway , 2010 .

[21]  K. Schaumann,et al.  Efficiency of uronic acid uptake in marine alginate-degrading fungi , 1995, Helgoländer Meeresuntersuchungen.

[22]  P. Maitra,et al.  A kinetic study of glycolytic enzyme synthesis in yeast. , 1971, The Journal of biological chemistry.

[23]  R. Lemoine,et al.  Identification of a Mannitol Transporter, AgMaT1, in Celery Phloem , 2001, Plant Cell.

[24]  Christine Nicole S. Santos,et al.  An Engineered Microbial Platform for Direct Biofuel Production from Brown Macroalgae , 2012, Science.

[25]  J. Preiss,et al.  Alginic acid metabolism in bacteria. II. The enzymatic reduction of 4-deoxy-L-erythro-5-hexoseulose uronic acid to 2-keto-3-deoxy-D-gluconic acid. , 1962, The Journal of biological chemistry.

[26]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[27]  Aie World Energy Outlook 2011 , 2001 .

[28]  Control of metabolic flux through the quinate pathway in Aspergillus nidulans. , 1996, The Biochemical journal.

[29]  Mohamed Ali Mekouar,et al.  15. United Nations Food and Agriculture Organization (FAO) , 2011 .