Presentation Information
[P02-281]Improving Mannitol Utilization in Saccharomyces cerevisiae for the Effective Use of Macroalgal Biomass
○Kan Iwata1, Kouichi Kuroda1 (1. Kyoto Institute of Technology (Japan))
Keywords:
Bioethanol,Marine biomass,Mannitol utilization,Saccharomyces cerevisiae
Macroalgae have attracted attention as a potential third-generation biomass feedstock because they can grow in seawater, do not compete with food crops, and do not contain lignin. In particular, brown macroalgae exhibit a high CO2 fixation capacity, and nearly half of their dry weight consists of carbohydrates such as alginate, mannitol, laminarin, and cellulose, with alginate and mannitol being especially abundant. Mannitol is one of the most prevalent soluble sugars in brown algae and is therefore considered an important target substrate for bioethanol production.
However, Saccharomyces cerevisiae, the primary microorganism used for industrial bioethanol fermentation, is inherently incapable of utilizing mannitol. Although S. cerevisiae possesses two endogenous mannitol dehydrogenase (MDH) genes, MAN2 and its paralog DSF1, their expression is typically repressed, and wild-type strains cannot assimilate mannitol. Our laboratory previously developed the yeast strain AM1, capable of producing ethanol from alginate and mannitol (1). AM1 acquired mannitol utilization ability through adaptive evolution by repeated serial cultivation in medium containing mannitol as the sole carbon source. To further improve AM1 and ensure stable mannitol utilization, we aimed to genetically introduce a mannitol assimilation pathway.
We first expressed the transporter genes HXT13 and HXT17, and the MDH genes MAN2 and DSF1, individually using multicopy plasmids in S. cerevisiae, but none of the single-gene expression strains were able to utilize mannitol. We then co-expressed HXT13 or HXT17 together with MAN2 or DSF1. All four co-expression combinations enabled mannitol utilization. Among them, the combination of HXT13 and DSF1 yielded the most favorable results. Therefore, this gene pair was subcloned into a genome-integrating plasmid. The resulting integrative transformants also exhibited growth and ethanol production in mannitol medium, demonstrating that this gene pair is sufficient to confer stable mannitol assimilation without the need for adaptive evolution.
This study provides a genetically stable platform for engineering yeast strains capable of efficiently converting brown algae–derived carbohydrates into bioethanol, contributing to the advancement of sustainable marine biomass utilization.
(1) Takagi, T., et al. Appl. Microbiol. Biotechnol. 101, 6627–6636 (2017).
However, Saccharomyces cerevisiae, the primary microorganism used for industrial bioethanol fermentation, is inherently incapable of utilizing mannitol. Although S. cerevisiae possesses two endogenous mannitol dehydrogenase (MDH) genes, MAN2 and its paralog DSF1, their expression is typically repressed, and wild-type strains cannot assimilate mannitol. Our laboratory previously developed the yeast strain AM1, capable of producing ethanol from alginate and mannitol (1). AM1 acquired mannitol utilization ability through adaptive evolution by repeated serial cultivation in medium containing mannitol as the sole carbon source. To further improve AM1 and ensure stable mannitol utilization, we aimed to genetically introduce a mannitol assimilation pathway.
We first expressed the transporter genes HXT13 and HXT17, and the MDH genes MAN2 and DSF1, individually using multicopy plasmids in S. cerevisiae, but none of the single-gene expression strains were able to utilize mannitol. We then co-expressed HXT13 or HXT17 together with MAN2 or DSF1. All four co-expression combinations enabled mannitol utilization. Among them, the combination of HXT13 and DSF1 yielded the most favorable results. Therefore, this gene pair was subcloned into a genome-integrating plasmid. The resulting integrative transformants also exhibited growth and ethanol production in mannitol medium, demonstrating that this gene pair is sufficient to confer stable mannitol assimilation without the need for adaptive evolution.
This study provides a genetically stable platform for engineering yeast strains capable of efficiently converting brown algae–derived carbohydrates into bioethanol, contributing to the advancement of sustainable marine biomass utilization.
(1) Takagi, T., et al. Appl. Microbiol. Biotechnol. 101, 6627–6636 (2017).
Comment
To browse or post comments, you must log in.Log in