Improvement of a Unified Saccharification and Fermentation System for Agaro-bioethanol Production in Yeast

The utilization of renewable resources is essential to achieve sustainable fuel production to solve the problem of excessive production of greenhouse gases that cause global warming [1]. Biomass is a renewable energy source, and bioethanol has attracted attention as a biomass-derived fuel. Among the many types of biomass, seaweed (marine biomass) is considered to be a promising raw material for bioethanol production owing to several advantages, including higher carbohydrate level and low lignin and hemicellulose content [2]. Specifically, marine red-algal biomass has emerged as a promising alternative for bioethanol production [3]. The major constituent of red macroalgal biomass is the recalcitrant agar polysaccharide. Agar is composed of agarose as the major component and agaropectin as the minor component. Agarose is the major cell wall component that consists of D-galactoses and 3,6-anhydro-L-galactoses (AHG) connected by alternating α-1,3 and β-1,4 linkages [4, 5]. In the practice of converting red algal biomass into bioenergy, the critical step in the agarose decomposition process is the release of fermentable monosugars (e.g. galactose). However, utilization of agarose as a biomass has been limited owing to a lack of established methods for pretreatment and an effective saccharification system. Kim et al. reported a sugar platform equipped with acetic acid, multiple agarases, and neoagarobiose hydrolase, and the enzyme system converted 79.1% of agarose to monosugars [6]. They used mild conditions for chemical liquefaction of agarose, and 4.4 g/l ethanol concentration was produced from 30 g/l agarose. In a previous study, we developed a unified enzymatic saccharification and fermentation (USF) system for the direct production We improved on a unified saccharification and fermentation (USF) system for the direct production of ethanol from agarose by increasing total agarase activity. The pGMFα-NGH plasmid harboring the NABH558 gene encoding neoagarobiose hydrolase and the AGAG1 and AGAH71 genes encoding β-agarase was constructed and used to transform Saccharomyces cerevisiae 2805. NABH558 gene transcription level was increased and total agarase activity was increased by 25 to 40% by placing the NABH558 gene expression cassette upstream of the other gene expression cassettes. In the 2805/pGMFα-NGH transformant, three secretory agarases were produced that efficiently degraded agarose to galactose, 3,6-anhydro-L-galactose (AHG), neoagarobiose, and neoagarohexaose. During the united cultivation process, a maximum of 2.36 g/l ethanol from 10 g/l agarose was produced over 120 h.