Enhancement of Galactose Uptake from Kappaphycus alvarezii Hydrolysate Using Saccharomyces cerevisiae Through Overexpres
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Enhancement of Galactose Uptake from Kappaphycus alvarezii Hydrolysate Using Saccharomyces cerevisiae Through Overexpression of Leloir Pathway Genes In Yung Sunwoo 1,2 & Pailin Sukwong 1 & Yu Rim Park 1 Soo Rin Kim 3 & Gwi-Teak Jeong 1 & Sung-Koo Kim 1
3
& Deok Yeol Jeong &
Received: 1 June 2020 / Accepted: 11 September 2020/ # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A total 42.68 g/L monosaccharide with 0.10 g/L HMF was obtained from 10% (w/v) Kappaphycus alvarezii with thermal acid hydrolysis using 350 mM HNO3 at 121 °C for 60 min and enzymatic saccharification with a 1:1 mixture of Viscozyme L and Celluclast 1.5 L for 72 h. To enhance the galactose utilization rate, fermentation was performed with overexpression of GAL1 (galactokinase), GAL7 (galactose-1-phosphate uridyltransferase), GAL10 (UDP-glucose-4-epimerase), and PGM2 (phosphoglucomutase 2) in Saccharomyces cerevisiae CEN.PK2 using CCW12 as a strong promoter. Among the strains, the overexpression of PGM2 showed twofold high galactose utilization rate (URgal) and produced ethanol 1.4-fold more than that of the control. Transcriptional analysis revealed the increase of PGM2 transcription level leading to enhance glucose-6phosphate and fructose-6-phosphate and plays a key role in ensuring a higher glycolytic flux in the PGM2 strain. This finding shows particular importance in biofuel production from seaweed because galactose is one of the major monosaccharides in seaweeds such as K. alvarezii. Keywords Kappaphycus alvarezii . Fermentation . CRISPR/Cas9 . PGM2 . Ethanol
In Yung Sunwoo and Pailin Sukwong contributed equally to this work.
* Sung-Koo Kim [email protected]
1
Division of Marine, Fisheries, and Life Science, Pukyong National University, Busan 48513, South Korea
2
Department of Chemistry, Umeå University, 90187 Umeå, Sweden
3
School of Food Science and Biotechnology, Kyungpook National University, Daegu 41566, South Korea
Applied Biochemistry and Biotechnology
Introduction Macroalgae are a promising bio-feedstock for renewable fuel production [1]. Bioethanol production using macroalgal biomass is advantageous as macroalgae grow faster and fix CO2 at a higher rate compared with terrestrial plants. In addition, in comparison with lignocellulosic biomass, macroalgae have a high level of carbohydrates without lignin and are easier to convert to monosaccharides [2]. The red microalga Kappaphycus alvarezii contains carrageenan and cellulose [3]. This species can be hydrolyzed to monosaccharides such as galactose and glucose to produce ethanol. The Cas9 protein, found in the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, is an RNA-guided DNA endonuclease, which can create double-strand breaks on chromosomes in vivo [4]. Owing to the homologous recombination ability of yeast, the precise engineering of genomes could be achieved using a short DNA fragment with the expression of Cas9 and a guide RNA (gRNA) [5]. Moreover, Zhang et al. have demonstrated that Cas9-based geno
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