• Journal of Microbiology and Biotechnology
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• 제31권7호
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• pp.1035-1043
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• 2021

Although engineered Saccharomyces cerevisiae fermenting cellobiose is useful for the production of biofuels from cellulosic biomass, cellodextrin accumulation is one of the main problems reducing ethanol yield and productivity in cellobiose fermentation with S. cerevisiae expressing cellodextrin transporter (CDT) and intracellular β-glucosidase (GH1-1). In this study, we investigated the reason for the cellodextrin accumulation and how to alleviate its formation during cellobiose fermentation using engineered S. cerevisiae fermenting cellobiose. From the series of cellobiose fermentation using S. cerevisiae expressing only GH1-1 under several culture conditions, it was discovered that small amounts of GH1-1 were secreted and cellodextrin was generated through trans-glycosylation activity of the secreted GH1-1. As GH1-1 does not have a secretion signal peptide, non-conventional protein secretion might facilitate the secretion of GH1-1. In cellobiose fermentations with S. cerevisiae expressing only GH1-1, knockout of TLG2 gene involved in non-conventional protein secretion pathway significantly delayed cellodextrin formation by reducing the secretion of GH1-1 by more than 50%. However, in cellobiose fermentations with S. cerevisiae expressing both GH1-1 and CDT-1, TLG2 knockout did not show a significant effect on cellodextrin formation, although secretion of GH1-1 was reduced by more than 40%. These results suggest that the development of new intracellular β-glucosidase, not influenced by non-conventional protein secretion, is required for better cellobiose fermentation performances of engineered S. cerevisiae fermenting cellobiose.

• Journal of Microbiology and Biotechnology
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• 제27권9호
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• pp.1649-1656
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• 2017

In simultaneous saccharification and fermentation (SSF) for production of cellulosic biofuels, engineered Saccharomyces cerevisiae capable of fermenting cellobiose has provided several benefits, such as lower enzyme costs and faster fermentation rate compared with wild-type S. cerevisiae fermenting glucose. In this study, the effects of an alternative intracellular cellobiose utilization pathway-a phosphorolytic pathway based on a mutant cellodextrin transporter (CDT-1 (F213L)) and cellobiose phosphorylase (SdCBP)-was investigated by comparing with a hydrolytic pathway based on the same transporter and an intracellular ${\beta}$-glucosidase (GH1-1) for their SSF performances under various conditions. Whereas the phosphorolytic and hydrolytic cellobiose-fermenting S. cerevisiae strains performed similarly under the anoxic SSF conditions, the hydrolytic S. cerevisiae performed slightly better than the phosphorolytic S. cerevisiae under the microaerobic SSF conditions. Nonetheless, the phosphorolytic S. cerevisiae expressing the mutant CDT-1 showed better ethanol production than the glucose-fermenting S. cerevisiae with an extracellular ${\beta}$-glucosidase, regardless of SSF conditions. These results clearly prove that introduction of the intracellular cellobiose metabolic pathway into yeast can be effective on cellulosic ethanol production in SSF. They also demonstrate that enhancement of cellobiose transport activity in engineered yeast is the most important factor affecting the efficiency of SSF of cellulose.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.846-853
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• 2016

We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd²⁺ in this study. The transformed yeast strains showed much higher resistance ability to Cd²⁺ compared with the control. Strikingly, their Cd²⁺ accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd²⁺ under a wide range of pH levels, from 3 to 7, without disturbing the Cu²⁺ and Hg²⁺. Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd²⁺-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

• Journal of Microbiology and Biotechnology
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• 제21권8호
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• pp.846-853
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• 2011

Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1912-1918
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• 2020

Hyper-thermal (HT) acid hydrolysis of red seaweed Gelidium amansii was performed using 12% (w/v) slurry and an acid mix concentration of 180 mM at 150℃ for 10 min. Enzymatic saccharification when using a combination of Celluclast 1.5 L and CTec2 at a dose of 16 U/ml led to the production of 12.0 g/l of reducing sugar with an efficiency of enzymatic saccharification of 13.2%. After the enzymatic saccharification, 2,3-butanediol (2,3-BD) fermentation was carried out using an engineered S. cerevisiae strain. The use of HT acid-hydrolyzed medium with 1.9 g/l of 5-hydroxymethylfurfural showed a reduction in the lag time from 48 to 24 h. The 2,3-BD concentration and yield coefficient at 72 h were 14.8 g/l and 0.30, respectively. Therefore, HT acid hydrolysis and the use of the engineered S. cerevisiae strain can enhance the overall 2,3-BD yields from G. amansii seaweed.

• Journal of Microbiology and Biotechnology
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• 제18권11호
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• pp.1797-1802
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• 2008

Saccharomyces cerevisiae was metabolically engineered to improve 1,2-propanediol production. Deletion of the tpil (triosephosphate isomerase) gene in S. cerevisiae increased the carbon flux to DHAP (dihydroxylacetone phosphate) in glycolysis, resulting in increased glycerol production. Then, the mgs and gldA genes, the products of which convert DHAP to l,2-propanediol, were introduced to the tpil-deficient strain using a multicopy plasmid. As expected, the intracellular level of methylglyoxal was increased by introduction of the mgs gene in S. cerevisiae and that of 1,2-propanediol by introduction of both the mgs and gldA genes. As a result, 1.11 g/l of 1,2-propanediol was achieved in flask culture.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.117-125
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• 2022

Until recently, four types of cellobiose-fermenting Saccharomyces cerevisiae strains have been developed by introduction of a cellobiose metabolic pathway based on either intracellular β-glucosidase (GH1-1) or cellobiose phosphorylase (CBP), along with either an energy-consuming active cellodextrin transporter (CDT-1) or a non-energy-consuming passive cellodextrin facilitator (CDT-2). In this study, the ethanol production performance of two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-2 (N306I) with GH1-1 or CBP were compared with two cellobiose-fermenting S. cerevisiae strains expressing mutant CDT-1 (F213L) with GH1-1 or CBP in the simultaneous saccharification and fermentation (SSF) of cellulose under various conditions. It was found that, regardless of the SSF conditions, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the best ethanol production among the four strains. In addition, during SSF contaminated by lactic acid bacteria, the phosphorolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-2 with CBP showed the highest ethanol production and the lowest lactate formation compared with those of other strains, such as the hydrolytic cellobiose-fermenting S. cerevisiae expressing mutant CDT-1 with GH1-1, and the glucose-fermenting S. cerevisiae with extracellular β-glucosidase. These results suggest that the cellobiose-fermenting yeast strain exhibiting low energy consumption can enhance the efficiency of the SSF of cellulosic biomass.

• Current Research on Agriculture and Life Sciences
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• 제32권4호
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• pp.199-202
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• 2014

The bioconversion of cellulosic biomass hydrolyzates consisting mainly of glucose and xylose requires the use of engineered Saccharomyces cerevisiae expressing a heterologous xylose pathway. However, there is concern that a fungal xylose pathway consisting of NADPH-specific xylose reductase (XR) and $NAD^+$-specific xylitol dehydrogenase (XDH) may result in a cellular redox imbalance. However, the glycerol biosynthesis and glycerol degradation pathways of S. cerevisiae, termed here as the glycerol cycle, has the potential to balance the cofactor requirements for xylose metabolism, as it produces NADPH by consuming NADH at the expense of one mole of ATP. Therefore, this study tested if the glycerol cycle could improve the xylose metabolism of engineered S. cerevisiae by cofactor balancing, as predicted by an in-silico analysis using elementary flux mode (EFM). When the GPD1 gene, the first step of the glycerol cycle, was overexpressed in the XR/XDH-expressing S. cerevisiae, the glycerol production significantly increased, while the xylitol and ethanol yields became negligible. The reduced xylitol yield suggests that enough $NAD^+$ was supplied for XDH by the glycerol cycle. However, the GPD1 overexpression completely shifted the carbon flux from ethanol to glycerol. Thus, moderate expression of GPD1 may be necessary to achieve improved ethanol production through the cofactor balancing.

• Biotechnology and Bioprocess Engineering:BBE
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• 제10권6호
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• pp.576-581
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• 2005

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. We have engineered the cell surface of the yeast. Saccharomyces cerevisiae, by anchoring active fungal phytase on its cell wall, in order to apply it as a dietary supplement containing bioconversional functions in animal foods and a whole cell bio-catalyst for the treatment of waste. The phytase gene (phyA) of Aspergillus niger with a signal peptide of rice amylase 1A (Ramy1A) was fused with the gene encoding the C-terminal half (320 amino acid residues from the C-terminus) of yeast ${\alpha}-agglutinin$, a protein which is involved in mating and is covalently anchored to the cell wall. The resulting fusion construct was introduced into S. cerevisiae and expressed under the control of the constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter. Phytase plate assay revealed that the surface-engineered cell exhibited a catalytically active opaque zone which was restricted to the margin of the colony. Additionally, the phytase activity was detected in the cell fraction, but was not detected in the culture medium when it was grown in liquid. These results indicate that the phytase was successfully anchored to the cell surface of yeast and was displayed as its active form. The amount of recombinant phytase on the surface of yeast cells was estimated to be 16,000 molecules per cell.

• Journal of Microbiology and Biotechnology
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• 제29권5호
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• pp.758-764
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• 2019

${\beta}$-Glucan is a chief structural polymer in the cell wall of yeast. ${\beta}$-Glucan has attracted intensive attention because of its wide applications in health protection and cosmetic areas. In the present study, the ${\beta}$-glucan biosynthesis pathway in S. Cerevisiae was engineered to enhance ${\beta}$-glucan accumulation. A newly identified bacterial ${\beta}-1$, 6-glucan synthase GsmA from Mycoplasma agalactiae was expressed, and increased ${\beta}$-glucan content by 43%. In addition, other pathway enzymes were investigated to direct more metabolic flux towards the building of ${\beta}$-glucan chains. We found that overexpression of Pgm2 (phosphoglucomutase) and Rho1 (a GTPase for activating glucan synthesis) significantly increased ${\beta}$-glucan accumulation. After further optimization of culture conditions, the ${\beta}$-glucan content was increased by 53.1%. This study provides a new approach to enhance ${\beta}$-glucan biosynthesis in Saccharomyces cerevisiae.