Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation

Accepted Manuscript Title: Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation Author: Won-Heong Lee Yong-Su Jin PII: DOI: Reference:

S0168-1656(17)30035-4 http://dx.doi.org/doi:10.1016/j.jbiotec.2017.01.018 BIOTEC 7779

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

23-9-2016 30-12-2016 27-1-2017

Please cite this article as: Lee, W.-H., Jin, Y.-S.,Improved ethanol production by engineered Saccharomyces cerevisiae expressing a mutated cellobiose transporter during simultaneous saccharification and fermentation, Journal of Biotechnology (2017), http://dx.doi.org/10.1016/j.jbiotec.2017.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights

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► Ethanol production from SSF can be improved by expression of a mutated cellobiose transporter in yeast

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in SSF without extracellular β-glucosidase

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► Engineered yeast expressing a cellobiose transporter can show efficient production of ethanol

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Monod constants (KS) for cellobiose than KS for glucose

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► Engineered yeast strains capable of fermenting cellobiose exhibited significantly lower

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Improved ethanol production by engineered Saccharomyces cerevisiae expressing a

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mutated cellobiose transporter during simultaneous saccharification and fermentation

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Won-Heong Lee and 1Yong-Su Jin*

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University of Illinois at Urbana-Champaign, Urbana, IL 61801, United States

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500-757, Korea

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Department of Food Science and Human Nutrition, and Institute for Genomic Biology,

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Department of Bioenergy Science and Technology, Chonnam National University, Gwangju

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Tel: 217-333-7981, Fax: 217-333-0508, E-mail: [email protected]

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Running title: Efficient ethanol production in SSF by engineered yeast

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Corresponding author: Prof. Yong-Su Jin

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Abstract

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Although simultaneous saccharification and fermentation (SSF) of cellulosic biomass can offer

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efficient hydrolysis of cellulose through alleviating feed-back inhibition of cellulases by glucose,

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supplementation of β-glucosidase is necessary because most fermenting microorganisms cannot

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utilize cellobiose. Previously, we observed that SSF of cellulose by an engineered

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Saccharomyces cerevisiae expressing a cellobiose transporter (CDT-1) and an intracellular β-

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glucosidase (GH1-1) without β-glucosidase could not be performed as efficiently as the

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traditional SSF with extracellular β-glucosidase. However, we improved the ethanol production

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from SSF of cellulose by employing a further engineered S. cerevisiae expressing a mutant

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cellobiose transporter [CDT-1 (F213L) exhibiting higher VMAX than CDT-1] and GH1-1 in this

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study. Furthermore, limitation of cellobiose formation by reducing the amounts of cellulases

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mixture in SSF could lead the further engineered strain to produce ethanol considerably better

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than the parental strain with β-glucosidase. Probably, better production of ethanol by the further

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engineered strain seemed to be due to a higher affinity to cellobiose, which might be attributed to

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not only 2-times lower Monod constant (KS) for cellobiose than KS of the parental strain for

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glucose but also 5-times lower KS than Michaelis-Menten constant (KM) of the extracellular β-

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glucosidase for glucose. Our results suggest that modification of the cellobiose transporter in the

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engineered yeast to transport lower level of cellobiose enables a more efficient SSF for

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producing ethanol from cellulose.

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Keywords: Cellulosic ethanol; simultaneous saccharification and fermentation; cellobiose

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transporter; intracellular β-glucosidase; engineered Saccharomyces cerevisiae 2

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1. Introduction Efficient production of ethanol from cellulosic biomass has been an outstanding issue for the economic production of renewable transportation fuels (Gray et al., 2006; Mussatto et al.,

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2010; Wyman, 2007). Simultaneous saccharification and fermentation (SSF) has been proposed

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for the production of cellulosic ethanol because it can provide several benefits: reduction of

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investment costs, attenuation of end-product inhibition on enzymatic hydrolysis of cellulosic

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biomass, and minimization of loss of sugars during separation and purification (Olofsson et al.,

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2008; Xu et al., 2009). Among the main fractions of cellulosic biomass, cellulose cannot be

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easily hydrolyzed to glucose because cellulose is made up of hard and compact linkages (β-1,4

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glycosidic bond) between glucose monomers. Therefore, considerable amounts of cellulolytic

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enzymes (such as endo- and exo-glucanase) need to be loaded for the hydrolysis of cellulose

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(Chauve et al., 2010; Gray et al., 2006; Martins et al., 2008). While fungal cellulases can be

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produced at high titers, most fungal cellulases have poor β-glucosidase activity and are severely

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inhibited by glucose. As a result, the complete hydrolysis of cellulose into glucose is difficult to

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achieve. Native S. cerevisiae cannot ferment the intermediate sugars (cellodextrins such as

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cellobiose, cellotriose, etc.) during the cellulose hydrolysis, which suggests that efficient SSF of

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cellulose cannot be accomplished without extra supplementation of β-glucosidase (Chauve et al.,

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2010; Martins et al., 2008). As such, numerous efforts to improve cellulase activities through

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enriching β-glucosidase without glucose inhibition have been made to produce glucose from

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cellulose economically (Rodrigues et al., 2015).

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As an alternative strategy to enable efficient SSF of cellulose, engineering of S. cerevisiae to directly ferment cellobiose has been attempted by many research groups. Along 3

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with an intracellular β-glucosidase (GH1-1) from Neurospora crassa, several cellobiose

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transporters such as CDT-1 and CDT-2 from N. crassa and HXT2.4 from Pichia stipitis have

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been introduced and functionally expressed in S. cerevisiae for cellobiose fermentation (Galazka

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et al., 2010; Ha et al., 2013b; Ha et al., 2011a; Kim et al., 2014). Co-expression of the putative

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sugar transporter (Pc_ST) from Penicillium chrysogenum and intracellular β-glucosidase

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(Tt_BG) from Thielavia terrestris led to engineered S. cerevisiae to ferment cellobiose as

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efficiently as co-expression of CDT-1 and GH1-1 from N. crassa (Bae et al., 2014). Based on the

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expression of GH1-1 from N. crassa, successful cellobiose fermentation of engineered S.

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cerevisiae was also achieved by introduction of cellobiose transporters (CltA and CltB) from

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Aspergillus nidulans as well as sugar transporter (Stp1) from Trichoderma reesei (Dos Reis TF

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et al., 2016; Zhang et al., 2013). With the introduction of lactose permease (Lac12) from

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Kluyveromyces lactis and CDT-1 from N. crassa, cellobiose was also fermented by engineered S.

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ceresivisiae expressing cellobiose phosphorylases including CepA from Clostridium

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stercorarium and CBP from Saccharophagus degradans, respectively (Sadie et al., 2011; Ha et

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al., 2013a).

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Integration of the intracellular cellobiose fermenting pathways into yeast was expected to

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allow economic ethanol production from SSF of cellulosic biomass. However, a previous SSF

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study revealed that the traditional SSF by wild type S. cerevisiae supplemented with extracellular

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β-glucosidase showed better performances than the engineered strain expressing GH1-1 and

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CDT-1 (Lee et al., 2013). This result proposed that further modifications of the intracellular

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cellobiose-assimilating pathway might be necessary to improve performance of the engineered

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strain during SSF. 4

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Previously, a mutant cellobiose transporter CDT-1 (F213L) exhibiting a higher VMAX was isolated from a laboratory evolution experiment of a cellobiose fermenting S. cerevisiae on

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cellobiose (Ha et al., 2013a). We hypothesized that improvement of ethanol production during

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SSF might be achieved by the engineered strain expressing GH1-1 and a mutant CDT-1 (F213L)

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as the F213L mutant transporter exhibited higher cellobiose (or cellodextrin) transporting

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capacity than the wild-type CDT-1 (Fig S1). A series of cellobiose fermentation and SSF of

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cellulose experiments confirmed that improved cellobiose uptake by the mutant cellobiose

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transporter can lead to enhanced performance of ethanol production in the engineered strain

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expressing the mutant cellobiose transporter during SSF.

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2. Materials and methods

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2.1. Strains, plasmids and culture conditions

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S. cerevisiae D452-2 (MATα, leu2, his3, ura3 and can1) (Hosaka et al., 1992) was used

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as a host strain for overexpressing the cellobiose transporter (CDT-1) or the mutant transporter

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[CDT-1 (F213L)] along with the intracellular β-glucosidase (GH1-1) from N. crassa FGSC 2489.

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Plasmids pRS425-gh1-1, pRS426-cdt1 and pRS426-cdt1 (F213L) were previously constructed

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for overexpression of GH1-1, CDT1 and CDT1 (F213L), respectively (Galazka et al., 2010; Ha

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et al., 2013a; Ha et al., 2011b). All strains and plasmids used in this study are listed in Table 1.

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A synthetic complete (SC) medium containing 6.7 g/L of Yeast Nitrogen Base (YNB)

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without amino acids, 0.625 g/L of Complete Supplement Mixture (CSM) without leucine,

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tryptophan and uracil, and 20 g/L of glucose (pH 6.0) was used for seed cultivation. Yeast

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extract-Peptone (YP) medium containing 10 g/L of yeast extract and 20 g/L of Bacto peptone at 5

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pH 6.7 with appropriate sugars (50 g/L of glucose or 50 g/L of cellobiose) was used for pre-

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cultivation. Yeast cells at exponential growth in the pre-cultivation were harvested and used in

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the cellobiose fermentation and SSF. Seed cultivation and pre-cultivation was carried out at 30°C

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and 250 rpm.

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2.2. Determination of growth kinetic parameters of engineered S. cerevisiae strains

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In order to determine growth kinetics of the parental strain and the engineered strains on cellobiose, yeast cells at exponential growth in the pre-cultivation (YPD for the parental strain

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and YPC for the engineered strains) were harvested and inoculated into 50 mL minimal (SC)

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medium (pH 6.0) with 0 to 2 g/L of cellobiose at initial OD600 of 0.05. The cultivation was

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carried out in 250 mL flasks at 30°C and 100 rpm. The OD values of culture broth were checked

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every two hours and used to determine the Monod constant (KS) and the maximum specific

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growth rate (µMAX). Growth kinetic parameters of the parental strain for glucose were also

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determined by the same procedure. The yeast cells at exponential growth in the pre-cultivation

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were harvested and inoculated into SC medium containing 0 to 2 g/L of glucose. All experiments

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were performed in triplicate.

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2.3. Culture conditions for cellobiose fermentation Pure sugar fermentation was carried out in 100 mL bottles containing 20 mL of YP

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medium with 85 g/L cellobiose at 30°C and 100 rpm. The initial concentrations of yeast cells

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were adjusted to 0.35 g/L for a low cell density fermentation and 10.5 g/L for a high cell density

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fermentation. Novozyme 188 [27 cellobiase unit (CBU)/g cellobiose (Sigma, St. Louis, MO)] 6

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was added for the cellobiose fermentation by the parental stain with extracellular β-glucosidase.

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Because CO2 production by yeast cells can represent ethanol production under anoxic condition,

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CO2 production was continuously monitored using bottles equipped with a wireless gas

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production measurement system (Ankom Technology, Macedon, NY). Cell culture in the bottle

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can be maintained at anoxic conditions because the gas-measuring module allows only release of

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CO2 without air intake. At the end of the fermentations, samples were taken to analyze remaining

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sugars and ethanol. All experiments were performed in duplicate.

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2.4. Culture conditions for SSF of cellulose

The pretreated corn stover (PCS) from the National Renewable Resource Laboratory

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(NREL) and Avicel PH-101 (Sigma) were used as substrates for SSF experiments. Pretreatment

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method of corn strove (treatment with dilute sulfuric acid and steam in pilot-scale continuous

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reactor) and the composition of PCS [3.6% ash, 29.8% lignin, 58.9% glucan, 3.4% xylan, 0.5%

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galactan and 0.7% arabinan (w/w) in solid fraction] are well described in a previous study

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reported by NREL (McMillan et al., 2011). In order to perform SSF without fermentation

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inhibitors, the pH value of PCS slurry was adjusted to 7.0 using NaOH and then washed with

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distilled water until the concentrations of monomeric sugars were below 0.1 g/L as described in

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the previous report (McMillan et al., 2011). After washing, PCS slurry was dried at 60°C for 24

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h. SSF was carried out under various conditions as follow: 1) 100 mL bottles containing 20 mL

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of YP, 10% (w/v) washed PCS, Celluclast 1.5L [50 filter paper unit (FPU)/g PCS, Sigma] with

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(or without) Novozyme 188 (27 CBU/ g PCS), 2) 100 mL bottles containing 20 mL of YP, 10%

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(w/v) washed PCS, Celluclast 1.5L (5.9 FPU/ g PCS) with (or without) Novozyme 188 (3.2

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CBU/ g PCS), and 3) 100 mL bottles containing 20 mL of YP, 13% (w/v) Avicel PH-101,

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Celluclast 1.5L (10 FPU/g cellulose) with (or without) Novozyme 188 (5.4 CBU/g cellulose).

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Celluclast 1.5L was used as the cellulase mixture for saccharification of cellulose and Novozyme

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188 was used as the extracellular β-glucosidase for conversion of cellobiose to glucose. The

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bottles were equipped with the gas-measuring module to monitor CO2 production continuously.

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Temperature and agitation speed were maintained at 30°C and 100 rpm. All experiments were

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performed in duplicate.

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2.5 Analytical methods

Cell concentration was measured using a spectrophotometer (Biomate 5, Thermo, NY).

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Glucose, cellobiose and ethanol concentrations were determined by high performance liquid

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chromatography (HPLC, Agilent Technologies 1200 Series) equipped with a refractive index

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detector using a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc., Torrance, CA).

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The column was eluted with 0.005 N of H2SO4 at a flow rate of 0.6 mL/min at 50˚C.

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3. Results

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3.1. Investigation of growth kinetic properties of the engineered S. cerevisiae

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It is generally known that the Monod constant (KS) of engineered yeast for cellobiose

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indicates not only binding affinity of the cell to cellobiose, but also efficiency of the cellobiose

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metabolism. Therefore, determination of KS of the engineered yeast cells for cellobiose was

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performed to evaluate the efficiency of the alternative SSF process with engineered yeast

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fermenting cellobiose directly. 8

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As shown in Fig 1, the specific growth rates of the parental strain containing empty plasmids (D-56) and two engineered strains, the D-BTw strain expressing the wild type

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cellobiose transporter (CDT-1) with the intracellular β-glucosidase (GH1-1) and the D-BTm

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strain expressing the mutant cellobiose transporter [CDT-1 (F213L)] with GH1-1, were

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measured with different cellobiose concentrations (0 to 2 g/L). To minimize cell growth by other

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nutrients in complex medium (e. g. yeast extract and peptone), minimal medium (SC) was used

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for measuring specific growth rates of the engineered strains. For comparison, the specific

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growth rates of the D-56 strain with glucose (0 to 2 g/L) were also measured. Determination of

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the specific growth rate was performed by calculating the slope of the linear line in the natural

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log scales of OD600 over time during the period of exponential growth (Fig S2). The KS and μMAX

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values were calculated by linear regression of Lineweaver-Burk plots between the specific

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growth rates and concentrations of initial substrate (Fig S3).

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Under cellobiose conditions, the µMAX and KS values of D-BTw (wild-type CDT-1

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expressing strain) were determined to be 0.17 h-1 and 0.04 g cellobiose/L, respectively. The µMAX

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and KS values of D-BTm [mutant CDT-1 (F213L) expressing strain] were determined to be 0.25

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h-1 and 0.06 g cellobiose/L, respectively. As expected, D-56 (the parental strain) did not grow

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under cellobiose condition. While both D-BTw and D-BTm showed the similar affinity to

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cellobiose (similar values of KS, 0.06 and 0.04 g cellobiose/L), the D-BTm strain showed higher

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maximum specific growth rate than the D-BTw strain, which suggests that D-BTm can grow

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faster than D-BTw with the aid of mutated cellobiose transporter.

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Under glucose conditions, the D-56 strain showed 0.43 h-1 of µMAX and 0.17 g glucose/L of KS. While the μMAX of the engineered strains (D-BTw and D-BTm) for cellobiose was 1.7 to 9

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2.5-times lower than the μMAX of the D-56 strain for glucose, both of the engineered strains (D-

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BTw and D-BTm) showed 3 to 5-times lower KS for cellobiose than KS of the parental strain (D-

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56) for glucose. Such lower KS values (0.06 and 0.04 g cellobiose/L) of the engineered strains for

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cellobiose than KS (0.17 g glucose/L) of the parental strain for glucose suggest that cellobiose

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can be more effectively eliminated by cellobiose-fermenting strains than glucose-fermenting

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strain combined with β-glucosidase in SSF. However, lower μMAX values (0.17 and 0.25 h-1) of

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the engineered strains on cellobiose than that (0.43 h-1) of the parental strain on glucose also

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suggest that cellobiose-fermenting strains might not perform ethanol production as efficiently as

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glucose-fermenting strain with external β-glucosidase when initial sugar concentration is higher

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than KS of glucose-fermenting strain for glucose because they exhibit slower specific growth

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rates (Fig 1).

3.2. Cellobiose fermentation by engineered S. cerevisiae

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In order to check the fermentation profile in detail, carbon dioxide production was

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continuously monitored with a gas measuring system described in the previous report (Hector et

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al., 2011). After inoculation of yeast cells into the bottles containing media, the bottles are tightly

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closed by the caps equipped with the gas monitoring modules, which allows only release of CO2

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without air inflow. During the fermentation, initial aerobic condition changes to anaerobic

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condition and CO2 formation becomes mainly dependent on the production of ethanol under the

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above condition (anoxic condition). Consequently, CO2 production monitored by measuring

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cumulative gas pressure can be expected to represent ethanol production by yeast cells under

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anoxic condition.

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Fermentation experiments with 8.5% cellobiose were performed and the rates of CO2 production were measured to confirm whether the cellobiose-fermenting strains can utilize

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cellobiose as fast as the glucose-fermenting strain with extracellular β-glucosidase at high

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concentrations of cellobiose. Also, fermentation experiments with different levels of initial cell

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concentrations were performed to check the effect of inoculum level on cellobiose fermentation.

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Gas production rates by the glucose-fermenting strain (D-56) only, the glucose-fermenting strain

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with extracellular β-glucosidase (D-56+188), the cellobiose-fermenting strain with wild type

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cellobiose transporter (D-BTw), and the cellobiose-fermenting strain with mutant cellobiose

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transporter (D-BTm) were compared during the fermentation of cellobiose. For the cellobiose

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fermentation by D-56+188, sufficient amount of β-glucosidase (27 CBU/g cellobiose of

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Novozyme 188) was supplemented.

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The profiles of CO2 accumulation during the cellobiose fermentation experiments with

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low initial cell concentration (0.35 g/L) are presented in Fig 2A. While the glucose-fermenting

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strain (D-56) could not produce gas at all, the cellobiose-fermenting strains (D-BTw and D-

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BTm) and the glucose-fermenting strain with β-glucosidase (D-56+188) produced gas

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continuously during the fermentation (0 to 48 h). Among the two cellobiose-fermenting strains,

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the engineered strain with the F213L mutant transporter (D-BTm) produced gas faster than the

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engineered strain with the wild type CDT-1 transporter (D-BTw). This difference in cellobiose

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fermentation rate by two cellobiose-fermenting strains might be due to the different specific

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growth rate observed in Fig 1. The glucose-fermenting strain with β-glucosidase (D-56+188)

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showed the fastest gas production rate among all tested strains. The gas production by the D-

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56+188 strain ended after around 20 h of the fermentation, perhaps due to sufficient amount of β-

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glucosidase supplied to degrade cellobiose to glucose. At the end of fermentation (48 h), the

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remaining sugars and ethanol were analyzed by HPLC. As expected, the D-56 strain could not

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consume cellobiose at all. The cellobiose-fermenting strain with the F213L mutant transporter

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(D-BTm) produced a similar amount (36.2 g/L) of ethanol as compared to the cellobiose-

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fermenting strain with wild type transporter (D-BTw) (35.4 g/L) and the glucose-fermenting

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strain with β-glucosidase (D-56+188) (36.5 g/L, Table 2).

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Fig 2B shows the profiles of CO2 accumulation during the cellobiose fermentation experiments with a higher cell concentration (10.5 g/L). Interestingly, both of the cellobiose-

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fermenting strains (D-BTw and D-BTm) showed faster gas production rates during the

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fermentation than the glucose-fermenting strain with β-glucosidase (D-56+188). In addition, gas

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production rate by D-56+188 did not increase substantially in spite of a higher inoculum, which

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is consistent with the results from the previous study (Lee et al., 2013) where the ethanol

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production rate in SSF of cellulose with a glucose-fermenting strain did not change after

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increasing the level of inoculum. The engineered strain with the mutant F213L transporter (D-

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BTm) exhibited faster gas production than the engineered strain with the wild type CDT-1

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transporter (D-BTw). This might be due to a higher specific growth rate of the D-BTm strain on

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cellobiose than that of the D-BTw strain. As a result, the D-BTm strain produced more ethanol

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(38.1 g/L) than other strains (D-BTw and D-56+188) (Table 2).

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3.3. SSF of washed cellulosic hydrolysate (pretreated corn stover) by cellobiose-fermenting S.

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cerevisiae

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In the fermentation of 8.5% cellobiose in Fig 2B, the cellobiose-fermenting strain with

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the wild type CDT-1 (D-BTw) showed faster gas production rate than the glucose-fermenting

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strain with β-glucosidase (D-56+188), indicating D-BTw produced ethanol faster than D-56+188

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when the fermentation was begun with a higher cell concentration. However, the previous study

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showed that D-BTw could not perform better ethanol production than D-56+188 even with

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higher inoculums in SSF of cellulose (Lee et al. 2013). Probably, cellobiose utilization by the

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engineered yeast strains in SSF might be affected by several factors, such as inefficient

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formation of cellobiose from cellulose, slower cellodextrin utilization by the engineered cells

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compared with cellobiose, and reduced cellobiose accessibility by the cells because of unknown

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inhibitory compounds.

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Hence, we investigated whether faster ethanol production can be obtained in SSF of cellulose by the further engineered cellobiose-fermenting strain (D-BTm) as compared with the

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glucose-fermenting strain with β-glucosidase (D-56+188). Ethanol production by the glucose-

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fermenting strain with β-glucosidase (D-56+188), and the cellobiose-fermenting strains (D-BTw

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and D-BTm) was compared during SSF of cellulose under anoxic conditions (performed by the

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continuous gas measuring system allowing only release of CO2 without air intake). In order to

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avoid limitations in enzymatic hydrolysis of cellulose and inhibitory effects of fermentation

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inhibitors on cell growth, pretreated and washed hydrolysate (washed PCS) was used as a

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substrate, and an excessive amount of cellulase mixture (50 FPU/g PCS) was loaded. For

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extracellular degradation of cellobiose to glucose (SSF by D-56+188), the same level of β-

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glucosidase as the 8.5% cellobiose fermentation (27 CBU/g PCS) was supplemented.

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The profiles of CO2 accumulation in SSF of washed PCS by the glucose and cellobiosefermenting yeast strains are illustrated in Fig 3A. Compared with SSF by the glucose-fermenting

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strains with β-glucosidase (D-56+188), the cellobiose-fermenting strains (D-BTw and D-BTm)

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showed faster gas production during the initial period (0-5 h) of SSF. After 5 h of the SSF,

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however, gas production by D-56+188 became faster than D-BTw and this pattern was

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maintained until the end (48 h) of SSF, which is in good accordance with the previous results

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from SSF of Avicel PH-101 with D-BTw (Lee et al. 2013). However, the D-BTm strain

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expressing the mutant F213L transporter continued faster gas production than D-56+188 from

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the beginning to the end of SSF. At the end of SSF (48 h), the concentrations of the remaining

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sugars and ethanol were analyzed and summarized in Table 3. The D-BTm strain produced the

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highest concentration of ethanol (22.0 g/L) but the D-BTw strain could not produce more ethanol

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than D-56+188 (20.5 g/L). In order to verify whether measurement of CO2 accumulation is

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consistent to direct measurement of ethanol in SSF, micro-aerobic SSF of washed PCS under the

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same conditions was also performed in the flask equipped with an air-lock device for minimizing

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air intake while releasing CO2. As expected, almost the same patterns of ethanol production (D-

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BTm > D-56+188 > D-BTw) were observed in the micro-aerobic SSF experiments, which

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suggests that monitoring cumulative pressure of CO2 is reliable to estimate the profile of ethanol

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production during SSF (Fig S4).

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In the SSF in Fig 2A, excessive amounts of cellulases and β-glucosidase were added in

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order to avoid limitation of cellulose hydrolysis. However, appropriate levels of cellulolytic

297

enzymes for hydrolysis of cellulose in SSF (Fox et al., 2012; Sun & Cheng, 2002; van Zyl et al.,

298

2011) are much smaller than the levels of the enzymes used in this study, which indicates that 14

Page 15 of 37

reduced (or optimum) levels of cellulolytic enzymes should be loaded in the SSF experiments. In

300

addition, it was observed that the cellobiose-fermenting strains can utilize cellobiose efficiently

301

even at very low level of cellobiose in Fig 1. Consequently, ethanol production by the cellobiose-

302

fermenting strains and the glucose-fermenting strain with β-glucosidase was compared in SSF

303

with 8.5-times reduced amounts of cellulolytic enzymes (5.9 FPU/g PCS of cellulases and 3.2

304

CBU/g PCS of β-glucosidase).

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The profiles of CO2 accumulation in SSF with lower loading of cellulase mixture by the glucose and cellobiose-fermenting yeast strains are illustrated in Fig 3B. Interestingly, both of D-

307

BTw and D-BTm showed faster gas production than D-56+188 until the end (72 h) of the SSF.

308

Similar to the previous SSF with higher loading of cellulase mixture, D-BTm exhibited the best

309

ethanol production performance. However, the yield of ethanol from cellulose was reduced to

310

65% of the previous SSF (Table 3), which might be due to the slow cellulose hydrolysis caused

311

by reduced amounts of cellulolytic enzymes. Nonetheless, the two cellobiose fermenting yeast

312

strains showed much faster ethanol production than the glucose-fermenting strain (D-56+188).

313

These results suggest that limitation of cellobiose formation for D-BTw and D-BTm (equal to the

314

limitation of glucose formation for D-56+188) can maximize the effect of the intracellular

315

cellobiose utilization of the cellobiose-fermenting strains because the cellobiose-fermenting

316

strains can eliminate cellobiose efficiently under very low cellobiose condition as observed in

317

Fig 1. The concentrations of the remaining sugars and ethanol at the end of the SSF were

318

summarized in Table 3.

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319 320

3.4. SSF of pure cellulose by cellobiose-fermenting S. cerevisiae 15

Page 16 of 37

321

SSF of washed PCS with loading of lower amounts of cellulase mixture suggested that beneficial effects of cellobiose utilization by the cellobiose-fermenting strains on cellulose

323

hydrolysis can be maximized when the amount of cellulase mixture is decreased. In order to

324

verify that effects of intracellular cellobiose utilization of the engineered strains could be

325

facilitated regardless of substrate types, SSF of pure cellulose (Avicel PH-101) was also

326

performed under the similar conditions. Initial concentration of cellulose was adjusted to 13%

327

and 10 FPU of cellulase mixture and 5.4 CBU of β-glucosidase were loaded to hydrolyze 1 g of

328

cellulose. In addition, SSF of Avicel PH-101 was maintained for 192 h (2.7-times longer than the

329

previous SSF of PCS) because SSF of pure cellulose takes longer time than SSF of pretreated

330

cellulose (phosphoric acid pretreated Avicel PH-101).

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The profiles of CO2 accumulation in SSF of Avicel PH-101 are illustrated in Fig 4. One interesting observation is that the glucose-fermenting strain with β-glucosidase (D-56+188)

333

began to produce more gas than the original cellobiose-fermenting strain with wild type

334

transporter (D-BTw) after 120 h of SSF. These gas production patterns were consistent with the

335

results from the previous SSF (Lee et al. 2013). In contrast, the superior cellobiose-fermenting

336

strain with mutant transporter (D-BTm) showed the faster gas production than the glucose-

337

fermenting strain with β-glucosidase (D-56+188). At the end of SSF (192 h), the concentrations

338

of ethanol and residual sugars were analyzed (Table 3). D-BTm produced the highest amount of

339

ethanol (37.3 g/L) and D-56+188 produced the second highest amount of ethanol (33.2 g/L).

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340 341

4. Discussion

16

Page 17 of 37

342

It was supposed that the cellobiose-fermenting S. cerevisiae with high KS for cellobiose might show inefficient uptake and incomplete (or slow) utilization of cellobiose unless cellobiose

344

concentration is maintained at higher value than KS. Additionally, if the cellobiose-fermenting

345

yeast exhibits higher KS than the Michaelis-Menten constant (KM, binding affinity of enzyme to

346

substrate) of exogenous β-glucosidase, the replacement of the traditional SSF (wild type S.

347

cerevisiae with extracellular β-glucosidase) with the alternative SSF (engineered yeast without

348

extracellular β-glucosidase) would not be feasible as described in the previous reports (Fox et al.,

349

2012; Jin & Cate, 2012). Considering that the traditional SSF process employs β-glucosidase

350

(such as Novozyme 188 from Aspergillus niger) exhibiting substantially low KM for cellobiose

351

(0.35 to 1.66 mM ≈ 0.12 to 0.57 g cellobiose/L) (Chauve et al., 2010; Fox et al., 2012; Jin & Cate,

352

2012; Teugjas & Valjamae, 2013), determination of KS of the cellobiose-fermenting yeast for

353

cellobiose was done to evaluate efficiency of the alternative SSF.

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From the results shown in Fig 1, we found that the growth kinetics of the cellobiose-

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fermenting strains were proportional to the transporting kinetics of the cellobiose transporters,

356

CDT-1 and CDT-1 (F213L); CDT-1 showed lower KM and VMAX than CDT-1 (F213L) when

357

transport kinetics were determined with isotope-labeled cellobiose (2.66 pmol/s of VMAX and

358

0.1888 mM of KM in CDT-1 (F213L) vs. 0.60 pmol/s and 0.0076 mM in CDT-1) (Ha et al.,

359

2013a). The results also suggest that the cellobiose-fermenting strains can reach the maximum

360

growth rate even with lower concentrations of cellobiose (similar situation to SSF of cellulose)

361

as compared with the glucose-fermenting strain because the cellobiose-fermenting strains (D-

362

BTm and D-BTw) showed similar specific growth rates to the glucose-fermenting strain (D-56)

363

when the initial sugar concentration was 0.1 g/L. In addition, Fig 1 suggests that the cellobiose-

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Page 18 of 37

fermenting strains can bind with cellobiose more efficiently than β-glucosidase at low level of

365

cellobiose because the KS values of the cellobiose-fermenting strains were 2 to 3-times lower

366

than KM of β-glucosidase (0.06 g/L in D-BTm and 0.04 g/L in D-BTw vs. 0.12 g/L in β-

367

glucosidase, the lowest value described in the previous reports) (Chauve et al., 2010; Jin & Cate,

368

2012; Teugjas & Valjamae, 2013).

cr

In the cellobiose fermentation with low level of inoculum, the cellobiose fermenting

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364

strains (D-BTw and D-BTm) showed the linear increase of gas production after longer lag time

371

(initial 15-16 hours), while the glucose-fermenting strain with β-glucosidase (D-56+188) strain

372

showed the linear increase of gas production after shorter lag time (initial 6 hours). Low

373

concentrations of the engineered strains (0.35 g/L) contained insufficient GH1-1 (intracellular β-

374

glucosidase) activity [1.2-1.3 CBU in 20 ml medium, calculated from the results of the previous

375

study (175-185 CBU/g cell) (Lee et al. 2013)] compared with the extracellular β-glucosidase

376

supplemented strain (D-56+188, 45.9 CBU in 20 mL medium), which may explain why the

377

engineered strains could not produce ethanol as fast as the parental strain with extracellular β-

378

glucosidase under lower inoculums. However, the cellobiose-fermenting strains with higher

379

inoculums showed faster ethanol production than the glucose-fermenting strain with β-

380

glucosidase, which is consistent to the results from the previous SSF study (Lee et al. 2013).

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In the SSF of pretreated PCS, D-BTw could not show better ethanol production than D-

382

56+188, even though pretreated substrate and considerably high amount of cellulases (50 FPU/g

383

Celluclast 1.5L) were used. Considering that cellodextrins are often accumulated by trans-

384

glycosylation activity of GH1-1 and they are re-utilized at a much slower rate than cellobiose by

385

D-BTw during the cellobiose fermentation (Ha et al., 2013a; Ha et al., 2011a; Kim et al., 2014), 18

Page 19 of 37

cellodextrins released from cellulose hydrolysis might be the limiting factors for cellobiose

387

utilization by the D-BTw strain during SSF. In contrast to the D-BTw strain, the D-BTm strain

388

exhibited 2-3 times faster growth rates for several cellodextrins such as cellotriose and

389

cellotetraose than the D-BTw strain when cellodextrins were used as the sole carbon source (Ha

390

et al., 2013a). Therefore, cellodextrins might be more rapidly eliminated by the D-BTm strain

391

than the D-BTw strain during SSF, which may explain why the fastest gas production was

392

observed in the SSF by the D-BTm strain among the four tested strains.

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386

Optimum levels of cellulolytic enzymes for economically feasible SSF processes are

394

reported to be about 2.5% of the total amount of cellulose (25 mg of enzymes/g of cellulose)

395

(Fox et al., 2012; Sun & Cheng, 2002; van Zyl et al., 2011). The amount of β-glucosidase is

396

known to represent 10% of the total cellulolytic enzymes (2.5 mg of β-glucosidase/25 mg of

397

cellulase mixture) (Banerjee et al., 2010; Fox et al., 2012). Previously, the specific activities of

398

Celluclast 1.5L and Novozyme 188 were determined to be 0.40 FPU/mg protein and 1.13

399

CBU/mg protein, respectively (Lee et al., 2013), which indicates that 125 mg of cellulase

400

mixture and 24 mg of β-glucosidase were used to degrade 1 g of PCS in the present study.

401

Considering that the glucan content of PCS from NREL is estimated to 58.9% (McMillan et al.,

402

2011), the actual amounts of cellulase mixture and β-glucosidase loaded to hydrolyze 1 g of

403

cellulose were 212 mg and 40 mg, respectively. These amounts of cellulase mixture and β-

404

glucosidase are corresponding to 8.5-times higher and 16-times higher than the amounts of

405

cellulase mixture and β-glucosidase employed in most of SSF studies (Fox et al., 2012; Sun &

406

Cheng, 2002; van Zyl et al., 2011). Considering that intracellular cellobiose utilization by the

407

engineered strains exhibited considerably high growth rates even when cellobiose concentrations

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19

Page 20 of 37

are extremely low (Ks = 0.04-0.06 g cellobiose/L), ethanol production by the cellobiose-

409

fermenting strains and the glucose-fermenting strain with β-glucosidase was compared in SSF

410

with reduced loading of cellulase mixture (25 mg cellulases/g cellulose = 10 FPU/g cellulose =

411

5.9 FPU/g PCS, 8.5-times lower than the previous SSF). The amount of β-glucosidase for SSF

412

by D-56+188 was also decreased to 4.8 mg/g cellulose (=5.4 CBU/g cellulose = 3.2 CBU/g PCS,

413

also 8.5-times lower than the previous SSF). As observed in Fig 3B, both D-BTw and D-BTm

414

showed faster gas production than D-56+188. These results can suggest that the effects of

415

intracellular cellobiose utilization in the engineered strain are maximized when cellobiose

416

formation is limited in the SSF. It might be because the cellobiose-fermenting strains could

417

eliminate cellobiose efficiently under low cellobiose condition as observed in Fig 1.

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Compared with the SSF of pretreated PCS, similar ethanol production pattern was not observed in SSF of Avicel (un-pretreated cellulose, slower hydrolysis rate than pretreated

420

substrate) with the D-BTw strain even when lower amount of cellulase mixture was loaded. Gas

421

production patterns in D-BTw and D-56+188 were consistent with the simulation patterns of the

422

previous SSF study (Fox et al., 2012). Probably, available energy source for cellobiose

423

transportation in the D-BTw strain might be depleted at the late period of SSF because CDT-1

424

expenses ATP to transport cellobiose into the cell (Kim et al. 2014), which may explain why gas

425

production of the D-BTw strain slowed down than D-56+188 after 120 h of SSF. However, the

426

D-BTm strain showed better gas production patterns than D-56+188 regardless of substrate types.

427

Even though the D-BTm strain also expenses ATP for cellobiose transportation, better

428

transportation activity on several cello-oligosaccharides might lead to continue gas production

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20

Page 21 of 37

429

rate until the end of SSF, which may be the reason that the D-BTm strain exhibited better gas

430

production than D-56+188 as shown in Fig 4.

432

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431

5. Conclusion

Optimal yeast strains for producing cellulosic ethanol have been developed to produce

434

ethanol efficiently from SSF of cellulose by introduction of intracellular cellobiose metabolic

435

pathway. In this study, the engineered S. cerevisiae capable of fermenting intracellular cellobiose

436

can facilitate efficient production of ethanol from SSF compared to the traditional SSF with the

437

parental S. cerevisiae capable of fermenting glucose only and additional supplementation of β-

438

glucosidase. Determination of growth kinetic parameters of the engineered S. cerevisiae strains

439

revealed that the engineered strains (cellobiose-fermenting strains) have higher affinity to

440

cellobiose than affinity of the parental strain (glucose-fermenting strain) to glucose. It also

441

revealed that the cellobiose-fermenting strain expressing the mutant cellobiose transporter

442

exhibits faster growth rate than the original strain expressing the wild type cellobiose transporter.

443

With the aid of enhanced growth kinetic properties, the cellobiose-fermenting strain with the

444

mutant transporter showed better ethanol production performance than the glucose-fermenting

445

strain with extracellular β-glucosidase in both cellobiose fermentation and SSF of cellulose.

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Our experimental results suggest that improvement of ethanol production in SSF can be

447

achieved by the modification of intracellular cellobiose metabolic pathway including engineering

448

of cellobiose transporters.

449 450

Acknowledgment 21

Page 22 of 37

451

This research was supported by funding from the Energy Biosciences Institute.

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References

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Bae, Y.H., Kang, K.H., Jin, Y.S., Seo, J.H., 2014. Molecular cloning and expression of fungal cellobiose transporters and β-glucosidases conferring efficient cellobiose fermentation in

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Saccharomyces cerevisiae. J. Biotechnol. 169, 34-41.

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Banerjee, G., Car, S., Scott‐Craig, J.S., Borrusch, M.S., Aslam, N., Walton, J.D., 2010. Synthetic

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enzyme mixtures for biomass deconstruction: production and optimization of a core set.

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Biotechnol. Bioeng. 106, 707-720.

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Chauve, M., Mathis, H., Huc, D., Casanave, D., Monot, F., Ferreira, N.L., 2010. Comparative kinetic analysis of two fungal β-glucosidases. Biotechnol. Biofuels 3, 3.

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Dos Reis, T.F., de Lima, P.B., Parachin, N.S., Mingossi, F.B., de Castro Oliveira, J.V., Ries,

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L.N., Goldman, G.H., 2016. Identification and characterization of putative xylose and

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cellobiose transporters in Aspergillus nidulans. Biotechnol. Biofuels. 9, 204.

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Fox, J.M., Levine, S.E., Blanch, H.W., Clark, D.S., 2012. An evaluation of cellulose

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saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of

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cellobiose and xylose utilization. Biotechnol. J. 7, 361-373.

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Galazka, J.M., Tian, C., Beeson, W.T., Martinez, B., Glass, N.L., Cate, J.H.D., 2010.

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Cellodextrin transport in yeast for improved biofuel production. Science 330, 84-86.

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Gray, K.A., Zhao, L., Emptage, M., 2006. Bioethanol. Curr. Opin. Chem. Biol. 10, 141-146.

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Ha, S.J., Galazka, J.M., Oh, E.J., Kordić, V., Kim, H., Jin, Y.S., Cate, J.H.D., 2013a. Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing

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cellobiose phosphorylase and mutant cellodextrin transporters. Metab. Eng. 15, 134-143.

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Ha, S.J., Kim, H., Lin, Y., Jang, M.U., Galazka, J.M., Kim, T.J., Cate, J.H.D., Jin, Y.S., 2013b.

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Single amino acid substitutions in HXT2. 4 from Scheffersomyces stipitis lead to improved

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cellobiose fermentation by engineered Saccharomyces cerevisiae. Appl. Environ. Microbiol.

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79, 1500-1507.

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Ha, S.J., Galazka, J.M., Rin Kim, S., Choi, J.H., Yang, X., Seo, J.H., Louise Glass, N., Cate,

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cellobiose and xylose fermentation. Proc. Natl. Acad. Sci. U.S.A. 108, 504-509.

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Ha, S.J., Wei, Q., Kim, S.R., Galazka, J.M., Cate, J.H.D., Jin, Y.S., 2011b. Cofermentation of

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cellobiose and galactose by an engineered Saccharomyces cerevisiae strain. Appl. Environ.

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Microbiol. 77, 5822-5825.

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Hector, R.E., Dien, B.S., Cotta, M.A., Qureshi, N., 2011. Engineering of industrial

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Saccharomyces cerevisiae strains for xylose fermentation and comparison for switchgrass

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conversion. J. Ind. Microbiol. Biotechnol. 38, 1193-1202.

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Hosaka, K., Nikawa, J., Kodaki, T., Yamashita, S., 1992. A dominant mutation that alters the

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regulation of INO1 expression in Saccharomyces cerevisiae. J. Biochem. 111, 352-358.

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Jin, Y.S., Cate, J.H.D., 2012. Model‐guided strain improvement: Simultaneous hydrolysis and

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co‐fermentation of cellulosic sugars. Biotechnol. J. 7, 328-329.

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Kim, H., Lee, W.H., Galazka, J.M., Cate, J.H.D, Jin, Y.S., 2014. Analysis of cellodextrin transporters from Neurospora crass in Saccharomyces cerevisiae for cellobiose fermentation.

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Appl. Microbiol. Biotechnol. 98, 1087-1094.

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Lee, W.H., Nan, H., Kim, H.J., Jin, Y.S. 2013., Simultaneous saccharification and fermentation

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by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase.

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Martins, L.F., Kolling, D., Camassola, M., Dillon, A.J.P., Ramos, L.P. 2008., Comparison of

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various cellulosic substrates. Bioresour. Technol. 99, 1417-1424.

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McMillan, J.D., Jennings, E.W., Mohagheghi, A., Zuccarello, M., 2011. Comparative

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performance of precommercial cellulases hydrolyzing pretreated corn stover. Biotechnol.

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Mussatto, S.I., Dragone, G., Guimarães, P.M.R., Silva, J.P.A., Carneiro, L.M., Roberto, I.C., Vicente, A., Domingues, L., Teixeira, J.A., 2010. Technological trends, global market, and

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challenges of bio-ethanol production. Biotechnol. Adv. 28, 817-830.

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Olofsson, K., Bertilsson, M., Lidén, G., 2008. A short review on SSF - an interesting process

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enzyme recycling by alkaline washing. Enzyme. Microb. Technol. 79-80, 70-77.

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Sadie, C.J., Rose, S.H., den Haan, R., van Zyl, W.H., 2011. Co-expression of a cellobiose

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phosphorylase and lactose permease enables intracellular cellobiose utilisation by

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Saccharomyces cerevisiae. Appl. Micribiol. Biotechnol. 90, 1373-1380.

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Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review.

Teugjas, H., Valjamae, P., 2013. Selecting beta-glucosidases to support cellulases in cellulose

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saccharification. Biotechnol. Biofuels 6, 105. van Zyl, J.M., van Rensburg, E., van Zyl, W.H., Harms, T.M., Lynd, L.R., 2011. A kinetic model

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for simultaneous saccharification and fermentation of avicel with Saccharomyces cerevisiae.

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Wyman, C.E., 2007. What is (and is not) vital to advancing cellulosic ethanol. Trends Biotechnol. 25, 153-157.

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Xu, Q., Singh, A., Himmel, M.E., 2009. Perspectives and new directions for the production of

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bioethanol using consolidated bioprocessing of lignocellulose. Curr. Opin. Biotechnol. 20,

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364-371.

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Zhang, W., Kou, Y., Xu, J., Cao, Y., Zhao, G., Shao, J., Wang, H., Wang, Z., Bao, X., Chen, G.,

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Liu, W., 2013. Two major facilitator superfamily sugar transporters from Trichoderma reesei

527

and their roles in induction of cellulase biosynthesis. J. Biol. Chem. 288, 32861-32872.

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Figure legends

529

Fig 1 Comparison of specific growth rates of the parental S. cerevisiae strain (D-56) and the

530

engineered strains (D-BTw and D-BTm) in minimal (SC) medium with different initial sugar

531

concentrations (A). D-56, D-BTw and D-BTm grown on cellobiose were designated as light grey

532

bar, white bar and black bar, respectively. D-56 grown on glucose was designated as dark grey

533

bar.

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528

534

Fig 2 Profiles of gas production in the anoxic 8.5% cellobiose fermentation with 0.35 g/L of

536

initial cell concentration (A) and 10.5 g/L of initial cell concentration (B). Cellobiose

537

fermentation was carried out at 30°C and 100 rpm. Yeast strains used in cellobiose fermentation

538

are as follows: D-56 (open circle), D-56+188 (closed circle), D-BTw (open triangle), D-BTm

539

(closed triangle).

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Fig 3 Profiles of gas production in the anoxic SSF of 10% washed PCS with excessive amount of

542

cellulase mixture (A) and SSF with limited amount of cellulase mixture (B). SSF was carried out

543

with 10.5 g/L of initial cell concentration at 30°C and 100 rpm. Celluclast 1.5L (50 FPU/g PCS

544

or 5.9 FPU/g PCS) was used for saccharification of PCS and Novozyme 188 (27 CBU/g PCS or

545

3.2 CBU/g PCS) was used for degradation of cellobiose to glucose. Yeast strains used in SSF are

546

as follows: D-56 (open circle), D-56+188(2.7 CBU, closed circle), D-BTw (open triangle), D-

547

BTm (closed triangle).

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548

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Page 29 of 37

Fig 4 Profiles of gas production in the anoxic SSF of 13% cellulose (Avicel PH-101) with

550

limited amount of cellulase mixture. SSF was carried out with 10.5 g/L of initial cell

551

concentration at 30°C and 100 rpm. Celluclast 1.5L (10 FPU/g cellulose) was used for

552

saccharification of Avicel PH-101 and Novozyme 188 (5.4 CBU/g cellulose) was used for

553

degradation of cellobiose to glucose. Yeast strains used in SSF are as follows: D-56 (open circle),

554

D-56+188 (closed circle), D-BTw (open triangle), D-BTm (closed triangle).

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Page 30 of 37

Table 1 The list of plasmids and S. cerevisiae strains used in this study Plasmids and strains

Sources and references

pRS425PGK

LEU2, PPGK-MCS-TCYC, 2µ origin, Ampr

(Galazka et al., 2010)

pRS426PGK

URA3, PPGK-MCS-TCYC, 2µ origin, Ampr

(Galazka et al., 2010)

pRS425-gh1-1

LEU2, PPGK-gh1-1-TCYC, 2µ origin, Ampr

pRS426-cdt1

URA3, PPGK-cdt1-TCYC, 2µ origin, Ampr

pRS426cdt1(F213L)

URA3, PPGK-cdt1 (F213L)-TCYC, 2µ origin, Ampr (Ha et al., 2013a)

558

(Galazka et al., 2010)

MATα, leu2, his3, ura3 and can1

(Hosaka et al., 1992)

D-56

D452-2/pRS425PGK /pRS426PGK

(Ha et al., 2011b)

D-56+188

D-56 with extracellular β-glucosidase

(Lee et al., 2013)

D-BTw

D452-2/ pRS425-gh1-1/pRS426-cdt1

(Ha et al., 2011b)

D-BTm

D452-2/ pRS425-gh1-1/pRS426-cdt1 (F213L)

(Ha et al., 2013a)

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D452-2

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an

Strains

556

(Galazka et al., 2010)

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Plasmids

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Relevant features

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Page 31 of 37

Table 2 Summarized results from cellobiose fermentation with engineered S. cerevisiae strains

Strains

Final ethanol (g/L)

D-56

0.0

D-56+188

36.5±0.35

D-BTw

35.4±0.47

0.43

D-BTm

36.2±0.43

0.44

0.0

0.00

37.8±0.13

0.44

D-BTw

37.7±0.95

0.43

D-BTm

38.1±0.19

0.44

0.35

D-56

an

D-56+188 10.5

M

8.5% cellobiose, anoxic 559

0.44

te Ac ce p

561

0.00

d

560

Ethanol yield from cellobiose (g/g)

ip t

8.5% cellobiose, anoxic

Initial cell concentration (g/L)

cr

Culture conditions

us

558

31

Page 32 of 37

562

Table 3 Summarized results from SSF of cellulose (PCS and Avicel PH-101) with engineered S.

563

cerevisiae strains

D-56

18.2±0.67

D-56+188

20.5±0.72

0.35

D-BTw

20.5±0.88

0.35

22.0±0.16

cr

ip t

Ethanol yield* from cellulose (g/g)

0.31

0.37

D-56

us

Limited loading [Celluclast 1.5L (10 FPU/g Avicel), Novozyme 188 (5.4 CBU/g Avicel)]

Ac ce p

13% Avicel PH-101, anoxic

6.9±0.11

0.12

D-56+188

11.6±0.35

0.20

D-BTw

12.3±0.39

0.21

D-BTm

14.2±0.41

0.24

D-56

22.8±0.61

0.19

D-56+188

33.2±0.62

0.28

D-BTw

30.3±0.61

0.26

D-BTm

37.3±0.92

0.32

D-BTm

an

Limited loading [Celluclast 1.5L (5.9 FPU/g PCS), Novozyme 188 (3.2 CBU/g PCS)]

Final ethanol (g/L)

M

10% washed PCS, anoxic

Sufficient loading [Celluclast 1.5L (50 FPU/g PCS), Novozyme 188 (27 CBU/g PCS)]

Strains

d

10% washed PCS, anoxic

Amount of enzymes loaded in SSF

te

Culture conditions

564

* Because cellulose content of PCS is known to be 58.9% (McMillan et al., 2011), overall yield

565

of ethanol from 10% PCS was calculated from dividing concentration of ethanol with estimated

566

concentration of cellulose (58.9 g/L). Because water content of Avicel was confirmed to be 9%,

567

overall yield of ethanol from 13% Avicel was calculated from dividing concentration of ethanol

568

with actual concentration of cellulose (118.3 g/L, 91% of 130 g/L cellulose).

32

Page 33 of 37

ip t cr us an

569

te

d

Fig 1

Ac ce p

571

M

570

33

Page 34 of 37

M

an

us

cr

ip t

(A)

572

Ac ce p

te

d

(B)

573 574

Fig 2 34

Page 35 of 37

M

an

us

cr

ip t

(A)

575

Ac ce p

te

d

(B)

576 577

Fig 3 35

Page 36 of 37

ip t cr us an M te

580

Fig 4

Ac ce p

579

d

578

36

Page 37 of 37