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22

Table 1 Characteristics of yeast strains and plasmids used in this study

Yeast strains and

Relevant genotype

Source

BY4741

MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

Life Technologies

BY-BG-SSS

BY4741/pIBG-SSS [SED1P–SED1SP–A. aculeatus BGL1–

Inokuma et al. (2016)

plasmids

S. cerevisiae

SED1A–SAG1T, leu2Δ0 met15Δ0 ura3Δ0]

BY-BG-SSSD

BY4741/pIBG-SSSD [SED1P–SED1SP–A. aculeatus BGL1–

This study

SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0]

ccw12-BGSD

BY-BG-SSSD CCW12Δ [SED1P–SED1SP–A. aculeatus

This study

BGL1–SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0 ccw12Δ]

ccw14-BGSD

BY-BG-SSSD CCW14Δ [SED1P–SED1SP–A. aculeatus

This study

BGL1–SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0 ccw14Δ]

ccw12/ccw14-BGSD

BY-BG-SSSD CCW12ΔCCW14Δ [SED1P–SED1SP–A.

This study

aculeatus BGL1–SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0

ccw12Δ ccw14Δ]

BY-EG-SSSD

BY4741/pIEG-SSSD [SED1P–SED1SP– T. reesei EGII–

This study

SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0]

ccw12/ccw14-EGSD

BY-EG-SSSD CCW12ΔCCW14Δ [SED1P–SED1SP– T.

This study

reesei EGII–SED1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0

ccw12Δ ccw14Δ]

BY-BG-SSAD

BY4741/pIBG-SSAD [SED1P–SED1SP–A. aculeatus

This study

BGL1–SAG1A–DIT1T, leu2Δ0 met15Δ0 ura3Δ0]

ccw12/ccw14-BGAD

BY4741/pIBG-SSAD CCW12ΔCCW14Δ [SED1P–SED1SP–

This study

A. aculeatus BGL1–SAG1A–DIT1T, leu2Δ0 met15Δ0

ura3Δ0 ccw12Δ ccw14Δ]

Plasmids

pIBG-SSS

HIS3 SED1P–SED1SP–A. aculeatus BGL1–SED1A–SAG1T

Inokuma et al. (2016)

pIBG-SSSD

HIS3 SED1P–SED1SP–A. aculeatus BGL1–SED1A–DIT1T

This study

pIEG-SSS

HIS3 SED1P–SED1SP–T. reesei EGII–SED1A–SAG1T

Inokuma et al. (2016)

pIEG-SSSD

HIS3 SED1P–SED1SP–T. reesei EGII–SED1A–DIT1T

This study

pIBG13

HIS3 TDH3P–A. aculeatus BGL1–SAG1A–SAG1T

Katahira et al. (2006)

pIBG-SSAD

HIS3 SED1P–SED1SP–A. aculeatus BGL1–SAG1A–DIT1T

This study

pGK415

CEN-ARS LEU2 PGK1P–PGK1T

(Ishii et al. 2009)

Cas9_Base

K. marxianus ARS7, K. marxianus CEN D, kanMX

(Nambu-Nishida et

PDC1P–Cas9–TDH3T

al. 2017)

pCL-Cas9

CEN-ARS LEU2 TEF1P–SV40NLS–Cas9–SV40NLS–CYC1T

This study

pGK426

2μ ori URA3 PGK1P–PGK1T

(Ishii et al. 2009)

pSUP4t

2μ ori URA3 SUP4T

This study

p2gRNA-CCW12

2μ ori URA3 SNR52P–gRNA for CCW12–SUP4T

This study

p2gRNA- CCW14

2μ ori URA3 SNR52P–gRNA for CCW14–SUP4T

This study

23

p2gRNA-

2μ ori URA3 SNR52P–gRNA for CCW12–SUP4T SNR52P–

CCW12/CCW14

gRNA for CCW14–SUP4T

This study

A. aculeatus, Aspergillus aculeatus; T. reesei, Trichoderma reesei; K. marxianus, Kluyveromyces

marxianus; P, promoter; SP, secretion signal peptide sequence; A, anchoring region; T, terminator;

NLS, nuclear localization signal.

24

Figure captions

Fig. 1 Sequence alignments of CCW12 and CCW14 compared to their respective dsOligos. The

sense strands of the target sites are shown. The green sequences of the wild-type reference sequence

denote the protospacer adjacent motif (PAM) sites. The red sequences indicate stop codons replacing

the PAM sites. Sequences present in the gRNAs are underlined.

Fig. 2 Effects of CCW12 and/or CCW14 knockout on BGL-displaying yeast. (a) Time-course of

cell-surface BGL activities. (b) Cell growth of BGL-displaying yeasts. (c) Comparison of transcript

levels of BGL1-encoding genes after cultivation in YPD medium for 48 h. The relative transcript

level of each gene is shown as a fold-change in mRNA levels relative to the average level detected

in the control strain (BY-BG-SSSD). (d) Relative quantification of BGL1 in the cell walls by nanoUPLC-MSE. The amount of BGL1 was normalized to the dry cell weight of each strain. Data are

presented as the means ± standard deviation (n = 3).

Fig. 3 Electron micrographs of ultrathin sections of (a) BY-BG-SSSD, (b) ccw12-BGSD, (c) ccw14BGSD, and (d) ccw12/14-BGSD cells.

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