Data for simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae expressing heterologous pathways for xylose, arabinose, and galacturonic acid metabolism has been constructed by a Cas9-based genome editing technology [1]. The fermentation performance of the final strain (YE9) was tested under various substrate conditions, and the fermentation parameters were calculated. The dataset can be used for designing bioprocesses for pectin-rich biomass.

Specifications Table

Subject	Applied Microbiology and Biotechnology
Specific subject area	Yeast metabolic engineering
Type of data	Tables and Figures
How data were acquired	The fermentation data were obtained by HPLC (Agilent Technologies 1260 series).
Data format	Raw and Analysed
Parameters for data collection	Fermentation conditions at 30oC and 130 rpm.
Description of data collection	Time series analysis of fermentation samples.
Data source location	Institution: Kyungpook National University City/Town/Region: Daegu Country: Korea
Data accessibility	With the article
Related research article	Author’s name: Deokyeol Jeong, Suji Ye, Heeyoung Park, and Soo Rin Kim Title: Simultaneous fermentation of galacturonic acid and five-carbon sugars by engineered Saccharomyces cerevisiae Journal: Bioresource Technology https://doi.org/10.1016/j.biortech.2019.122259

Value of the Data •The dataset contains the construction strategy and fermentation data for the engineered strain simultaneously fermenting representative three carbon sources (xylose, arabinose, galacturonic acid) in pectin-rich biomass. •The fermentation data of the YE9 strain expressing the three pathways can be useful for process design utilizing pectin-rich biomass consisting mainly of galacturonic acid and arabinose. •Based on the fermentation data of the YE9 strain, feasible options for strain engineering can be broadened for industrial bioprocesses.

1. Data

This dataset contains 1) the construction of engineered Saccharomyces cerevisiae strain (YE9) capable of fermenting galacturonic acid, arabionse, and xylose, and 2) its fermentation data with different carbon sources (galacturonic acid, arabinose, xylose, galactose, glucose, and fructose) and their mixtures, all of which present in pectin-rich biomass. In Fig. 1, the fermentation patterns of the YE9 strain with natively fermentable sugars (glucose, fructose, and galactose) as a sole carbon source are presented. In Table 1, the fermentation profiles of the YE9 strain with xylose, arabinose, and galacturonic acid in comparison to its wild type strain (D452-2). In Fig. 2, the YE9 strain was tested for xylose and galacturonic acid consumption rates in a mixture of 40 g/L xylose and various galactornic acid concentrations. In Table 2, the fermentation parameters of the YE9 strain with a mixture of galacturonic acid and co-substrates.

Fig. 1.

Fermentation profiles of the YE9 strain in a complex medium containing (A) 40 g/L d-glucose, (B) 40 g/L d-fructose, and (C) 40 g/L d-galactose as the sole carbon sources. Fermentations were performed under oxygen-limited conditions (130 rpm) with a starting cell density of 25 g/L. All experiments were performed in biological triplicate, and the error bars indicate the standard deviations.

Table 1.

Fermentation profiles of the native S. cerevisiae strain (D452-2) and engineered strain (YE9) expressing heterologous pathways for metabolizing d-xylose, l-arabinose, and d-galacturonic acid (galUA).

Strain			Substrate	Substrate consumed (g/L)	Substrate consumption rate (g/L/h)	Products (g/L)		Parametersb)
						Glycerol	Ethanol	YGlycerol	YEthanol	PEthanol∗
D452-2			D-xylosea)	5.9 ± 0.2	0.19 ± 0.01	0.3 ± 0.0	n. d.	0.07 ± 0.02	n. d.	n. d.
			l-arabinose	1.3 ± 0.6	0.08 ± 0.03	n. d.	n. d.	n. d.	n. d.	n. d.
			galUA	< 0.0	< 0.00	n. d.	n. d.	n. d.	n. d.	n. d.
YE9			d-xylose	33.7 ± 0.5	1.41 ± 0.02	0.6 ± 0.1	11.3 ± 0.1	0.02 ± 0.00	0.34 ± 0.01	0.05 ± 0.00
			l-arabinose	30.2 ± 0.1	0.63 ± 0.07	n. d.	1.9 ± 0.1	n. d.	0.07 ± 0.00	<0.00
			galUA	6.7 ± 0.7	0.27 ± 0.01	0.3 ± 0.1	0.3 ± 0.0	0.04 ± 0.01	0.08 ± 0.02	< 0.00

^a)Fermentations were performed in a complex medium containing 40 g/L d-xylose, 40 g/L l-arabinose, or 20 g/L d-galacturonic acid under oxygen-limited conditions (130 rpm) with a starting cell density of 25 g/L. Substrate consumption rate was calculated for 24 h and the others were calculated for 72 h.

^b)Y_Glycerol, glycerol yield (g glycerol/g substrate); Y_Ethanol, ethanol yield (g ethanol/g substrate); P_Ethanol∗, specific ethanol productivity (g ethanol/g cell/h); n. d., not detected.

Fig. 2.

Effect of d-galacturonic acid on the rate of d-xylose consumption in the YE9 strain. Consumption rate of d-xylose (A) and d-galacturonic acid (B) was evaluated under 40 g/L D-xylose and different d-galacturonic acid concentrations (0–100 g/L). All experiments were performed in biological triplicate, and error bars indicate standard deviations and were not visible when smaller than the symbol size.

Table 2.

Fermentation profiles of mixed culture by engineered S. cerevisiae YE9 strain expressing heterologous pathways metabolizing d-xylose, l-arabinose, and d-galacturonic acid (galUA).

Mediuma)	Substrate consumed (g/L)		galUA consumption rate (g/L/h)	Products (g/L)		Parametersb)
	galUA	Sugars		Glycerol	Ethanol	YGlycerol	YEthanol	PGlycerol∗	PEthanol∗
galUA	6.7 ± 0.7	–	0.27 ± 0.01	0.3 ± 0.1	0.3 ± 0.0	0.04 ± 0.01	0.08 ± 0.02	< 0.00	< 0.00
galUA + Glucose	3.3 ± 0.2	36.7 ± 0.1	0.14 ± 0.01	2.4 ± 0.3	16.9 ± 0.2	0.06 ± 0.01	0.40 ± 0.01	0.06 ± 0.00	0.66 ± 0.01
galUA + Fructose	4.5 ± 0.3	36.1 ± 0.8	0.18 ± 0.02	2.9 ± 0.1	16.9 ± 0.5	0.07 ± 0.00	0.36 ± 0.01	< 0.00	0.65 ± 0.03
galUA + Galactose	4.6 ± 1.2	25.4 ± 7.3	0.17 ± 0.03	1.6 ± 0.7	2.4 ± 1.1	0.04 ± 0.01	0.05 ± 0.02	< 0.00	< 0.00
galUA+ Xylose	13.1 ± 0.4	33.3 ± 0.5	0.49 ± 0.02	4.5 ± 0.1	12.8 ± 0.3	0.08 ± 0.00	0.23 ± 0.01	0.01 ± 0.00	0.04 ± 0.00
galUA+ Arabinose	11.9 ± 0.7	28.4 ± 0.1	0.32 ± 0.03	4.2 ± 0.2	4.1 ± 0.5	0.11 ± 0.01	0.11 ± 0.02	< 0.00	< 0.00
galUA +Xylose (X) +Arabinose (A)	15.3 ± 0.6	33.7 ± 0.1 (X) 25.9 ± 4.4 (A)	0.49 ± 0.04	5.3 ± 0.6	16.5 ± 1.2	0.07 ± 0.00	0.22 ± 0.01	< 0.00	0.02 ± 0.00

^a)Fermentations were performed in a complex medium containing 20 g/L d-galacturonic acid (galUA) and 40 g/L sugar (d-glucose, d-fructose, d-galactose, d-xylose, l-arabinose, and mixture of d-xylose and l-arabinose) under oxygen-limited conditions (130 rpm) with a starting cell density of 25 g/L. d-galacturonic acid consumption rate was calculated for 24 h and the others were calculated for 72 h.

^b)Y_Glycerol, glycerol yield (g glycerol/g substrates); Y_Ethanol, ethanol yield (g ethanol/g substrates); P_Glycerol∗, specific glycerol productivity (g glycerol/g cell/h); P_Ethanol∗, specific ethanol productivity (g ethanol/g cell/h).

2. Experimental design, materials, and methods

2.1. Strain construction by Cas9-based genome editing

To construct the YE9 strain, four consecutive transformations were performed as summarized in Fig. 3 using strains listed in Table 3. Briefly, the strain construction includes three parts: 1) guide RNA (gRNA) plasmid construction, 2) donor DNA preparation, and 3) yeast transformation.

• 1)Guide RNA (gRNA) plasmid construction

Fig.3.

Construction of engineered S. cerevisiae YE9 strains expressing heterologous d-xylose, d-galacturonic acid, and l-arabinose pathways. (A) Strain construction using Cas9-based in vivo assembly and genome integration strategy. (B) Confirmation primers for correct assembly and integration by yeast colony PCR. The primer sequences are listed in Table S5.

Table 3.

Saccharomyces cerevisiae strains used for the construction of YE9.

Strains	Description/relevant genotypea	Ref.
D452-2	Wild type; Matα leu2 his3 ura3	[7]
DY02	Expressing the heterologous d-xylose pathway; D452-2 ald6::TDH3P-XYL1-TDH3T-PGK1P-XYL2-PGK1Tpho13::TEF1P-XYL3-TEF1T
YE3	DY02 int#4::CCW12P-gaaA-CCW12T
YE4	DY02 int#4::PGK1P-lgd1-PGK1T
YE5	DY02 int#4::TDH3P-gaaC-TDH3T
YE6	Expressing the heterologous D-xylose and d-galacturonic acid pathway; DY02 int#4::CCW12P-gaaA-CCW12T-PGK1P-lgd1-PGK1T-TDH3P-gaaC-TDH3T
YE6 YPR1	YE6 CCW12P-YPR1
YE6 gaaD	YE6 int#6::CCW12P-gaaD-CCW12T
YE01	Expressing the heterologous d-xylose, and l-arabinose pathway; D452-2 ald6::TDH3P-XYL1-TDH3T-PGK1P-XYL2-PGK1Tint#1::TEF1P-XYL3-TEF1Tsor1::FBA1P-LAD1-FBA1T-PGK1P-ALX1-CYC1T	[8]
YE9	Expressing the heterologous d-xylose, l-arabinose, and d-galacturonic acid pathway;YE6 int#7::FBA1P-lad1-FBA1T-PGK1P-alx1-CYC1T

^aXYL1, XYL2, and XYL3 are derived from Pichia stipitis; gaaA, gaaC, and gaaD are derived from Aspergillus niger; lgd1 and lad1 are derived from Trichoderma reesei; alx1 is derived from Ambrosiozyma monospora.

gRNA sequences are designed to be target cut site-specific and 20-bp long, as listed in Table 4. The plasmids expressing each gRNA sequence were constructed by the fast cloning method [2], which is a PCR-based protocol for plasmid mutagenesis. To construct the pRS42H-ALD6.1 plasmid, for example, the pRS42H-GND1.1 plasmid (a template plasmid) [3] was amplified with the primers Kim044/Kim045 (Table 5). The PCR products were treated with DpnI and used to transform E. coli TOP10 (Invitrogen, Carlsbad, CA, USA). The transformants were selected on an LBA (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 100 μg/mL ampicillin) agar plate. The gRNA sequence of the resulting plasmid was confirmed by Sanger sequencing using a universal primer for the T3 promoter. All other gRNA plasmids were constructed using the same procedure but different primers, as listed in Table 5.

• 2)Donor DNA preparation

Table 4.

Guide RNA (gRNA) plasmids.

gRNA	Target cut site	gRNA and PAM sequences (5’-)	Plasmid name
ALD6.1	ALD6	GTCAAGATCACACTTCCAAA TGG	pRS42H-ALD6.1
PHO13.1	PHO13	TCCCTTATCTATTAACTTTC CGG	pRS42H-PHO13.1
YPR1.1	YPR1	CATGGTAGATTATTATCTGT GGG	pRS42H-YPR1.1
INT#4	Intergenic region upstream ASF1	CTCTCGAAGTGGTCACGTGC GGG	pRS42H-INT#4
INT#6	Intergenic region upstream ATG33	TTGTCACAGTGTCACATCAG CGG	pRS42H-INT#6
INT#7	Intergenic region downstream YGR190C	GATACTTATCATTAAGAAAA TGG	pRS42H-INT#7

Table 5.

Primers used for construction of guide RNA plasmids.

Plasmid name	Primers	Sequences (5’-)
pRS42H-ALD6.1	Kim044	AAGATCACACTTCCAAAGTTTTAGAGCTAGAAATAGCAAG
	Kim045	TTGGAAGTGTGATCTTGACGATCATTTATCTTTCACTGCG
pRS42H-PHO13.1	Kim624	CTTATCTATTAACTTTCGTTTTAGAGCTAGAAATAGCAAG
	Kim625	AAAGTTAATAGATAAGGGAGATCATTTATCTTTCACTGCG
pRS42H-YPR1.1	Kim535	GGTAGATTATTATCTGTGTTTTAGAGCTAGAAATAGCAAG
	Kim536	CAGATAATAATCTACCATGGATCATTTATCTTTCACTGCG
pRS42H-INT#4	Kim310	TCGAAGTGGTCACGTGCGTTTTAGAGCTAGAAATAGCAAG
	Kim311	CACGTGACCACTTCGAGAGGATCATTTATCTTTCACTGCG
pRS42H-INT#6	Kim314	TCACAGTGTCACATCAGGTTTTAGAGCTAGAAATAGCAAG
	Kim315	TGATGTGACACTGTGACAAGATCATTTATCTTTCACTGCG
pRS42H-INT#7	Kim486	AGGAATTATGTTCGCCCGTTTTAGAGCTAGAAATAGCAAG
	Kim487	GGCGAACATAATTCCTTACGATCATTTATCTTTCACTGCG

Donor DNA fragments were prepared by PCR using the primers listed in Table 6. Each of the fragments was flanked by 40–50 bp to allow in vivo assembly and genome integration through homologous recombination. Each assembly was an expression cassette of a heterologous gene as described in Fig. 3A. The donor DNAs for the xylose expression cassettes were designed to achieve complete removal of a target gene when genome integrated. On the other hand, the expression cassettes of the arabinose pathway and galacturonic acid pathway were integrated into an intergenic region without interfering neighboring genes.

• 3)Yeast transformation

Table 6.

Primers used for construction of donor DNA fragments.

Template genomic DNAa	Donor DNA fragments	Primers	Sequences (5’-)
XYL1 and XYL2 expression cassettes for deleting ALD6 (ald6::TDH3P-XYL1-TDH3T-PGK1P-XYL2-PGK1T)
S. cerevisiae	TDH3P	Kim626	TAACATACACAAACACATACTATCAGAATACACTATTTTCGAGGACCTTGTC
		SOO384	TCAACTTAATAGAAGGCATTTTTAGATCTCCTAGGTTTGTTTGTTTATGTGTGTTTAT TC
P. stipitis	XYL1	SOO385	ATAAACACACATAAACAAACAAACCTAGGAGATCTAAAAATGCCTTCTATTAAGTTGA AC
		SOO386	AAT GCAAGATTTAAAGTAAATTCACTGTTAACGCATGCTTAGACGAAGATAGGAATCTTG
S. cerevisiae	TDH3T	SOO387	GGA CAAGATTCCTATCTTCGTCTAAGCATGCGTTAACAGTGAATTTACTTTAAATCTTGC
		SOO388	ATTCTTTGAAGGTACTT CTTCGAAAAATTCGCGTCTGCTAGCTCCTGGCGGAAAAAATTC
S. cerevisiae	PGK1P	SOO389	TTTTAAAGTTTACAAAT GAATTTTTTCCGCCAGGAGCTAGCAGACGCGAATTTTTCGAAG
		SOO390	CACCAA GGAAGGGTTAGCAGTCATTTTTTCTAGATGTTTTATATTTGTTGTAAAAAGTAG
P. stipitis	XYL2	SOO391	AATTAT CTACTTTTTACAACAAATATAAAACATCTAGAAAAAATGACTGCTAACCCTTCC
		SOO392	AAAAAATTGAT CTATCGATTTCAATTCAATTCAATACTAGTTTACTCAGGGCCGTCAATG
S. cerevisiae	PGK1T	SOO393	GTCAAGTGTCT CATTGACGGCCCTGAGTAAACTAGTATTGAATTGAATTGAAATCGATAG
		Kim627	GTATATGACGGAAAGAAATGCAGGTTGGTACA AAATAATATCCTTCTCGAAAG
XYL3 expression cassette for deleting PHO13 (pho13::TDH3P-XYL1-TDH3T-PGK1P-XYL2-PGK1T)
S. cerevisiae	TDH3P	Kim628	ATGTGACATCTTTACTATTCTCCAGCACGTTT CTTCATCGGTATCTTCGC
		SOO374	AA TGGGGTAGTGGTCATTTTTAAGCTTGAATTCTTTGTAATTAAAACTTAGATTAGATTG
P. stipitis	XYL3	SOO375	AT CTAATCTAAGTTTTAATTACAAAGAATTCAAGCTTAAAAATGACCACTACCCCATTTG
		SOO376	GCAACTA GAAAAGTCTTATCAATCTCCGTCGACATCGATTTAGTGTTTCAATTCACTTTC
S. cerevisiae	TDH3T	SOO377	CAAGATG GAAAGTGAATTGAAACACTAAATCGATGTCGACGGAGATTGATAAGACTTTTC
		Kim629	CTATAACTCATTATTGGTTAAGGTGTAGATG AAGTTGGGTAACGCCAGG
gaaA expression cassette (int#4::CCW12P-gaaA-CCW12T)
S. cerevisiae	CCW12P	Kim379	TTCCTCGGGCAGAGAAACTCGCAGGCAACTTG CACGCAAAAGAAAACCTT
		Kim380	TCAACA CAGCTGGGGGAGCCATTTTTTATTGATATAGTGTTTAAGCGAAT
A. niger	gaaA	Kim381	TCTGTC ATTCGCTTAAACACTATATCAATAAAAAATGGCTCCCCCAGCTG
		Kim382	TAGA ATGTATAAATAATAATAAACTAAGTCTACTTCAGCTCCCACTTTCC
S. cerevisiae	CCW12T	Kim383	GGAT GGAAAGTGGGAGCTGAAGTAGACTTAGTTTATTATTATTTATACAT
		Kim384	TGTGAGGGCCGATTATGCAGGCCTAGA TGTTCTAGTGTGTTTATATTATC
lgd1 expression cassette (int#4::PGK1P-lgd1-PGK1T)
S. cerevisiae	PGK1P	Kim385	CCTCGGGCAGAGAAACTCGCAGGCAACTTG GTGAGTAAGGAAAGAGTGAG
		Kim386	GTGATGGTGACTTCAGACATTTTTTGTTTTATATTTGTTGTAAAAAGTAG
T. reesei	lgd1	Kim387	CTACTTTTTACAACAAATATAAAACAAAAAATGTCTGAAGTCACCATCAC
		Kim388	ATTGATCTAT CGATTTCAATTCAATTCAATTCAGATCTTCTCTCCGTTCA
S. cerevisiae	PGK1T	Kim389	CTGCCCATCT TGAACGGAGAGAAGATCTGAATTGAATTGAATTGAAATCG
		Kim390	CTCTGTGAGGGCCGATTATGCAGGCCTAGA AAATAATATCCTTCTCGAAA
gaaC expression cassette (int#4::TDH3P-gaaC-TDH3T)
S. cerevisiae	TDH3P	Kim391	CTCGGGCAGAGAAACTCGCAGGCAACTTG GAATAAAAAACACGCTTTTTC
		Kim392	GACTCCGGGGCG GAGCGGGGTAAAAGGCATTTTTTTTGTTTGTTTATGTGTGTT
A. niger	gaaC	Kim393	TTCGAATA AACACACATAAACAAACAAAAAAAATGCCTTTTACCCCGCTC
		Kim394	ATTTAAAT GCAAGATTTAAAGTAAATTCACCTAAGCAATATCCGGCAACG
S. cerevisiae	TDH3T	Kim395	TGAGAAGT CGTTGCCGGATATTGCTTAGGTGAATTTACTTTAAATCTTGC
		Kim396	CCTCTGTGAGGGCCGATTATGCAGGCCTAGA ATCCTGGCGGAAAAAATTC
gaaA, lgd1, and gaaC expression cassettes (int#4::CCW12P-gaaA-CCW12T-PGK1P-lgd1-PGK1T-TDH3P-gaaC-TDH3T)
S. cerevisiae YE3	CCW12P-gaaA-CCW12T	Kim410	TCTTTAGGTTAATTGTCGCTGTTATTGTCTA GATTTTTTCTCGGAGATGG
		Kim411	TAGTTC CTCACTCTTTCCTTACTCACTGTTCTAGTGTGTTTATATTATCC
S. cerevisiae YE4	PGK1P-lgd1-PGK1T	Kim412	AGCCAA GGATAATATAAACACACTAGAACA GTGAGTAAGGAAAGAGTGAG
		Kim413	AAACTCGAA CTGAAAAAGCGTGTTTTTTATTCCCGATTATGCAGGCCTAG
S. cerevisiae YE5	TDH3P-gaaC-TDH3T	Kim414	TATTATTTT CTAGGCCTGCATAATCGGGAATAAAAAACACGCTTTTTCAG
		Kim415	CTACTCTCTTCCTAGTCGCCCGGTTGTT GAAAGTTTAATTGTGGGTTTTC
lad1 and alx1 expression cassettes (int#7::FBA1P-lad1-FBA1T-PGK1P-alx1-CYC1T)
S. cerevisiae YE01	FBA1P-lad1-FBA1T-PGK1P-alx1-CYC1T	Kim553	CTTACACTTGTGTAATGACAAATGTTTTT TGAACAACAATACCAGCCTTC
		Kim554	TGTTTCACGTTATCAAGATTATGTCATCTATT GGCCGCAAATTAAAGCCT
Overexpression of YPR1 (CCW12P-YPR1)
S. cerevisiae	CCW12P	Kim537	GTAACTTTGCAATATAATCAGGTCGCAAATAT CACGCAAAAGAAAACCTT
		Kim538	GAAGAATTCTTTAACGTAGCAGGCAT TATTGATATAGTGTTTAAGCGAAT
gaaD expression cassette (int#6::CCW12P-gaaD-CCW12T)
S. cerevisiae	CCW12P	Kim541	CGGAGGAGACCGCTATAACCGGTTTGAATTTA CACGCAAAAGAAAACCTT
		Kim542	TA ACCTTCTTTCCGAGAGACATTTTTTATTGATATAGTGTTTAAGCGAAT
A. niger	gaaD	Kim543	TC ATTCGCTTAAACACTATATCAATAAAAAATGTCTCTCGGAAAGAAGGT
		Kim544	GT ATAAATAATAATAAACTAAGTTTATTAAACAATCACCTTATGACCAGC
S. cerevisiae	CCW12T	Kim545	TG GTCATAAGGTGATTGTTTAATAAACTTAGTTTATTATTATTTATACAT
		Kim546	CTTGCTTGCTGTCAAACTTCTGAGTTG TGTTCTAGTGTGTTTATATTATC

The flanking region is underlined.

^aSaccharomyces cerevisiae D452-2; Pichia stipitis CBS 6054; Aspergillus niger CBS 120.49; Trichoderma reesei ATCC 5676.

For yeast transformation, a gRNA plasmid (4 μg) and donor DNA fragments (4 μg each) were used to transform a designated strain harboring pRS41N-Cas9 [3]. The resulting transformants were selected on a YPD agar plate supplemented with 100 μg/mL nourseothricin sulfate (Gold Biotechnology, St. Louis, MO, USA) and 300 μg/mL hygromycin B (Invitrogen, Carlsbad, CA, USA). Selected transformants were serially sub-cultured in YPD medium supplemented with 100 μg/mL nourseothricin sulfate to only remove the existing gRNA plasmids. Correct assembly and integration was then confirmed by yeast colony PCR with the primers listed in Table 7. Through four consecutive transformations, as described in Fig. 3, the YE9 strain was finally constructed.

Table 7.

Primers used for confirmation of correct assembly and integration.

Primers	Sequences (5’-)	Primers	Sequences (5’-)
Introduction of d-xylose pathway		Introduction of d-galacturonic acid pathway
Kim049	GGAACGGTGAGTGCAACG	Kim322	GCGCATCTATTTGCCGTC
Kim427	AAACTGTTCACCCAGACACC	Kim397	GCTGGGGGAGCCATTTTTTATTG
Kim194	AGCGCAACTACAGAGAACAGG	Kim398	GTGGGAGCTGAAGTAGACTTAG
Kim100	CGGCACCGTCGAACAATCTG	Kim323	TCACGACACACCTCACTG
Kim101	CCGCTTACTCTTCGTTCGGTCC	Kim399	CCTGTGATGGTGACTTCAGAC
Kim193	CTCAGCATCCACAATGTATCAG	Kim401	GAACGGAGAGAAGATCTGAATTG
Kim426	GCGCTATTGCATTGTTCTTGTC	Kim400	ACAGCCTGTTCTCACACAC
Kim547	AGGTATGCGATAGTTCCTCAC	Kim402	GCGGGGTAAAAGGCATTTTTTTTG
Kim125	TGCAGCTTCCAATTTCGTCAC	Kim408	GCCGGATATTGCTTAGGTG
Kim630	GAGGTGACACCCTTACCAAC
Kim631	CTGCTACTCACACCTTCAACTC	Introduction of l-arabinose pathway
Kim632	CGCTGAACCCGAACATAGAAATATC	Kim490	GGCACTAGGAGCATTTGTCG
Kim633	TCGATATTTCTATGTTCGGGTTCAG	Kim304	GCTTCGCTAATCCAGAGGTC
Kim078	GATTGGAATTGGTTCGCAGTG	Kim400	ACAGCCTGTTCTCACACAC
Kim048	GAGGAAGACGTTGAAGGTGG	Kim491	GTCCCTTAGGGTGCGTATAATG
Kim149	TTTGAAGTGGTACGGCGATG
Kim577	CACCCAAGCACAGCATAC	Overexpression of YPR1
Kim634	TGGCTCGATAACGAAGATTCAG	Kim539	CAATTCCGTGAAACCCTTTTCTT
Kim635	GTCTTGTAGATTGAGAACTGGTCC	Kim540	CTGCCAACTTCTTCTTCATTCAA
Kim636	TCTATGAGGCAAGTAAGAGGCAC
Kim492	AACAGGCGACAGTCCAAATG	Introduction of gaaD gene cassette
Kim077	TTGGAGTTCAAACTGGCGAG	Kim326	GGTTCTGACTCCTACTGAGC
		Kim093	GCAAAGATAGCGGCGTAGGTG
		Kim549	GCATCCTTTGCCTCCGTTC
		Kim327	AGCATCGAGTACGGCAGTTC

2.2. Fermentation

For fermentation of the YE9 strain, one colony was pre-cultured in YP medium (10 g/L yeast extract and 20 g/L peptone) supplemented with 20 g/L of glucose for 36 h at 30^oC and 250 rpm. Cells were centrifuged, washed twice, and re-suspended in YP medium supplemented with desired carbon sources. The initial cell density of fermentation was 25 g/L dry weight, which corresponds to approximately 125 g/L wet weight, and this conversion factor was obtained from a prior study [4]. In the industrial bioethanol processes, >90% cells are recycled in repeated batch-type fermentation; therefore, very high cell density of up to 170 g/L wet weight [5] is often achieved. The concentrations of the carbon sources were selected to reflect the typical chemical composition of pectin-rich biomass (Table 8).

Table 8.

Chemical composition of pectin-rich biomass.

Source	Arabinose	Galacturonic acid	Ratio	Reference
Orange peel hydrolysate (g/L, ∼ 10% solid loading)	32.6	13.2	2.47	[9]
Sugar beet pulp hydrolysate (g/100 g dry matter)	22.5	22.5	1.00	[10]

2.3. HPLC analysis

Quantitation of glucose, fructose, galactose, xylose, arabinose, galacturonic acid, glycerol, and ethanol was performed by high-performance liquid chromatography (HPLC; Agilent Technologies, 1260 series, USA) device equipped with a RI detector and a Rezex-ROA Organic Acid H+ (8%) (150 mm × 4.6 mm) column (Phenomenex Inc., Torrance, CA, USA). The column was eluted with 0.005 N H₂SO₄ at 0.6 mL/min and 50^oC [1,6].

Conflict of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: