Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Abstract

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L⁻¹ glucose and 95 g L⁻¹ xylose generated 120.6 g L⁻¹ ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g⁻¹, and ethanol productivity of 5.26 g L⁻¹·h⁻¹.

1. Introduction

Ethanol can be produced from renewable resources such as crops or agricultural waste. It is therefore a sustainable and clean fuel. Further growth in bioethanol production largely depends on the effective conversion of lignocellulosic feedstock such as agricultural and forestry wastes to bioethanol because they are the most abundant polymers of fermentable sugars [1,2,3].

Saccharomyces cerevisiae is the most effective microorganism for fermenting sugars to ethanol due to its rapid sugar consumption rate, high sugar and ethanol tolerance, and resistance to biomass-derived inhibitors [4,5]. Much research has been done to genetically engineer S. cerevisiae strains for xylose fermentation [6,7,8,9,10].

Two xylose-assimilating pathways were heterologously engineered in S. cerevisiae for xylose-fermenting yeast construction. One focused on the D-xylose isomerase (XI) pathway [11,12], the other focused on the D-xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway [10,13,14,15]. In the XI pathway, xylose is first isomerized into xylulose by XI and xylulose was then phosphorylated into xylulose 5-phosphate by xylulokinase. Xylulose is subsequently metabolized to ethanol through glycolysis in the pentose phosphate pathway [11]. As no xylitol is produced in this pathway, much research focused on the XI pathway construction in xylose-fermenting yeast [12].

However, for all the engineered strains developed, rapid glucose and xylose co-fermentation is still challenging, in particular when sugar concentration is high [4,8,9,10]. Because fermentation time on mixed-substrate hydrolysates is still not cost-effective, strategies in evolutionary engineering were used to improve fermentation kinetics [16,17,18] and much research focused on the search for new or engineered sugar transporters [19,20,21]. More recently, robust diploid S. cerevisiae strains were developed for rapid xylose-fermentation [22,23,24,25].

This study aims to develop a recombinant S. cerevisiae strain for rapid glucose and xylose co-fermentation through metabolic engineering, evolutionary engineering and strain mating. Firstly, four episomal plasmids containing the two-copy codon-optimized Piromyces sp. E2 XI gene (PirXylA, GenBank accession number AJ249909.1), one-copy codon-optimized Orpinomyces sp. ukk1 XI gene (OrpXylA, GenBank accession number EU411046), one-copy PirXylA and OrpXylA in tandem, and two-copy OrpXylA, were individually constructed. They were subsequently transformed to an evolved respiration-deficient yeast strain. Four engineered strains with episomal XI genes were generated and optimal XI functional expression was identified. Afterwards, PirXylA and OrpXylA were separately integrated into the genome of two evolved S. cerevisiae strains (one respiration-efficient and the other respiration-deficient) in multiple copies according the methods described previously [26,27]. Four engineered yeasts with integrated XI genes were generated and screened for xylose fermentation or glucose/xylose co-fermentation. In the end, a diploid recombinant xylose-fermenting S. cerevisiae was constructed by mating a respiration-efficient haploid strain with a respiration-deficient haploid strain. To the best of our knowledge, this is the first report on diploid xylose-fermenting yeast strain construction by such strain mating. The resulted diploid yeast strain displayed superior glucose and xylose co-fermentation performance, which far exceeded that by engineered S. cerevisiae reported to-date [28,29].

2. Materials and Methods

2.1. Plasmid Construction

All plasmids used in this work are listed in

Table 1. Strains and plasmids used in this study.

Strains/Plasmid	Genotype/Phenotype	References
Saccharomyces cerevisiae strains
Saccharomyces cerevisiae ATCC 24860	Obtained from American Type Culture Collection (ATCC)
JUK36α	S. cerevisiae ATCC 24860 segregant; MAT; ura3::loxP; TKL1::RKIt-RKI1-ADH1p-RPE1t-RPE1-TPI1p-loxP-XKS1t-XKS1-PGK1p-PDC1p-TAL1-TAL1t-FBA1p; gre3::loxP	[12]
JUK39a	S. cerevisiae ATCC 24860 segregant; MATa; ura3::loxP; TKL1::RKIt-RKI1-ADH1p-RPE1t-RPE1-TPI1p-loxP-XKS1t-XKS1-PGK1p-PDC1p-TAL1-TAL1t-FBA1p; gre3::loxP; cyc3::loxP	[12]
JUK51a_2	JUK36α derivative; {pJFX11}/(BvuXylA, XK, PPP, gre3∆)	[12]
39aXpXp	JUK39a derivative; {pPYXpXp}/(two-copy PirXylA, XK, PPP, gre3∆, cyc3∆)	This work
39aXpXp2415	39aXpXp derivative; {pPYXpXp}/(two-copy PirXylA, XK, PPP, gre3∆, cyc3∆, AE)	This work
36α2	Isolate from chemostat anaerobic and adaptive evolution at a dilution rate of 0.15 h−1 on xylose of JUK51a_2 and loss of plasmid pJFX11	This work
39a2	Isolate from 39aXpXp2415 and loss of plasmid pPYXpXp	This work
39a2XpXp	39a2 derivative; {pPYXpXp}/(two-copy PirXylA, XK, PPP, gre3∆, cyc3∆)	This work
39a2XpXo	39a2 derivative; {pPYXpXo}/(OrpXylA, PirXylA, XK, PPP, gre3∆, cyc3∆)	This work
39a2Xo	39a2 derivative; {pPYXo}/(OrpXylA, XK, PPP, gre3∆, cyc3∆)	This work
39a2XoXo	39a2 derivative; {pPYXoXo}/}/(two-copy OrpXylA, XK, PPP, gre3∆, cyc3∆)	This work
36α2XpXpUN	36α2 derivative;NTS2-2::two-copy PirXylA, ura3, XK, PPP, gre3∆	This work
36α2XoNK	36α2 derivative;NTS2-2::OrpXylA-KanMX4, XK, PPP, gre3∆, ura3∆	This work
39a2XpXpUN	39a2 derivative; NTS2-2::two-copy PirXylA, ura3, XK, PPP, gre3∆, cyc3∆	This work
39a2XoNK	39a2 derivative; NTS2-2::OrpXylA-KanMX4, XK, PPP, gre3∆, cyc3∆, ura3∆	This work
STXQ	Isolate from mating of 36α2XpXpUN with 39a2XoNK	This work
Plasmids
pUG6	E. coli plasmid with segment loxP–KanMX4–loxP	[30]
pJFX11	YEp, TEF1p-BvuXylA-CYC1t	[12]
pPY1	pPYES2; GAL1p replaced by PGK1p	This work
pPYXo	pPY1; PGK1p-OrpXylA-CYC1t	This work
pPYXpXp	pPY1; 2 copies of PGK1p-PirXylA-CYC1t in tandem	This work
pPYXpXo	pPY1; PGK1p-PirXylA-CYC1t-PGK1p-OrpXylA-CYC1t	This work
pPYXoXo	pPY1; 2 copies of PGK1p-OrpXylA-CYC1t in tandem	This work
XXUN	pPYXpXp-based yeast integration plasmid; 2 μ and ura3 were replaced with ura3 and NTS2-2 partial fragment	This work
XoNK	pUC19-based yeast integration plasmid; loxP-KanMX4-loxP-pNTS-PGK1p-OrpXylA-CYC1t	This work

. All primers used in this study are listed in Table S1.

E. coli strain DH5α (Life Technologies, Rockville, MD, USA) were used as the transformation host for plasmid construction. E. coli were grown in LB medium containing 100 μg/mL ampicillin at 30 °C or 37 °C for plasmid maintenance [25]. The PGK1 promoter was amplified from genomic DNA of S. cerevisiae strain ATCC 24860 and was ligated to pYES2 (Thermo-Fischer Scientific, Singapore) to replace the GAL1 promoter, resulting in pPY1. PirXylA and OrpXylA were synthesized by Integrated DNA Technologies Singapore). Cassettes PGK1p-OrpXylA-CYC1t, PGK1p-PirXylA-CYC1t-PGK1p-OrpXylA-CYC1t, PGK1p-OrpXylA-CYC1t-PGK1p-OrpXylA-CYC1t and PGK1p-PirXylA-CYC1t-PGK1p-PirXylA-CYC1t were cloned into pPY1 individually, resulting in plasmids pPYXo, pPYXpXo, pPYXoXo and pPYXpXp (Figure 1A–D).

NTS2-2 partial fragment (pNTS) was obtained by overlap extension polymerase chain reaction (OE-PCR) of S. cerevisiae ATCC 24860 genomic DNA over 274 bp upstream and 245 bp downstream homologous regions of NTS2-2. The XXUN plasmid (Figure 1E) is an integrating yeast plasmid based on pPYXpXp whereby the 2 µ origin was replaced with pNTS. The loxP-KanMX4-loxP cassette was amplified from the plasmid pUG6 [30]. Cassettes PGK1p-OrpXylA-CYC1t and loxP-KanMX4-loxP-pNTS were obtained by OE-PCR and then subsequently constructed into plasmid pUC19 [12], resulting in plasmid XoNK (Figure 1F). Both plasmids were digested with SwaI for XI genome integration using NTS2-2 homologous recombinant arms.

2.2. Strain Construction and Adaptive Evolution

All strains used in this work are listed in Table 1. S. cerevisiae haploid strains JUK36α and JUK39a were isolated from the diploid strain S. cerevisiae ATCC 24860. They were both overexpressed with the non-oxidative pentose phosphate pathway (PPP) genes and xylulokinase gene, XKS1. URA3 and GRE3 genes were disrupted in both haploid strains. In addition, the CYC3 gene, encoding cytochrome c heme lyase, was knocked out in strain JUK39a to eliminate respiration [12].

Plasmid pPYXpXp was transformed into strain JUK39a using the LiAc/SS carrier DNA/PEG method [31], resulting in recombinant S. cerevisiae 39aXpXp. The respiration-deficient strain 39aXpXp was evolved by continuous transfer and cultivation in a synthetic medium (SM) containing 6.7 g L⁻¹ yeast nitrogen base without amino acids (YNB) (Difco Laboratories Inc., Detroit, MI, USA) and 20 g L⁻¹ xylose (SMX) under oxygen-limited conditions according to the protocols described in our earlier report [12]. After 75-day continuous transfer, cell doubling time was reduced from 15.9 h to 6.4 h. Samples were taken on day 75 and streaked on SMX plates containing 20 g L⁻¹ xylose and 20 g L⁻¹ agar. Fifteen large single colonies were selected. They were then incubated in 50 mL SMX medium in 100 mL Erlenmeyer flasks capped with rubber stoppers, shaken at 200 rpm and 30 °C. Weight loss of the cultures from the 15 colonies was individually measured on Day 4. The best ethanol-producing strain was indicated by the highest weight loss [18]. This strain was denoted 39aXpXp2415, and plasmid pPYXpXp in this strain was removed by streaking the culture on 5-FOA plates [12]. This generated the background strain 39a₂ (Table 1). On the other hand, strain JUK51a_2 (Table 1) was evolved anaerobically in a chemostat with an increase in the dilution rate from 0.01 to 0.14 h⁻¹ on xylose using the method described in our earlier report [12]. The fastest growing strain was selected, and the plasmid pJFX11 was removed according to the above-described method on 5-FOA plates. The background strain 36α₂ was later obtained (Table 1).

Plasmids pPYXo, pPYXpXo, pPYXoXo and pPYXpXp were individually transformed into 39a₂, resulting in strains 39a₂Xo, 39a₂XpXo, 39a₂XoXo and 39a₂XpXp, respectively. Plasmids XoNK and XXUN were digested with SwaI and linearized. The linearized fragments were integrated into the genome of 39a₂ and 36α₂ at the NTS2-2 site, individually, resulting in recombinant strains 39a₂XoNK, 39a₂XpXpUN, 36α₂XoNK and 36α₂XpXpUN. For each plasmid transformation, a pool of transformants were generated. The best transformant was isolated based on its cell growth and ethanol production in xylose medium as described in the isolation of 39aXpXp2415. Recombinant strains, 36α₂XpXpUN and 39a₂XoNK, were later mated to obtain strain STXQ through screening on SMX agar plates containing 20 g L⁻¹ xylose at pH 6 followed by fermentation in SMX medium containing up to 250 g L⁻¹ xylose.

2.3. Enzyme Activity Assay

Cells were grown to the exponential phase in SMX medium containing 20 g L⁻¹ xylose. After centrifugation, cells were washed twice with chilled distilled water, and then lysed in chilled extraction buffer (100 mM Tris-HCl, 10 mM MgSO₄, pH 7.5) by vortex mixing using a Vortex Mixer (Mixer UZUSIO, Tokyo, Japan) with 0.5 mg of 0.5 mm glass beads (Sigma-Aldirich, Singapore). Protease inhibitor cocktail set V (Merck Millipore, Singapore) was added to inhibit serine and cysteine generated in the lysis process. Cell debris was removed by centrifugation (Microcentrifuge D3024, DR. LAB Technology Hong Kong, Hong Kong, China) at 4000× g for 10 min at 4 °C, and the crude extract was stored for enzyme activity assay. The protein concentration of the cell extract was determined by the Bradford Assay using a Coomassie Protein Assay Kit (Thermo Scientific, Singapore), and bovine serum albumin (BSA) was used as the standard. Extraction of raw proteins from the yeast strains was performed in duplicate.

The in-vitro XI activity was determined by measuring NADH absorbance using sorbitol dehydrogenase (SDH) (Sigma Aldrich, Singapore). The assay mixture (1 mL) contained extraction buffer, 0.15 mM NADH, 1 U SDH, and 50 μL crude extract. It was equilibrated at 30 °C for 2 min. The reaction was started by the addition of d-xylose to a final concentration of 500 mM. The change of NADH concentration within 3 min was detected using a UV-visible spectrophotometer (Shimadzu, Tokyo, Japan) at wavelength 340 nm, and the specific activity of XI in the recombinant strains was determined [32]. A molar extinction coefficient of 6.25 (mM cm)⁻¹ at 340 nm for NADH was used to calculate specific activity. Specific activity was expressed as units per mg protein. One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of coenzyme/min, under the specified reaction conditions [12].

2.4. Glucose and Xylose Fermentation by the Recombinant Strains

The preculture of the evolved engineered strains was prepared by growing them in 40 mL SMX medium containing 20 g L⁻¹ xylose in 100 mL Erlenmeyer flaks at 200 rpm and 30 °C for 24 h. The oxygen-limited conditions in the flasks was maintained by capping the flasks with rubber stoppers pierced with a needle to allow the release of CO₂. Cells in the exponential phase were harvested by centrifugation (Microcentrifuge D3024, DR. LAB Technology Hong Kong, Hong Kong, China) at 14,000× g for 1 min. Cell pellets were washed twice and were then inoculated into SM medium supplemented with 20 g L⁻¹ xylose with (SMGX) or without 20 g L⁻¹ glucose (SMX). The initial optical cell density at 600 nm (OD600) of the culture was about 2 unless otherwise stated. Fermentation was conducted in 100 mL Erlenmeyer shaking flasks under oxygen-limited conditions with a working volume of 40 mL at 200 rpm and 30 °C. The pH value was adjusted at 5.0–6.0 using 3 M NaOH during fermentation. All fermentation experiments were performed in duplicate. Samples were taken periodically to measure OD600, sugar and metabolite concentration.

2.5. Analytical Methods

Cell densities (OD600) were determined using a UV-visible spectrophotometer (Shimadzu, Tokyo, Japan). Fifty-mL cell cultures with varying OD600 (1–5) were filtered with 0.22 μm glass fiber filter membrane (Merck Millipore, Singapore) using Aspirator A-3S (Fisher Scientific, Tokyo, Japan). Cells were washed twice with distilled water, dried at 105 °C in an oven for 24 h, and then weighed. One OD600 unit corresponded to 0.241 g L⁻¹ dry cell weight (DCW). Concentrations of glucose, xylose, xylitol, acetate, glycerol and ethanol produced in fermentation were determined by Agilent 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive index detector RID-10A using an Aminex HPX-87H ion exchange column (Bio-Rad Laboratories, Woodinville, WA, USA). The column was eluted at 60 °C with 5 mM of sulfuric acid as the mobile phase at a flow rate of 0.6 mL min⁻¹.

2.6. Quantitative Reserve Transcription Polymerase Chain Reaction (RT-PCR)

Recombinant yeast strains 39a₂Xo, 39a₂XpXo, 39a₂XoXo and 39a₂XpXp were individually cultivated in 40 mL SMX medium containing 20 g L⁻¹ xylose under oxygen-limited conditions at 200 rpm and 30 °C. The expression of XI gene transcripts was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Primers for RT-PCR are listed in Table S1. Samples were taken at 24 h, and cells were harvested by centrifuging 2-mL culture at 14,000× g and 4 °C for 1 min. Cell pellets were washed twice using double distilled water and total RNA was isolated by using the E.Z.N.A™ Yeast RNA Kit (Omega Bio-tek, Norcross, GA, USA). First-strand cDNA was obtained by using the SuperScript^® First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA). Such cDNA was then used as the template for qRT-PCR using iCycler iQ™ Real-time PCR Detection System (Bio-Rad Laboratories, Woodinville, WA, USA) and FastStart Universal SYBR Green Master (Roche Applied Science, Penzberg, Germany). The cycle threshold values were calculated with the Optical System Software Version 3.1 (Bio-Rad Laboratories, Woodinville, WA, USA), and the detection threshold over the cycle range was set at 2 to 10. Each PCR was carried out in duplicate. All kits were used under conditions recommended by the manufacturers. The 2^−ΔΔCt method [33] was used to analyze the relative changes in gene expression using the housekeeping ACT1 gene as the reference.

2.7. Biomass Hydrolysate Fermentation Using Strain S. cerevisiae STXQ

The diploid recombinant S. cerevisiae strain STXQ (Table 1) was inoculated into YP medium (10 g L⁻¹ yeast extract, 20 g L⁻¹ peptone, pH 5.0) containing 132 g L⁻¹ xylose or mixture of 162 g L⁻¹ glucose and 95 g L⁻¹ xylose at an initial OD600 of about 13. Fermentation was conducted in 40 mL YP medium in 100 mL shaking flasks under oxygen limited conditions at 200 rpm and 30 °C.

Oil palm empty fruit bunch (OPEFB) hydrolysate was obtained using crude cellulase from Trichoderma reesei Rut-C30 according to the protocols described in our earlier report [34]. OPEFB hydrolysate was sterilized using 0. 22 μm filter membrane (Merck Millipore, Singapore) and was supplemented with 7 g L⁻¹ yeast extract 2 g L⁻¹ peptone, 2 g L⁻¹ (NH₄)₂SO₄, 2.05 g L⁻¹ KH₂PO₄, and 0.25 g L⁻¹ Na₂HPO₄. The diploid recombinant S. cerevisiae strain STXQ was inoculated into the above OPEFB hydrolysate medium with an initial OD600 about 5. Fermentation was carried out in 40 mL fermentation medium in 100 mL shaking flasks under oxygen-limited conditions at 200 rpm and 30 °C with an initial pH of 4.48. Samples were taken periodically for OD600, sugar and metabolite analysis. Experiments were conducted in duplicate.

3. Results

3.1. Expression of XIs with Various Combinations

XI activities were assayed for 39a₂ recombinant strains with episomal XI genes (

Table 2. Specific activity of xylose isomerase in the recombinant strains.

Strains	Specific Activity (U mg−1 Protein)
39a2XpXp	0.10 ± 0.003
39a2XpXo	0.11 ± 0.019
39a2Xo	0.12 ± 0.041
39a2XoXo	0.30 ± 0.079
36α2XpXpUN	0.11 ± 0.007
36α2 XoNK	0.04 ± 0.005
39a2XpXpUN	0.26 ± 0.004
39a2XoNK	0.72 ± 0.006

The results represent the mean ± standard deviation of duplicate independent experiments.

). XI activity of 39a₂XoXo was three times higher than that of 39a₂XpXp and 2.5 times higher than that of 39a₂Xo. Strain 39a₂XoNK presented the highest XI activity among all the yeast strains with integrated XI genes, followed by 39a₂XpXpUN. Quantitative RT-PCR showed that the transcription level of XI gene in the engineered strains significantly increased compared to that in the parent strain 39a₂ (

Table 3. Fold-change in xylose isomerase (XI) mRNA expression.

Strain	Fold-Change a
	PirXylA	OrpXylA
39a2XpXp	50.21 (47.81–52.74)	nil
39a2XpXo	29.86 (28.43–31.36)	11.71 (10.62–12.92)
39a2Xo	nil	59.71 (51.98–68.59)
39a2XoXo	nil	59.71 (55.72–64)
36α2XpXpUN	51.98 (49.50–54.60)	nil
39a2XoNK	nil	84.45 (59.71–119.43)

^a Fold-change of XI mRNA level in the recombinant yeast strains compared to the parent strain 39a₂. Results were based on duplicate independent real-time RT-PCR reactions.

). In 36α₂XpXpUN and 39a₂XoNK, respective 1.04-fold and 1.41-fold increases in XI gene transcription levels were observed compared to those in 39a₂XpXp and 39a₂XoXo. Inconsistent XI activity and transcription level were observed.

3.2. Glucose and Xylose Fermentation by the Engineered 39a₂ Strains Harboring Episomal XI Genes

Fermentation performance of 39a₂ recombinant strains with episomal XI genes was tested in SMX medium containing 20 g L⁻¹ xylose under oxygen-limited conditions. Apparently, strain 39a₂XpXp displayed the fastest xylose utilization rate, followed by 39a₂XoXo (Figure 2). Strain 39a₂XpXo utilized xylose more slowly than 39a₂XpXp, though it demonstrated almost the same XI activity (Table 2). On the other hand, strain 39a₂XoXo showed a faster xylose utilization rate than strain 39a₂Xo; however, the former displayed much higher activity than the latter. On the contrary, xylose utilization results accorded quite well with the results of qRT-PCR analysis showing that strain 39a₂XpXp had higher XI gene transcription levels than strain 39a₂XpXo (Table 3) and a faster xylose utilization rate (Figure 2). However, strains 39a₂XoXo and 39a₂Xo displayed identical XI gene transcription levels (Table 3), almost the same xylose consumption rate (Figure 2) and xylose conversion (

Table 4. Glucose and xylose fermentation by recombinant xylose-fermenting yeast strains.

Strains	Initial glucose (g L−1)	Initial Xylose (g L−1)	Xylose Conversion (%)	Ethanol Yield (g g−1)	Ethanol Productivity (g h−1 L−1)	Specific Growth Rate (h−1)
39a2xpXp a	-	20	98% ± 0.004	0.472 ± 0.013	0.098 ± 0.004	0.014 ± 0.001
39a2XpXo a	-	20	89.8% ± 0.011	0.434 ± 0.017	0.098 ± 0.004	0.011 ± 5.89 × 10−6
39a2Xo a	-	20	97.3% ± 0.0138	0.428 ± 0.026	0.084 ± 0.001	0.011 ± 0.0003
39a2XoXo a	-	20	98.5% ± 0.0057	0.449 ± 0.038	0.108 ± 0.001	0.013 ± 2.29 × 10−5
36α2XpXpUN a	-	20	86.4% ± 0.052	0.318 ± 0.01	0.069 ± 0.007	0.014 ± 0.0003
36α2XoNK a	-	20	6.42% ± 0.057	ND	ND	0.003 ± 0.0006
39a2XpXpUNa	-	20	67.0% ± 0.089	0.398 ± 0.048	0.067 ± 0.001	0.013 ± 0.0002
39a2XoNK a	-	20	99.0% ± 0.002	0.368 ± 0.029	0.107 ± 0.007	0.0098 ± 0.0003
39a2XpXp a	20	20	22.0% ± 0.003	0.444 ± 0.029	0.221 ± 0.020	0.020 ± 0.0007
39a2XpXo a	20	20	77.5% ± 0.017	0.392 ± 0.001	0.081 ± 0.006	0.022 ± 0.0001
39a2Xo a	20	20	84.3% ± 0.0007	0.428 ± 0.01	0.238 ± 0.003	0.021 ± 0.0004
39a2XoXo a	20	20	96.7% ± 0.011	0.449 ± 0.022	0.213 ± 0.004	0.021 ± 0.001
36α2XpXpUN a	20	20	73.8% ± 0.011	0.348 ± 0.049	0.225 ± 0.007	0.029 ± 0.0006
36α2XoNK a	20	20	2.62% ± 0.000	0.390 ± 0.043	0.222 ± 0.003	0.016 ± 0.0013
39a2XpXpUN a	20	20	25.6% ± 0.097	0.405 ± 0.049	0.213 ± 0.002	0.013 ± 0.0002
39a2XoNK a	20	20	97.3% ± 0.006	0.387 ± 0.002	0.243 ± 0.003	0.020 ± 0.0005
36α2XpXpUN b	-	40	75.9% ± 0.041	0.384 ± 0.033	0.108 ± 0.011	0.013 ± 0.0004
39a2XoNK b	-	40	81.6% ± 0.004	0.421 ± 0.004	0.131 ± 0.000	0.010 ± 0.0003
STXQ b	-	40	93.3% ± 0.019	0.393 ± 0.024	0.146 ± 0.009	0.017 ± 0.0002
STXQ b	-	132	100%	0.498 ± 0.006	1.13 ± 0.01	0.014 ± 0.0004
STXQ c	162	95	99.27% ± 0.002	0.475 ± 0.01	5.24 ± 0.02	0.024 ± 0.0001

^a Fermentation in SM medium in 72 h; ^b Fermentation in YP medium in 102 h; ^c Fermentation in YP medium. The results represent the mean ± standard deviation of duplicate independent experiments.

). Interestingly, strain 39a₂XpXp also exhibited the maximal ethanol yield of 0.472 g g⁻¹ (Table 4). Such results suggest that expression of two-copy PirXylA is favorable for xylose alcoholic fermentation when xylose is used as the sole carbon source.

Co-fermentation of 20 g L⁻¹ glucose and 20 g L⁻¹ xylose was carried out in SM medium under oxygen-limited conditions. It can be seen that for all strains, glucose was almost completely consumed at 24 h (Figure 2). However, xylose utilization varied greatly for the four engineered 39a₂ strains. Noticeably, in the presence of 20 g L⁻¹ glucose, strain 39a₂XpXp utilized xylose quite slowly and about 22% xylose was consumed at 72 h (Table 4). On the other hand, xylose utilization was improved to 77.5% by strain 39a₂XpXo. Moreover, xylose consumption was improved to 84.3% by strain 39a₂Xo and it was further improved to 97% by 39a₂XoXo (Table 4). It is worth noting that strain 39a₂XoXo presented the highest ethanol yield (0.449 g g⁻¹). The above results suggest that the expression of two-copy of OrpXylA is beneficial for glucose and xylose co-fermentation.

3.3. Glucose and xylose Fermentation by the Engineered Yeast Strains Harboring Integrated XI Genes

Fermentation performance of 39a₂ recombinant strains with integrated XI genes was tested in SM medium containing 20 g L⁻¹ xylose (SMX) or 20 g L⁻¹ glucose and 20 g L⁻¹ xylose (SMGX) under oxygen-limited conditions (Figure 3). In SMX fermentation, the specific growth rate of 36α₂XpXpUN on xylose was 0.014 h⁻¹, much higher than that of 36α₂XoNK (0.003 h⁻¹), whereas xylose conversion by strain 39a₂XoNK was 99.0%, much higher than that by 39a₂XpXpUN (67.0%) (Table 4). In SMGX fermentation, the specific growth rate of 36α₂XpXpUN on xylose was 0.029 h⁻¹, much higher than that of 36α₂XoNK (0.016 h⁻¹), whereas xylose conversion by strain 39a₂XoNK was 97.30%, much higher than that by 39a₂XpXpUN (73.8%) (Table 4). In both SMX and SMGX fermentation, strain 36α₂XpXpUN presented the best cell growth, whereas strain 39a₂XoNK displayed the best xylose conversion. Furthermore, strain 39a₂XoNK exhibited the highest ethanol productivity in both SMX and SMGX fermentation. Despite the possible XI gene copy number variation in these strains, the above results demonstrated that the elimination of respiration was favorable for xylose fermentation, which corroborated quite well with previous reports [12,14]. Strains 36α₂XpXpUN and 39a₂XoNK were therefore selected for mating to generate the diploid recombinant strain STXQ to attain good cell growth and xylose fermentation.

3.4. Glucose and xylose Fermentation by Diploid Recombinant Strain STXQ

Fermentation performance of STXQ was tested in YP medium containing 40 g L⁻¹ xylose under oxygen-limited conditions. As expected, strain STXQ inherited the advantages of both parent strains, 36α₂XpXpUN and 39a₂XoNK. It presented 93.3% xylose conversion and a specific growth rate of 0.017 h⁻¹; both were higher than those of its parent strains (Table 4). In addition, ethanol productivity was greatly improved (Figure 3, Table 4). A very minimum amount of glycerol was produced by STXQ, although glycerol production was notable for the respiration-deficient parent strain 39a₂XoNK (Figure 3). Moreover, production of xylitol and acetate was almost undetectable.

Fermentation performance of strain STXQ was further invested in YP medium containing high-concentration sugar. With an initial OD600 of about 13, strain STXQ consumed 100% xylose in fermenting 132 g L⁻¹ xylose and produced 65.8 g L⁻¹ ethanol at 46 h. The corresponding ethanol yield was 0.498 g g⁻¹ (Figure 4A, Table 4). In fermentation of 162 g L⁻¹ glucose and 95 g L⁻¹ xylose with about the same initial OD600, more than 99% glucose and xylose were co-utilized within 23 h (Figure 4B, Table 4). Ethanol titer reached 120.6 g L⁻¹ corresponding to an ethanol volumetric productivity of 5.26 g L⁻¹ h⁻¹. These are so far the highest values compared to those reported in the literature. The above results indicate that the strain development strategy elaborated in this study is efficient in generating a robust S. cerevisiae strain with improved xylose fermentation capabilities.

3.5. Oil Palm Empty Fruit Bunch Hydrolysate Fermentation by Diploid Recombinant Strain STXQ

Fermentation performance of strain STXQ was further tested in OPEFB hydrolysate containing 41.81 g L⁻¹ glucose, 30.00 g L⁻¹ xylose, 7 g L⁻¹ yeast extract, 2 g L⁻¹ peptone, 2 g L⁻¹ (NH4)₂SO₄, 2.05 g L⁻¹ KH₂PO₄, and 0.25 g L⁻¹ Na₂HPO₄ under oxygen-limited conditions with an initial OD600 of about 10. Strain STXQ consumed 95.3% glucose and 88.9% xylose (Figure 5). The pH value decreased from 4.48 to 4.00 within 72 h. Strain STXQ consumed 94.0% total sugar from the OPEFB hydrolysate without detoxification. The specific cell growth rate (μ_max) reached 0.013 h⁻¹ and the ethanol yield was 0.420 g g⁻¹. Glucose was quickly consumed, and glucose-xylose co-fermentation was clearly observed within 24 h. Subsequently, ethanol concentration kept increasing with almost the sole consumption of xylose until it reached the final titer of 28.4 g L⁻¹ at 72 h.

4. Discussion

Episomal plasmids containing OrpXylA, PirXylA and OrpXylA in tandem, two-copy PirXylA and two-copy OrpXylA expression cassettes (Figure 1) were transformed into the background strain 39a₂ individually. Among all the 39a₂ strains harboring episomal XI genes, strain 39a₂XpXp presented the fastest rate of xylose utilization when xylose was used as the sole carbon source. However, it did not exhibit the highest XI activity and transcription level (Table 2 and Table 3). Inconsistent XI activity and transcription level in recombinant xylose-fermenting S. cerevisiae strain was reported and it could be due to rearrangement of pentose phosphate pathway (PPP) genes, decreased glycolysis activity, repressed respiration activity, and enhanced gluconeogenesis [35]. Besides XI activity, the enhanced xylose utilization and fermentation could be associated with the elevated expression of sugar transporter genes, non-oxidative pentose phosphate pathway (PPP) genes such as TAL1, TKL1, RKI1, and RPE1 and xylulokinase gene, XKS1 [19,20,25,36].

For glucose and xylose co-fermentation, strain 39a₂XoXo presented the best glucose and xylose co-utilization (Figure 2) and exhibited the highest xylose conversion (96.7%) (Table 4). S. cerevisiae does not contain specific xylose transporters. Xylose was therefore transported by glucose transporters. Xylose affinity for the glucose transporters was very low at a high glucose concentration; however, it could increase to a similar level of glucose affinity at low glucose concentration [19,20]. As a result, diauxic lag is still a practical problem associated with mixed sugar utilization by xylose-fermenting yeasts. However, the diauxic growth was not significant for strains expressing OrpXylA, 39a₂Xo and 39a₂XoXo (Figure 2). The above results and analysis suggest that expression of OrpXylA is beneficial for glucose and xylose co-fermentation. This is consistent with a previous report [37].

Chromosomal integration of the XI gene into the S. cerevisiae genome has received a significant amount of attention in recent years [25,26]. It allows the recombinant strain to retain its physiological characteristics in nonselective medium. In the present study, multiple copies of XI genes were integrated at the 18S rDNA sites based on random homologous recombination. Such genome integration led to stable recombinant yeast strains (Figure S1) and boosted XI activity to 0.72 U mg⁻¹ protein in 39a₂XoNK (Table 2). Such improvement might be associated with the multiple copies of XI integrated in the yeast genome [25]. This further led to high XI transcription levels (Table 3), high xylose conversion and ethanol production (Table 4). Such results accorded quite well with earlier reports [25,26,36]. Notably, among these strains, 39a₂ strains with integrated XI genes presented higher XI activity and transcription level than 36α₂ strains (Table 2 and Table 3). This further confirmed that the elimination of respiration was favorable for xylose fermentation [12,14].

S. cerevisiae strains are regarded as industrial working horses for ethanol production owing to their high ethanol titer and sugar tolerance. Mating is one of the traditional yeast breeding methods to develop improved S. cerevisiae strains without genetic modifications [38]. Through strain mating, robust diploid S. cerevisiae strains were developed for enhanced xylose-fermentation and inhibitor resistance [23,24]. In the present work, a recombinant diploid S. cerevisiae strain STXQ was obtained by mating respiration-dependent strain 36α₂XpXpUN with the respiration-deficient strain 39a₂XoNK. Such strain mating enabled strain STXQ to present improved cell growth, xylose utilization and ethanol production (Figure 4, Table 4).

For glucose-xylose co-fermentation by engineered laboratory S. cerevisiae strains, higher ethanol concentration of ~60 g L⁻¹ was reported by Ho et al. [39] (

Table 5. Comparison of fermentation performance of engineered xylose-fermenting S. cerevisiae.

Strain	Description	Inoculum Biomass (g DCW L−1)	Initial Glucose (g L−1)	Initial Xylose (g L−1)	Final Ethanol (g L−1)	Ethanol Yield (g g−1)	Volumetric Ethanol Productivity (g L−1·h−1)	Reference
CIBTS0735	PirXylA; XKS1; PPP; ciGXF1; adaptive evolution	Rich medium, initial inoculum size at 0.63 g DCW L−1		40	17.47	0.44	1.09	[18]
		Rich medium, initial inoculum size at 0.63 g DCW L−1	80	40	53	0.45	2.22
RWB218	PirXylA; XKS1; PPP; gre3∆; adaptive evolution)	defined synthetic medium, initial inoculum size at 1.1 g DCW L−1	100	25	47.5	0.38	1.98	[40]
1400 (pLNH32)	XR; XDH; XK; adaptive evolution	Rich medium; OD 40–45	50		24	0.48	0.52	[39]
		Rich medium; OD 40–45	80	40	60	0.45	1.3
H31-A3-ALCS	PirXylA; XKS1; PPP; gre3∆; adaptive evolution	Defined medium;initial inoculum size at 0.05 g DCW L−1		40	16.4	0.41	0.55	[28]
GS1.11-26	cpXylA; XKS1; PPP; HXT7; AraT; AraA; AraB; AraD; TAL2; TKL2 mutagenesis; genome shuffling; adaptive evolution	Rich medium; initial inoculum size at 1.3 g DCW L−1	36	37	33.6	0.46	2.58	[18]
LF1	Ru-XylA, XKS1, PPP, gre3::MGT05196N360F, adaptive evolution	Rich medium, initial inoculum size at 1.00 g DCW L−1	80	42	58.0	0.47	3.60	[25]
STXQ	OrpXylA, PirXylA, XKS1, PPP, gre3∆; adaptive evolution	Rich medium, initial inoculum size at 3.13 g DCW L−1		132	65.8	0.50	1.13	This study
		Rich medium, initial inoculum size at 3.43 g DCW L−1	162	95	120.6	0.48	5.24	This study

). An ethanol titer of 47.5 g L⁻¹ was obtained by recombinant S. cerevisiae RWB218 expressing PirXylA [40]. About 53 g L⁻¹ ethanol titer was reported by Diao and his colleagues using the diploid recombinant S. cerevisiae strain CIBTS0735 [22]. Demeke et al. obtained an inhibitor-resistant recombinant S. cerevisiae through metabolic engineering and adaptive evolution [18]. It utilized glucose and xylose rapidly with ethanol titer up to 46 g L⁻¹ and ethanol productivity of 2.58 g L⁻¹·h⁻¹. This is the highest volumetric ethanol productivity reported to-date in the literature. More recently, about 58 g L⁻¹ ethanol titer was obtained by a diploid recombinant yeast strain LF1 developed from a wild-type S. cerevisiae strain in YP medium [25]. Strain STXQ yielded 65.8 g L⁻¹ ethanol with an ethanol yield of 0.50 g g⁻¹ at 56 h in YPX medium containing 132 g L⁻¹ xylose (Figure 4A, Table 4). Further glucose-xylose co-fermentation produced 120.6 g L⁻¹ ethanol with an ethanol yield of 0.48 g g⁻¹ at 23 h in YP medium containing 162 g L⁻¹ glucose and 95 g L⁻¹ xylose. The corresponding volumetric ethanol productivity reached 5.24 g L⁻¹·h⁻¹. (Figure 4B, Table 4). Both ethanol titer and volumetric productivity far exceeded the results in previous reports.

The OPEFB hydrolysate fermentation result demonstrated that strain STXQ could co-ferment glucose and xylose without detoxification, with 95.3% glucose conversion and 88.9% xylose conversion at 72 h (Figure 5). Ethanol titer reached 28.4 g L⁻¹ at 72 h with an ethanol yield of 0.420 g g⁻¹. Strain STXQ has potential in the conversion of lignocellulosic biomass hydrolysate to ethanol. These results suggest that the strain development protocols outlined in this study are effective in obtaining robust xylose-fermenting yeast strains for industrial applications.

5. Conclusions

A recombinant haploid strain containing plasmids harboring two-copy PirXylA presented the best xylose utilization among the engineered yeast strains harboring episomal XI genes when xylose was used as the sole carbon source. On the other hand, the strain containing plasmids with two-copy OrpXylA exhibited the best glucose and xylose co-fermentation. Respiration-deficient 39a₂ strains harboring the OrpXylA gene were favorable for xylose fermentation and glucose-xylose co-fermentation in the engineered yeast strains. Chromosome integration of XI genes in S. cerevisiae resulted in high XI activity, high XI transcription levels, and improved xylose fermentation. Mating the respiration-efficient strain 36α₂XpXpUN with the respiration-deficient strain 39a₂XoNK resulted in a diploid recombinant S. cerevisiae strain STXQ with enhanced cell growth and xylose fermentation. Strain STXQ demonstrated superior glucose and xylose co-fermentation performance. It produced 120.6 g L⁻¹ ethanol with a volumetric productivity of 5.24 g L⁻¹ h⁻¹, the highest among those reported to-date. Such superior performance by strain STXQ is largely associated with its development process, in particular with strain adaptive evolution, XI gene chromosome integration and strain mating.