Scholarly article on topic 'Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae'

Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae Academic research paper on "Industrial Biotechnology"

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{Xylose / Fermentation / Biofuels / "Xylose isomerase" / "Xylose reductase" / "Xylitol dehydrogenase"}

Abstract of research paper on Industrial Biotechnology, author of scientific article — Xin Li, Annsea Park, Raissa Estrela, Soo-Rin Kim, Yong-Su Jin, et al.

Abstract Economical biofuel production from plant biomass requires the conversion of both cellulose and hemicellulose in the plant cell wall. The best industrial fermentation organism, the yeast Saccharomyces cerevisiae, has been developed to utilize xylose by heterologously expressing either a xylose reductase/xylitol dehydrogenase (XR/XDH) pathway or a xylose isomerase (XI) pathway. Although it has been proposed that the optimal means for fermenting xylose into biofuels would use XI instead of the XR/XDH pathway, no clear comparison of the best publicly-available yeast strains engineered to use XR/XDH or XI has been published. We therefore compared two of the best-performing engineered yeast strains in the public domain—one using the XR/XDH pathway and another using XI—in anaerobic xylose fermentations. We find that, regardless of conditions, the strain using XR/XDH has substantially higher productivity compared to the XI strain. By contrast, the XI strain has better yields in nearly all conditions tested.

Academic research paper on topic "Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae"

Accepted Manuscript

Title: Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae

Author: Xin Li Annsea Park Raissa Estrela Soo-Rin Kim Yong-Su Jin Jamie H.D. Cate

PII: DOI:

Reference:

S2215-017X(16)30002-9 http://dx.doi.org/doi:10.1016/j.btre.2016.01.003 BTRE 137

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Received date: 1-10-2015

Revised date: 4-1-2016

Accepted date: 20-1-2016

Please cite this article as: Xin Li, Annsea Park, Raissa Estrela, Soo-Rin Kim, Yong-Su Jin, Jamie H.D.Cate, Comparison of xylose fermentation by two highperformance engineered strains of Saccharomyces cerevisiae, Biotechnology Reports http://dx.doi.org/10.10167j.btre.2016.01.003

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Comparison of xylose fermentation by two high-performance engineered strains of

Saccharomyces cerevisiae

Xin Lim, Annsea Park11, Raissa Estrela1, Soo-Rin Kim2#, Yong-Su Jin2,3, Jamie H. D. Cate1,4,5* jcate@lbl.gov

department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA

2Institute for Genomic Biology, University of Illinois, Urbana, IL 61801, USA

3Department of Food and Science and Human Nutrition, University of Illinois, Urbana, IL 61801, USA

4Department of Chemistry, University of California, Berkeley, CA 94720, USA

5Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

Corresponding author.

^Present Address: Impossible Foods, Inc., Redwood City, CA 94063, USA

#Present Address: School of Food Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea

* These authors contributed equally to this work.

Graphical Abstract

Highlights

• Publicly available xylose-fermenting Saccharomyces cerevisiae were compared.

• Yeast using xylose reductase/xylitol dehydrogenase had higher ethanol production.

• Yeast using xylose isomerase had better ethanol yields from xylose.

• Yeast using xylose reductase/xylitol dehydrogenase finish fermentations faster.

• Comparisons between the two strains provide benchmarks for future engineering.

Abstract

Economical biofuel production from plant biomass requires the conversion of both cellulose and hemicellulose in the plant cell wall. The best industrial fermentation organism, the yeast Saccharomyces cerevisiae, has been developed to utilize xylose by heterologously expressing either a xylose reductase/xylitol dehydrogenase (XR/XDH) pathway or a xylose isomerase (XI) pathway. Although it has been proposed that the optimal means for fermenting xylose into biofuels would use XI instead of the XR/XDH pathway, no clear comparison of the best publicly-available yeast strains engineered to use XR/XDH or XI has been published. We therefore compared two of the best-performing engineered yeast strains in the public domain-one using the XR/XDH pathway and another using XI-in anaerobic xylose fermentations. We find that, regardless of conditions, the strain using XR/XDH has substantially higher productivity compared to the XI strain. By contrast, the XI strain has better yields in nearly all conditions tested.

Keywords: xylose; fermentation; biofuels; xylose isomerase; xylose reductase; xylitol dehydrogenase.

Xylose is the second most abundant sugar next to glucose in plant cell wall hydrolysates (Kotter and Ciriacy, 1993). Therefore, efficient and rapid utilization of xylose along with glucose is essential for economic and sustainable production of fuels and chemicals from lignocellulosic biomass (Stephanopoulos, 2007). Although Saccharomyces cerevisiae has been exclusively used for producing bioethanol, this yeast cannot ferment xylose. In order to confer xylose-fermenting capabilities into S. cerevisiae, two metabolic pathways from other microorganisms have been introduced (Hahn-Hagerdal et al., 2007, van Maris et al., 2007). An oxidoreductase pathway consisting of xylose reductase (XR) and xylitol dehydrogenase (XDH) (Kotter and Ciriacy, 1993; Tantirungkij et al., 1993), produces reduced byproducts such as xylitol and glycerol during anaerobic xylose fermentation, likely due to the difference in cofactor specificities of XR and XDH (Bruinenberg et al., 1983; Kotter and Ciriacy, 1993). XR uses NADPH, whereas XDH uses NAD+. Therefore, xylose fermentation facilitated by the XR/XDH pathway under anaerobic conditions can lead to surplus NADH production (Bruinenberg et al., 1983; Tantirungkij et al., 1993), eliciting the production of glycerol and xylitol. As a solution of the redox imbalance problem, the second pathway involving xylose isomerase (XI) pathway was proposed (Walfridsson et al., 1996). Several XIs from anaerobic fungi and bacteria have been identified that are functionally expressed in S. cerevisiae, allowing construction of xylose-fermenting S. cerevisiae using the XI pathway (Brat et al., 2009; Ha et al., 2011; Madhavan et al., 2009; Kuyper et al., 2005).

In addition to the introduction and functional expression of two different metabolic pathways for xylose consumption (XR/XDH and XI), rational and evolutionary metabolic engineering strategies have been used to construct efficient xylose-fermenting S. cerevisiae strains (Jin et al., 2005; Kim et al., 2013; Kuyper et al., 2005; Lee et al., 2014). An outstanding

question in the field is whether one pathway or the other would be preferred for large-scale fermentation (van Maris et al., 2007). However, there has not been a direct comparison of the best performing strains with each pathway available in the public domain. Such a comparison would benefit future efforts to develop bioethanol production from lignocellulosic biomass.

Combined rational and evolutionary engineering efforts have led to the generation of strains SR8 (Kim et al., 2013) and SXA-R2P-E (Lee et al., 2014), two of the best-performing strains reported to utilize XR/XDH and XI pathway, respectively. SR8 contains two or more chromosomal copies of S. stipitis XYL1 (XR), XYL2 (XDH) and XYL3 (xylulokinase, XK) each with optimized expression, along with deletion of acetaldehyde dehydrogenase, ALD6. It was evolved on xylose in the laboratory, which resulted in the loss of phosphatase Pho13 function. It was subsequently made auxotrophic for uracil (ura3-52) for additional engineering (SR8u) (Kim et al., 2013). SXA-R2P-E, also evolved on xylose, has two copies of Piromyces sp. xylA3* (an improved XI mutant), S. stipitis TAL1 (transaldolase) integrated, native XKS1 (XK) overexpressed, and GRE3 (aldose reductase) and PHO13 deleted (Lee et al., 2014). It has been recently reported that PHO13 deletion leads to up-regulation of the pentose phosphate genes including TAL1 (Kim et al., 2015).

The xylose fermentation performance of the two strains, SR8u and SXA-R2P-E, was compared in various anaerobic batch culture conditions (Table 1). Over a range of cell loadings (5-20 Optical Density (OD) at 600 nm), strain SR8u expressing XR/XDH always displayed higher rates of ethanol production when compared to strain SXA-R2P-E expressing XI (Figure 1A). This result was also true for seed cultures prepared from mid-log cultures (Figure 1B) and for fermentation carried out in both oMM and YSC media, which was used to evolve strain SXA-R2P-E (Figure 2). While xylose consumption and ethanol production rates were higher in

strain SR8u expressing the XR/XDH pathway, strain SXA-R2P-E had slightly higher ethanol yields and produced less xylitol as a byproduct (Table 1). Overall, strain SR8u showed 16-104% higher productivities (g ethanol/OD^h) and 5-19% lower yields (g ethanol/g xylose) than strain SXA-R2P-E in the tested xylose fermentation conditions.

Acetic acid is present in cellulosic hydrolysates due to the acetylation of hemicellulose in the plant cell wall (Palmqvist and Hahn-Hagerdal, 2000). However, its toxicity has inhibitory effects on fermentation (Klinke et al., 2004, Wei et al., 2013). We therefore compared xylose fermentation in the presence of 1 g/L of acetate, and found that strain SR8u consumed xylose and produced ethanol faster than strain SXA-R2P-E (Figure 3). It is notable that no acetate reduction pathway was used in these experiments. This suggests that an acetate reduction pathway could be coupled with the XR/XDH pathway to improve ethanol productivity and yield even further by not only resolving the problematic redox cofactor imbalance but also using it to drive co-consumption of xylose and acetate (Wei et al., 2013).

Here we found that in two high-performance engineered yeast strains, the XR/XDH xylose utilization pathway was capable of higher xylose consumption and ethanol production rates compared to the XI pathway, despite formation of more xylitol as a byproduct. It is worth noting that the XR/XDH pathway has been shown to produce much less xylitol or no xylitol when xylodextrins are used rather than xylose in fermentations (Li et al., 2015), or when undetoxified lignocellulose hydrolysate is used (Karhumaa et al., 2007). Thus, xylodextrin consumption by means of the XR/XDH pathway could result in yeast strains with both high ethanol productivity and yield, without the drawback of xylitol byproduct formation. Here we used media conditions chosen previously in the development of these strains, to enable comparisons to previously published results. In the future, our results can serve as a baseline for

optimizing industrially relevant media, and for in-depth metabolic engineering of industrial yeast strains. It will be important to continue benchmarking high-performance engineered yeast strains with these alternative xylose utilization pathways, to identify and remove remaining bottlenecks in yeast strains to be used in industrial applications.

Materials and Methods

Anaerobic xylose fermentation

Yeast strains SR8u or SXA-R2P-E were pre-grown aerobically to mid-log or late-log phase in oMM or YSC medium containing 2% glucose, washed 3 times with water, and re-suspended in either oMM or YSC medium, depending on the batch fermentation. Media containing 4% w/v xylose (5% in Figure 3) was inoculated with a range of starting OD values, as indicated in the figure panels, and then purged with N2. Anaerobic batch fermentations were carried out in 50 mL of oMM or YSC medium in 125 mL serum bottles, closed with butyl rubber stoppers, shaking at 220 rpm in a 30 °C shaker. At the indicated time points, 1 mL samples were removed with a syringe and pelleted. 5 ^L supernatants were analyzed by ion-exclusion HPLC to determine xylose, xylitol, glycerol, and ethanol concentrations.

During the anaerobic fermentations, we collected OD values at every time point and used the average value of each time interval for the rate calculations. Xylose consumption rate (g xylose/OD*h) and ethanol production rate (g ethanol/OD*h) were calculated from the steepest slope of the curve using the average OD of that time interval. Ethanol yield (g ethanol/g xylose) was calculated using grams of xylose and ethanol directly measured at the initial and final time points.

To account for variations from the stated initial xylose concentration of 40 g/L (50g/L in Figure 3), rate and yield calculations in Table I and the text used values measured by HPLC, including values measured for the time = 0 h time point. This addressed some variation that occurred due to the time needed to set up fermentations in parallel. Experiments to directly compare SR8u and SXA-R2P-E in a given condition and with a given OD were prepared from the same batches of reagents.

In the experiment in which strains were pre-grown to mid-log phase (Figure 1B), we observed ethanol production over the theoretical maximum. We believe this violation likely arose from cell loading of glucose, as the cells were pre-grown on media containing glucose.

Optimal minimal medium (oMM) contained 1.7 g/L YNB (Y1251, Sigma, Saint Louis, MO, USA), two-fold complete CSM, 10 g/L (NH4)2SO4, 1 g/L MgSO^7H2O, 6 g/L KH2PO4, 100 mg/L adenine hemisulfate, 10 mg/L inositol, 100 mg/L glutamic acid, 20 mg/L lysine, 375 mg/L serine and 0.1 M 2-(#-morpholino) ethanesulfonic acid (MES) pH 6.0. Yeast synthetic complete (YSC) medium contained 6.7 g/L YNB and appropriate CSM dropout mixture.

Acknowledgements

We thank Prof. Hal Alper for sharing strain SXA-R2P-E. This work was funded by the Energy Biosciences Institute.

References

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Figure Captions

Figure 1. Comparison of XI and XR/XDH pathway fermentation performance. Xylose fermentations using strain SR8u (XR/XDH pathway) and strain SXA-R2P-E (XI pathway) with (A) different starting cell loadings (5-20 OD600), (B) mid-log phase cultures as fermentation seeds. The strain and starting OD600 values are indicated above each panel. The medium used in these fermentations was oMM. Error bars represent the standard deviation of biological triplicates. Xylose fermentation samples were resolved as previously described (Li et al., 2015).

Figure 2. Xylose fermentation performance of strains SR8u (XR/XDH) and SXA-R2P-E (XI), using either oMM or YSC medium. The media, strain, and starting OD600 values are given above each panel. Error bars represent the standard deviation of biological triplicates.

Figure 3. Xylose fermentation performance of strains SR8u (XR/XDH) and SXA-R2P-E (XI) in the presence of acetate. The strain and starting OD600 values are given above each panel. The medium used in these fermentations was oMM. Error bars represent the standard deviation of biological triplicates.

Tables

Table I. Fermentation performance of the two xylose-utilizing S. cerevisiae strains.

Medium1 Seed Strain2 Xylose Ethanol Ethanol yield4

condition consumption rate3 (g xylose* OD-1*h-1) [OD]5 production rate3 (g ethanol* OD-1*h-1) [OD]5 (g ethanol*g xylose-1)

oMM late-log phase, 5 OD SR8u 0.127 [5.9] 0.063 [5.9] 0.369

oMM late-log phase, 5 OD SXA-R2P-E 0.072 [8.3] 0.038 [6.6] 0.402

oMM late-log phase, 10 OD SR8u 0.112 [12.6] 0.052 [12.6] 0.378

oMM late-log phase, 10 OD SXA-R2P-E 0.085 [14.4] 0.045 [14.8] 0.419

oMM late-log phase, 20 OD SR8u 0.102 [24.4] 0.044 [24.4] 0.390

oMM late-log phase, 20 OD SXA-R2P-E 0.046 [26.0] 0.022 [26.0] 0.430

oMM mid-log phase, 20 OD SR8u 0.097 [23.7] 0.049 [23.7] 0.392

oMM mid-log phase, 20 OD SXA-R2P-E 0.052 [21.8] 0.024 [24.6] 0.412

YSC late-log phase, 10 OD SR8u 0.129 [14.7] 0.046 [14.7] 0.378

YSC late-log phase, 10 OD SXA-R2P-E 0.077 [15.7] 0.033 [15.7] 0.453

oMM+ late-log SR8u 0.087 [11.0] 0.042 [11.0] 0.374

0.1% Ac phase, 10 OD

oMM+ late-log SXA-R2P-E 0.062 [14.6] 0.031 [14.6] 0.403

0.1% Ac phase, 10 OD

fermentations were carried out anaerobically in batch conditions using serum bottles and 40 g/L xylose.

2Strain characteristics: (1) SR8u: XYL1, XYL2, XYL3, Aald6, ura3-52, evolved, (2) SXA-R2P-E: xylA3*, XKS1, TALI, Agre3, Aphol3, evolved.

3Maximal values in g of xylose consumed or ethanol produced per OD per hour. 4Grams of ethanol yield per g of xylose.

5OD value at the maximum rate of xylose consumption or ethanol production.