Causative genes conferring acetic acid tolerance in yeast

Abstract

The present invention relates to the use of GLO1 to modulate acetic acid tolerance in yeast. More specifically, it relates to the use of a specific GLO1 allele to confer tolerance to acetic acid, and to improve the fermentation performance of yeast in the presence of acetic acid.

Claims

1-10. (canceled)

11. A xylose fermenting yeast strain comprising a GLO1 allele, which confers on the strain improved fermentation performance in the presence of at least 0.4% acetic acid in the culture medium as compared to performance of a control strain that is genetically identical except for the GLO1 allele, wherein the improved fermentation performance is manifest as a faster fermentation rate or a shorter lag period.

12. The yeast strain according to claim 11, wherein the improved fermentation performance occurs in the presence of at least 0.5%, acetic acid in the medium.

13. The yeast strain according to claim 11 wherein the improved fermentation occurs in the presence of at least 0.6% acetic acid in the medium.

14. The yeast strain according to claim 11 wherein the improved fermentation occurs in the presence of at least 0.7% acetic acid in the medium.

15. The yeast strain according to claim 11 wherein the improved fermentation occurs in the presence of at least 0.8% acetic acid in the medium.

16. The A xylose fermenting yeast strain according to claim 11, wherein the GLO1 allele is overexpressed.

17. The yeast strain according to claim 16, wherein the GLO1 allele is overexpressed as a result of: (a) incorporating more than one copy of the allele in the strain, and/or (b) the coding sequence of the allele being under control of a strong promoter.

18. The yeast strain according to claim 11 that is selected from the group consisting of Saccharomyces sp., Pichia sp., Candida sp., Pachysolen sp. and Spathaspora sp.

19. The yeast strain according to claim 18 that is a member of the species Saccharomyces cerevisiae.

20. The yeast strain according to claim 11, wherein the GLO1 allele: (a) encodes a polypeptide the amino acid sequence of which is SEQ ID NO:2; or (b) comprises a nucleic acid sequence which is SEQ ID NO:1.

21. A process for producing bioethanol, comprising culturing the yeast strain according to claim 11, in the presence of xylose to produce ethanol.

22. The process according to claim 21, wherein the bioethanol is produced from a hydrolysate of lignocellulose.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1: Small scale fermentation of RHA strains for GLO1. The sugar conversion is expressed as % weight loss.

[0018] FIG. 2: Replacement of GLO1 in ER18 by GLO1 from 16D (A) replacement of GLO1 in ER18 by a kanamycin marker; (B) removal of the kanamycin cassette (C) Tagging of GLO1 in 16D by a kanamycin cassette and isolation of the GLO1 kanamycin fragment by PCR (D) Transformation of the tagged fragment in ER18 and removal of the kanamycin cassette.

[0019] FIG. 3: Fermentation performance of the parental ER18 strain, compared with the ER18 variant in which the original inferior GLO1 allele has been replaced by a superior 16D allele

EXAMPLES

Material and Methods to the Examples

Strains Used in the Study

[0020] Ethanol red is a diploid industrial strain, and was obtained from Fermentis. The strain was sporulated, and the haploid segregants ER18 and 16D have been isolated on the base of the difference in their acetic acid resistance (see Table I)

TABLE-US-00001 TABLE I Stains used for RHA analysis GLO1 Name Genotype Stocknumber ER18 Inferior parent JT 24050 16D Superior parent JT 24211 ER18 16D Hybrid of ER18 and 16D JT 24198 BY4741 glo1::KanMX4 Isogenic to BY4741; except glo1 ::KANMX JT_a.390 16D glo1::KanMX4 colony1 Isogenic to 16D; except glo1 ::KANMX PV_T1 16D glo1::KanMX4 colony2 Isogenic to 16D; except glo1 ::KANMX PV_T2 ER18 glo1::KanMX4 colony1 Isogenic to ER18; except glo1 ::KANMX PV_T3 ER18 glo1::KanMX4 colony2 Isogenic to ER18; except glo1 ::KANMX PV_T4 ER18 16D glo1::KanMX4 colony1 Hybrid of ER18 and 16D glo1::KanMX4 colony1 PV_T5 ER18 16D glo1::KanMX4 colony2 Hybrid of ER18 and 16D glo1::KanMX4 colony2 PV_T6 ER18 glo1::KanMX4 16D colony1 Hybrid of 16D and ER glo1::KanMX4 colony1 PV_T7 ER18 glo1::KanMX4 16D colony2 Hybrid of 16D and ER glo1::KanMX4 colony2 PV_T8

Construction of RHA Strains

[0021] The reciprocal deletions were engineered in the haploid strains, after which the proper haploids were crossed to obtain the diploid hybrids. The haploid deletion strains were created by gene targeting in the parental strains 16D and ER18. Deletion cassettes were PCR amplified from genomic DNA of strain BY4741 glo1::KANMX4 (JT_a.390), obtained from the deletion collection (Winzeler et al., 1999), and primers B-2344 and B-2345. After io transformation with the lithium acetate method (Gietz et al., 1995), transformants were selected on YPD plates containing geneticin (200 mg/l). Deletion of GLO1 was confirmed by PCR with primercouple A-3863/B-2612. Of each transformed strain, two transformants were selected and subsequently crossed with the corresponding parental strain to construct the hybrid diploid strains. Mating type of the diploids was confirmed by diagnostic PCR for the MAT locus (Huxley et al., 1990).

Assessment of Acetic Acid Tolerance

[0022] Acetic acid tolerance in media containing acetic acid and glucose was evaluated by determination of the fermentation performance of yeast strains in small scale, near anaerobic batch fermentations. Yeast strains were pre-cultured in YPD medium (30 C., static incubation and 60 hour). After collection (1700 g, 2 minutes) and washing of the cells with Milli-Q water, cylindrical glass tubes containing 100 ml of YP medium supplemented with 4% w/v D-glucose and a range of acetic acid, adjusted to pH=4 with HCL or KOH, were inoculated at an OD600 of 0.3. The culture was agitated continuously at 120 rpm using a magnetic rod. The fermentations were performed at 30 C. The course of the fermentation was monitored by weighing the fermentation tubes at regular intervals.

Example 1: Screening for Superior Acetic Acid Tolerance

[0023] Ethanol Red is a diploid yeast strain that is being used for bio-ethanol production at high temperatures, showing ethanol yields of up to 18%. However, the fermentation performance of this industrial yeast strain is severely affected by acetic acid, a weak organic acid present in high quantities in lignocellulosic hydrolysates. Haploid segregants were isolated from this yeast strain and scored on acetic acid tolerance by fermentation in YPD medium supplemented with various concentrations of acetic acid. It was observed that the maximum tolerance of Ethanol io Red towards acetic acid was 0.6% (v/v) in YPD medium at a pH of 4.0. However, the lag phase was significantly prolonged by adding acetic acid to the growth medium, with a lag phase of approximately 30 hours at concentrations of 0.5% and 0.6%. The haploid Ethanol Red segregant #18 (named ER18) showed similar tolerance to acetic acid and was therefore selected for further experiments.

[0024] In order to obtain a yeast strain with high acetic acid tolerance, the in-house yeast collection and the yeast collection from the Fungal Biodiversity Centre (CBS-KNAW, Utrecht, The Netherlands) were screened under acetic acid conditions. More than 1000 yeast strains were assessed, from which strain JT 22689 showed the best performance under fermentative conditions at high acetic acid concentrations, being able to ferment glucose in the presence of 0.9% acetic acid without a lag phase (not shown). Also from this strain a haploid segregant, named 16D, could be isolated that showed a similar phenotype in terms of acetic acid tolerance.

Example 2: QTL Mapping with Pooled F1 Segregants

[0025] Mapping the genetic determinants that are responsible for the high acetic acid tolerance of 16D was initiated by crossing the haploid segregants ER18 and 16D. The resulting hybrid strain was subsequently sporulated to obtain segregants that contain a mixture of the parental genomes. Obtained segregants were subsequently screened for high acetic acid tolerance, resulting in the identification of 27 (out of 288) segregants that were able to ferment glucose in the presence of 0.9% acetic acid, which is comparable with the tolerance observed for the superior parent strain. These 27 segregants were therefore selected for pooled-segregant whole-genome sequencing analysis. Genomic DNA isolated from the two parent strains, a pool of the 27 selected segregants and a control pool of 27 randomly selected segregants was sent for custom sequencing analysis using the Illumina HiSeq2000 technology (BGI, Hong Kong, China). The sequence reads from parent strains ER18 and 16D were aligned with the reference sequence from strain S288C. A total number of 23,150 SNPs between ER18 and 16D could be identified, which were subsequently filtered according to the method described by Duitama et al. (2012). The SNP variant frequencies were calculated by dividing the number of the alternative variant by the total number of aligned reads. The calculated variant frequencies were subsequently plotted against the respective chromosomal positions. The underlying structure in the SNP variant frequencies scatterplot of a given chromosome was identified by fitting smoothing splines in the generalized linear mixed model framework, as described by Claesen et al. (2013). Variant frequencies that significantly deviate from 50% (random segregation) are indicative of genetic linkage to the phenotype.

[0026] The results from the QTL mapping show two loci on the genome with a strong linkage to the superior segregant 16D: QTL1 on chromosome XIII and a second QTL on chromosome XVI. The statistical significance of QTL1 was confirmed using the Hidden Markov Model described previously, stretching from position 181019-294166. Both QTLs were further investigated by scoring selected SNPs in the 27 individual segregants in order to precisely determine the SNP variant frequencies and the statistical significance of the genetic linkage. Using a binomial test previously described (Swinnen et al., 2012; Claessen et al., 2013), both loci were found to be statistically significant. Furthermore, the size of both QTLs could be decreased to regions stretching from roughly 224000-277000 for QTL1 on chromosome XIII, and 568000-615000 for QTL2 on chromosome XVI.

[0027] GLO1 was confirmed as causative gene for acetic acid tolerance by RHA

Example 3: Fermentation Assay of RHA Strains

[0028] FIG. 1 shows the fermentation profiles of the RHA strains for GLO1. Every point represents the average of two biological repeats. The error bars indicate the standard error of the mean.

[0029] The strains with at least one allele originating from the 16D strain show superior fermentation performance in presence of acetic acid.

Example 4: Replacement of the GLO1 Allele from Strain ER18 Byt the Allele From Stain 16D

[0030] In order to upgrade the GLO1 allele of ER18, a fragment comprising the ORF, 631 bp upstream and 44 bp downstream of the ORF of GLO1 was replaced by its 16D counterpart. The method to replace the allele comprises three steps:

[0031] 1. Deletion of the region containing the ORF of GLO1, 631 bp upstream and 44 bp downstream in ER18. Primers B-2610 and B-2609 are used to amplify the deletion cassette from plasmid pJET1,2-AttB-KANMX-AttP.

[0032] Both primers contain a 19 bp region, binding to pJET1,2-B-KANMX-P and 50 bp tails that are homologous to the nucleotides flanking the region that needs to be deleted. In the schematic representation of FIG. 2A, these homologous regions are shown as light grey boxes. After transformation, colonies will be selected on YPD plates containing geneticin (200mg/I). Hereafter, colonies were confirmed by PCR with primer couple A-3863/B-2612.

[0033] Hereafter, strain ER18 glo1::KANMX4 was transformed with plasmid pBEVY-nat-Phic31integrase. After selection on YPD plates containing nourseothricin (100 mg/l), the kanamycin marker was removed due to the action of the phage derived phiC31 integrase, leaving an AttL sequence at the recombination site (FIG. 2B).

[0034] After confirming of the loss of the KANMX marker by checking the lack of growth on YPD geneticin plates, the strain was cured of the plasmid by growing several rounds in liquid YPD medium.

[0035] 2. Next, 16D was tagged by a kanamycin marker, 631 bp upstream of GLO1. As in step 1, primers were used that contain a 19 bp region binding to pJET1,2-B-KANMX-P and 50 bp tails that are homologous to the regions flanking the location where the marker needs to be inserted. The primers used for amplification of the cassette from pJET1,2-B-KANMX-P are B-2610 and B-2827.

[0036] After transformation of this fragment, selection on YPD plates containing geneticin and confirmation of the colonies by PCR with primer couple A-3863/B-2612, genomic DNA of this strain was used as a template for amplification of the tagged GLO1_16D allele. Primers B-2965 and B-2611 were used for amplification of the tagged GLO1_16D allele. (FIG. 2C)

[0037] 3. Finally, the PCR product of the tagged GLO1_16D allele, containing the GLO1 allele of 16D linked to a KANMX cassette, was transformed in ER18 glo1::AttL, the strain obtained after step 1. After transformation of this fragment, selection on YPD plates containing geneticin and confirmation of the colonies by PCR with primer couple A-3863/B-2612, the KANMX cassette is removed by the action of the phiC31 integrase (described previously). (FIG. 2D)

Example 5: The GLO1 Allele from Strain 16D is Needed and Sufficient to Confer Acetic Acid Tolerance

[0038] The fermentation profiles of the ER18 parental strain, and the ER18 strain in which the original GLO1 allele has been replaced by an 16D GLO1 allele are shown in FIG. 3. Every point represents the average of two biological repeats. The error bars indicate the standard error of the mean. ER18 is the original inferior parent. ER18 glo1::GLO1_16D is the ER18 strain in which the GLO1 gene comprising the ORF, 631 bp upstream and 44 bp downstream of the ORF of GLO1 were replaced by its 16D counterpart.

TABLE-US-00002 TABLE II Presence of non-synonymous mutations and the corresponding codons and encoded amino acids in GLO1 from different S. cerevisiae strains for which the whole genome sequence is available. GLO1 nt nt (+106-108) aa (36) (+964-966) aa (322) ER18 ACC T CAT H 16D GCT A TAT Y S288C ACC T CAT H AWRI1631 GCT A TAT Y AWRI796 GCT A TAT Y BY4741 ACC T CAT H BY4742 ACC T CAT H CBS7960 GCT A CAT H CEN.PK113 ACC T CAT H CLIB215 GCT A TAT Y EC1118 GCT A TAT Y EC9-8 GCT A TAT Y FL100 ACC T CAT H FostersB GCT A CAT H FostersO GCT A CAT H JAY291 GCT A CAT H Kyokai7 ACC T CAT H LalvinQA23 GCT A PW5 ACT T CAT H RM11-1a GCT A TAT Y Sigma1278b ACC T CAT H T7 ACT T CAT H UC5 ACC T CAT H VL3 TAT Y Vin13 GCT A TAT Y W303 ACC T CAT H YJM269 ACC T CAT H YJM789 ACT T CAT H ZTW1 ACC T TAT Y GLO1 of strain LalvinQA23 has an early stop codon resulting in a truncated ORF that lacks amongst others nt 964-965 and codes for a truncated protein that lacks amongst others aa 322. GLO1 of strain VL3 lacks nucleotides 1-58 of the ORF and starts with the ATG at position 559-561 resulting in a shortened protein. Hence, it lacks nt 106-108 and aa 36, but not nt 964-966 and aa 322.

TABLE-US-00003 TABLE III GLO1 Promoter mutations: comparison of strains 782 775 645b 645a 562 559 531 460 431 385b 385a 384 273 230 219 135 77 64 48 ER18 A A T G C C A A T T C A C C G T 16D G G C A C A C T G A T T T T T G A G S288C A G C G T T G C C A C C A A T AWRI1631 G G C A C A C T G A T T T T T G A G AWRI796 G G C A C A C T G A T T T T T G A G T CBS7960 G G C A C A C T G A T T T A C G A G CEN. A G C G T T G C C A C C A A T PK113 CLIB215 G G C A C A C T G A T T T T T G A G EC1118 G G C A C A C T G A T T T T T G A G EC9-8 G G C A C A C T G A T T T T T G A G FL100 A G C A C T G A T T T T T G A A T FostersB G G C A C A C T G A T T T W* Y** G A G FostersO G G C A C A C T G A T T T W* Y** G A G JAY291 G G C A C A C T G A T T T A C G A G Kyokai7 A A T G C C G A T T C A C C A G T LalvinQA23 G G C A C A C T G A T T T T T G A G PWS A G C A C T G A T T T A C G A G RM11-1a G G C A C A C T G A T T T T T G A G Sigma1278b A G C G T T G C C A C C A A T T7 A G C A C T G A T T T A C G A G UC5 A A T G C C G A T T C A C C A G T VL3 G G C A C A C T G A T T T T T G A G Vin13 G G C A C A C T G A T T T T T G A G W303 A G C G T T G C C A C C A A T YJM269 A G C G T T G C C A C C A G T YJM789 A G C A C T G A T T T A C G A G ZTW1 A A T G C C G A T T C A C C A G T *W: A, T or U **Y: C, T or U

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