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. 2013 Apr 3;6(1):48.
doi: 10.1186/1754-6834-6-48.

Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

Daniel González-Ramos  1 , Marcel van den Broek, Antonius Ja van Maris, Jack T Pronk, Jean-Marc G Daran
Affiliations

Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

Daniel González-Ramos et al. Biotechnol Biofuels. .

Abstract

Background: n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance.

Results: By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols.

Conclusions: This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance.

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Figures

Figure 1
Figure 1
Reverse engineering cycle for butanol tolerance in S. cerevisiae. The figure represents the evolutionary engineering strategy used in this study. It comprises three phases a) generation of biodiversity by evolutionary engineering and screening for improved isolates (1- and 2-), b) analysis of evolved genomes and identification of genetic variations (SNV: Single Nucleotide Variation, INDELS: INsertion - DELetion and CNV: Copy Number Variation) (3-) and c) reverse genetic engineering of detected variation in a “naive” genetic background and characterization of the engineered strain (5-, 6-). Prioritization of the variations prior reintroduction in naive reference was guided by sequence analysis of offspring from three consecutive back crossing (4-).
Figure 2
Figure 2
Specific growth rate and final OD660 measured after 48 h of incubation of the reference strains BY4741 (A) and CEN.PK113-7D (B) in 96 well plates in synthetic medium containing n-butanol concentrations ranging from 0 to 1.9% (v/v). □: OD660, ▲: μ (h-1). The data presented are average and standard deviation of at least eight biological replicates.
Figure 3
Figure 3
An example of screening for n-butanol tolerance of a single deletion strain in the CEN.PK background, comparing IMK356 ( pre9 Δ) (●) and CEN.PK113-7D (□). The strains were grown in 96 well plates in synthetic medium containing different concentrations of n-butanol, and the OD660 was measured after 48 h. Each point represents the average final OD660 and standard deviation for each n-butanol concentration, calculated from at least 16 independent cultures.
Figure 4
Figure 4
Hierarchical map of the GO complete categories found enriched in the set of 35 genes whose deletion leads to higher n-butanol sensitivity phenotype in both BY4741 and CEN.PK113-7D strains. Pink boxes denote enriched (QFDR< 0.05) GO term categories related to formation of multivesicular bodies, and the purple boxes enriched (QFDR< 0.05) GO term categories related to ubiquitin-proteasome system based on Fisher exact statistics.
Figure 5
Figure 5
Growth of the strains IMI088 (overexpressing YLR224W) (●) and CEN.PK113-7D (□) in the presence of n-butanol. A: Growth rate in synthetic medium containing n-butanol concentrations ranging from 1% to 1.7%. The data represent the average apparent growth rate μ (h-1) and standard deviation of a minimum of four independent cultures. B: Growth in the presence of 1.33% n-butanol. The values correspond to the average final OD660 of three independent cultures and the standard deviation of replicate cultures.
Figure 6
Figure 6
Evolutionary engineering of n-butanol tolerance in sequential shake flask cultivation. A: OD660 measured at the end of each batch throughout the evolution process. After 55 batches (vertical line) the n-butanol concentration was raised to 1.25%. The laboratory evolution was stopped after 83 batches. B: n-Butanol tolerance of the evolved strains IMS0351 (○) and IMS0344 (●) and the reference strain CEN.PK113-7D (□). The strains were grown in 96 well plates in synthetic medium in the presence of n-butanol concentrations ranged from 0 to 1.9%. The data represent the average and the standard deviation of the biomass yield (OD660) measured after 48 h from 16 independent cultures. C: Growth (OD660) in anaerobic pH controlled-bioreactor of the strains IMS0351 (○) and CEN.PK113-7D (□) in the presence of 1.5% n-butanol. The concentration of n-butanol throughout the experiments is shown for IMS0351 (----) and CEN.PK113-7D (····).
Figure 7
Figure 7
Tolerance of the strains IMS0344 (●) and IMS0351 (○) and CEN.PK113-7D (□) to different alcohols. The strains were grown in 96 well plates containing synthetic medium with increasing concentrations of isobutanol, 2-butanol, propanol and ethanol. The strains were grown in 96 well plates in synthetic medium in the presence of different concentration of alcohols. The data represent the average and the standard deviation of the biomass yield (OD660) measured after 48 h from 16 independent cultures. A: Growth in the presence of isobutanol, B: Growth in the presence of 2-butanol, C: Growth in the presence of propanol, D: Growth in the presence of ethanol.
Figure 8
Figure 8
Segregation of the mutations found in the evolved n-butanol tolerant strains IMS0344 and IMS0351 and the F1, F2 and F3 generation of back crossing. The evolved strains IMS0344 and IMS0351 were crossed with IMK439 (MATα isogenic of CEN.PK113-7D and deleted in URA3) to create a diploid. The diploid was sporulated, one haploid segregant with the same tolerance as the evolved strain was selected and the cycle repeated was repeated three times.
Figure 9
Figure 9
n-Butanol tolerance of the strains IMI218 (containing the rpn4-1 allele) (▲), IMI238 (containing the rtg1-1 allele) (◊), CEN.PK113-7D (□) and the evolved strain IMS0344 (●). The data represent the average and the standard deviation of the biomass yield (OD660) measured after 48 h from at least 16 independent cultures in presence of different concentration of n-butanol.
Figure 10
Figure 10
Protein degradation under butanol stress. The gene deletion that resulted in strains with a reduced butanol tolerance are shown in red. After their ubiquitination, membrane proteins are internalized via endocytosis and endocytic vesicles fuse with the membrane of Multivesicular Bodies (MBVs). In the MBVs, proteins are deubiquitinated and sorted into vesicules. When the membrane of the MBVs fuses with the vacuole, releases the vesicles that are degraded by vacuolar hydrolases. Ubiquitinated cytosolic proteins are degraded in the proteasome, producing small peptides and free ubiquitin. Rpn4p induces the expression of proteasome genes including PRE9, and was found to be relevant for butanol tolerance in the evolutionary engineering approach.
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