Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

TLDR: These genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi and suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass.

Abstract: Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

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Table 1 Assembly and gene model statistics 23

Fig. 6). The fact that these organisms have maintained a diverse array of GH61 genes throughout 3

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Lawrence Berkeley National Laboratory
Recent Work
Title
Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora
thermophila and Thielavia terrestris
Permalink
https://escholarship.org/uc/item/90w6c49d
Authors
Berka, Randy M.
Grigoriev, Igor V.
Otillar, Robert
et al.
Publication Date
2011-10-02
eScholarship.org Powered by the California Digital Library
University of California

Comparative genomic analysis of the thermophilic biomass-degrading fungi
Myceliophthora thermophila and Thielavia terrestris
Randy M Berka
1,15
, Igor V Grigoriev
2,15
, Robert Otillar
2
, Asaf Salamov
2
, Jane Grimwood
3
, Ian
Reid
4
, Nadeeza Ishmael
4
, Tricia John
4
, Corinne Darmond
4
, Marie-Claude Moisan
4
, Bernard
Henrissat
5
, Pedro M Coutinho
5
, Vincent Lombard
5
, Donald O Natvig
6
, Erika Lindquist
2
,
Jeremy Schmutz
3
, Susan Lucas
2
, Paul Harris
1
, Justin Powlowski
4
, Annie Bellemare
4
, David
Taylor
4
, Gregory Butler
4
, Ronald P de Vries
7,8
, Iris E Allijn
7
, Joost van den Brink
7
, Sophia
Ushinsky
4
, Reginald Storms
4
, Amy J Powell
9
, Ian T Paulsen
10
, Liam D H Elbourne
10
, Scott E
Baker
11
, Jon Magnuson
11
, Sylvie LaBoissiere
12
, A John Clutterbuck
13
, Diego Martinez
6, 14
,
Mark Wogulis
1
, Alfredo Lopez de Leon
1
, Michael W Rey
1
& Adrian Tsang
4,15
1
Novozymes, Inc., Davis, California, USA.
2
US Department of Energy Joint Genome Institute,
Walnut Creek, California, USA.
3
HudsonAlpha Institute for Biotechnology, Huntsville,
Alabama, USA.
4
Centre for Structural and Functional Genomics, Concordia University,
Montreal, Quebec, Canada.
5
Architecture et Fonction des Macromolécules Biologiques,
CNRS/Universités de Provence/Université de la Mediterranée, Marseille, France.
6
Department of Biology, University of New Mexico, Albuquerque, New Mexico, USA.
7
CBS-
KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.
8
Microbiology and Kluyver
Centre for Genomics of Industrial Fermentation, Utrecht University, Utrecht, The
Netherlands.
9
Sandia National Laboratory, Albuquerque, New Mexico, USA.
10
Department of
Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
11
Fungal
Biotechnology Team, Pacific Northwest National Laboratory, Richland, Washington, USA.
12
McGill University and Génome Québec Innovation Centre, Montreal, Canada.
13
University
of Glasgow, Glasgow, UK.
14
Present address: Broad Institute of MIT & Harvard, Cambridge,
Massachusetts USA.
15
These authors contributed equally to this work.
October 2011
The work conducted by the U.S. Department of Energy Joint Genome Institute is supported
by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-
05CH11231

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and opinions of authors expressed herein do not necessarily state or reflect those of the
United States Government or any agency thereof or The Regents of the University of
California.

1
1
2
3
4
Comparative genomic analysis of the thermophilic biomass-degrading 5
fungi Myceliophthora thermophila and Thielavia terrestris 6
Randy M Berka
1,15
, Igor V Grigoriev
2,15
, Robert Otillar
2
, Asaf Salamov
2
, Jane Grimwood
3
, Ian Reid
4
, Nadeeza 7
Ishmael
4
, Tricia John
4
, Corinne Darmond
4
, Marie-Claude Moisan
4
, Bernard Henrissat
5
, Pedro M Coutinho
5
, Vincent 8
Lombard
5
, Donald O Natvig
6
, Erika Lindquist
2
, Jeremy Schmutz
3
, Susan Lucas
2
, Paul Harris
1
, Justin Powlowski
4
, 9
Annie Bellemare
4
, David Taylor
4
, Gregory Butler
4
, Ronald P de Vries
7,8
, Iris E Allijn
7
, Joost van den Brink
7
, Sophia 10
Ushinsky
4
, Reginald Storms
4
, Amy J Powell
9
, Ian T Paulsen
10
, Liam D H Elbourne
10
, Scott E Baker
11
, Jon 11
Magnuson
11
, Sylvie LaBoissiere
12
, A John Clutterbuck
13
, Diego Martinez
6, 14
, Mark Wogulis
1
, Alfredo Lopez de 12
Leon
1
, Michael W Rey
1
& Adrian Tsang
4,15
13
1
Novozymes, Inc., Davis, California, USA.
2
US Department of Energy Joint Genome Institute, Walnut Creek, 14
California, USA.
3
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, USA.
4
Centre for Structural and 15
Functional Genomics, Concordia University, Montreal, Quebec, Canada.
5
Architecture et Fonction des 16
Macromolécules Biologiques, CNRS/Universités de Provence/Université de la Mediterranée, Marseille, France. 17
6
Department of Biology, University of New Mexico, Albuquerque, New Mexico, USA.
7
CBS-KNAW Fungal 18
Biodiversity Centre, Utrecht, The Netherlands.
8
Microbiology and Kluyver Centre for Genomics of Industrial 19
Fermentation, Utrecht University, Utrecht, The Netherlands.
9
Sandia National Laboratory, Albuquerque, New 20
Mexico, USA.
10
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia. 21
11
Fungal Biotechnology Team, Pacific Northwest National Laboratory, Richland, Washington, USA.
12
McGill 22
University and Génome Québec Innovation Centre, Montreal, Canada.
13
University of Glasgow, Glasgow, UK. 23
14
Present address: Broad Institute of MIT & Harvard, Cambridge, Massachusetts USA.
15
These authors contributed 24
equally to this work. Correspondence should be addressed to A.T. (tsang@gene.concordia.ca
). 25
26
Received 16 May; accepted 18 August; published online 02 October 2011; doi:10.1038/nbt1976 27
28
Thermostable enzymes and thermophilic cell factories may afford economic advantages in 29
the production of many chemicals and biomass-based. Here we describe and compare the 30
genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To 31

2
our knowledge, these genomes are the first described for thermophilic eukaryotes and the 1
first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and 2
experimental data suggest that both thermophiles are capable of hydrolyzing all major 3
polysaccharides found in biomass. Examination of transcriptome data and secreted 4
proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose 5
and xylan but distinct mechanisms in pectin degradation s. Characterization of the 6
biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are 7
highly efficient in biomass decomposition at both moderate and high temperatures. 8
Furthermore, we present evidence suggesting that aside from representing a potential 9
reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using 10
classical and molecular genetics. 11
Rapid, efficient and robust enzymatic degradation of biomass-derived polysaccharides is 12
currently a major challenge for biofuel production. A prerequisite is the availability of enzymes 13
that hydrolyze cellulose, hemicellulose and other polysaccharides into fermentable sugars at 14
conditions suitable for industrial use. The best studied and most widely used cellulases and 15
hemicellulases are produced by Trichoderma, Aspergillus and Penicillium species, and they are 16
most effective over a temperature range from 40 °C to ~50 °C. At these temperatures, complete 17
saccharification of biomass polysaccharides (>90% conversion to fermentable sugars) requires 18
long reaction times, during which hydrolysis reactors are susceptible to contamination. One way 19
to overcome these obstacles is to raise the reaction temperature, thereby increasing hydrolytic 20
rates and reducing contamination risks. However, implementing higher reaction temperatures 21
requires the deployment of enzymes that are more thermostable than the available preparations 22
from mesophilic fungi. Additional advantages of elevated hydrolysis temperatures include 23
enhanced mass transfer, reduced substrate viscosity, and the potential for enzyme recycling
1
. 24
Thermophilic fungi represent a potential reservoir of thermostable enzymes for industrial 25
applications. They can also potentially be developed into cell factories to support production of 26
chemicals and materials at elevated temperatures. Enzymes from thermophilic fungi often 27
tolerate higher temperatures than enzymes from mesophilic species, and some show stability at 28
70–80 °C
1,2
. Notably, it has been reported the cellulolytic activity of some thermophilic species 29
was several times higher than that of the most active cellulolytic mesophiles
3
. Furthermore, 30
biomass-degrading enzymes from thermophilic fungi consistently demonstrate higher hydrolytic 31

Frequently asked questions

Q1. What was the function to determine if the splice junctions were overlapping?

To pass the filter, the splice junctions needed to have introns with GT-AG, GC-AG, or AT-AC donor-acceptor pairs, and to score above 0.5 in a discriminant function that combined the donoracceptor type, the number and distribution of reads spanning the junction, the number of reads mapping inside the intron relative to the number of spanning reads, and the presence of overlapping splice junctions. 

Q2. Why was a single representative model chosen for each locus?

Because 6multiple gene models were generated for each locus, a single representative model was 7algorithmically chosen based on model quality. 

Q3. What are the roles of chitinases in fungi?

In fungi, enzymes which break down chitin (collectively termed chitinases) are believed to have autolytic, nutritional, morphogical and mycoparasitic roles. 

Q4. How many peptidases were identified in each genome?

A total of approximately 150 peptidase sequences were identified in each genome (143 in T. terrestris and 159 in M. thermophila). 

Q5. What are the major cell wall polysaccharides in A. nidul?

In filamentous ascomycetes the major cell wall polysaccharides include chitin, 1,3-β-glucan, 1,3- β-/1,4-β-glucan, and 1,3-α-glucan. 

Q6. What was the common method of predicting multigene families?

Multigene families were predicted with the Markov clustering algorithm (MCL) 46, using BLASTp alignment scores between proteins as a similarity metric. 

Q7. how many chromatin remodeling proteins are found in m. thermophila?

Forty-six chromatin remodeling proteins were identified for both M. thermophila and T. terrestris, ten members of the SWI/SNF complex, four condensins, ten SAGA complex factors, six INO80 complex factors and one from the FACT complex 9, 10, 11. 

Q8. What are the common strategies for adaptation of the lipid membrane to tolerance of high or low temperatures?

Common strategies for adaptation of the lipid membrane to tolerance of high or low temperatures include changes in sterol content, the ratio of saturated to unsaturated fatty acids, or alterations to fatty acid chain length 18, 19, 20. 

Q9. What was the recombination of the cDNAs used to transform A.?

The amplified 6cDNAs were cloned into the A. niger expression vector using the Gateway recombination 7method (Invitrogen) and used to transform A. niger. 

Q10. How many SOD genes are in C. globosum?

There are a total of six SOD genes in both C. globosum and T. terrestris, whereas, M. thermophila has five, missing the Cu-Zn SOD orthologue that is predicted to be secreted. 

Q11. How many substitutions were found when comparing thermophilic pairs?

the authors also found 20 significantly asymmetric substitutions when comparing thermophilic pairs: M. thermophila and T. terrestris. 

Q12. What is the way to analyze amino acid adaptations in thermophiles?

Another approach to analyze the potential amino acid adaptations in thermophiles is to align closely related thermophilic and mesophilic proteins to detect substitutional asymmetry, i.e., when certain aligned amino acids appear to occur substantially more often in either mesophilic or thermophilic proteins 4, 5, 6. 

Q13. How many pairs of substitutions were found in the BLAST?

For each thermophilic - mesophilic pair (M.t - C.g and T.t - C.g) the authors found correspondingly 29 and 36 pairs of substitutions (out of 190) with significant deviation from an expected 1:1 ratio (Bonferroni-corrected chi-square test P < 10-5). 

Q14. How many additional sequences were identified in each genome?

There were approximately 50 additional sequences in each genome identified as peptidases only by INTERPROSCAN and not by MEROPS batch BLAST. 

Q15. What are the reasons why a cluster may have more than one consensus sequence?

Clusters may have more than one consensus sequence for various reasons to include; the clone has a long insert, clones are splice variants or consensus sequences are erroneously not assembled. 

Q16. Why is there less number of transporters in M. thermophila than in T. terre?

The lower number of transporters encoded by M. thermophila compared with T. terrestris is largely due to decreased numbers of paralogues in large transporter families, for example, 86 M. thermophila MFS transporters compared to 221 members in T. terrestris. 

Q17. How many of the 40 M. thermophila proteins had signal sequences?

In contrast, only a quarter of serine peptidases, a fifth of metallo-peptidases and a tenth of cysteine peptidases had signal sequences. 

Q18. Why do the authors find asymmetric substitutions in the Trichoderma species?

Because none of the Trichoderma species are thermophilic and because many asymmetric substitutions coincide in the two analyses, the authors conclude that most of asymmetric substitutions are probably not related to high temperature adaptability. 

Q19. How many transporters are encoded by M. thermophila?

there are less than half as many ATP Binding Cassette (ABC) Superfamily transporters encoded by the M. thermophila genome (19 versus 44).