10 Filamentous Fungi as Hosts for Heterologous Production of
Proteins and Secondary Metabolites in the Post-Genomic Era
JAKOB K. H. RENDSVIG
1
, MALGORZATA E. FUTYMA
1 , ZOFIA D. JARCZYNSKA
1
,
UFFE H. MORTENSEN
1
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
II. Expression Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 228
A. Construction of Simple Gene-Expression
Cassettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
B. Introducing Gene-Expression Cassettes into
Fungal Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
C. Bio-Block-Based Multi-GOI Expression
Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
III. Bio-Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
A. Gene of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
B. Selection Markers . . . . . . . . . . . . . . . . . . . . . . . . . 234
C. Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
D. Terminators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
E. Protein Tags and Linkers . . . . . . . . . . . . . . . . . . 242
IV. Heterologous Protein Production in
Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
A. Engineering the Secretory Pathway . . . . . . . . 244
C. Building Blocks for Protein Synthesis . . . . . 248
D. Protease-Deficient Strains . . . . . . . . . . . . . . . . . 249
V. Heterologous Production of Secondary
Metabolites in Filamentous Fungi . . . . . . . . . . . 249
A. Challenges in Heterologous Secondary
Metabolite Production . . . . . . . . . . . . . . . . . . . . 250
B. Secondary Metabolite Discovery via
Different Gene-Expression Systems . . . . . . . . 252
VI. Concluding Remarks and Perspectives . . . . . . 254
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
I. Introduction
Fungi are well-established producers of foods,
food additives, industrial enzymes, and pharmaceutical drugs and contribute significantly
to human health and economy. Production of
industrial enzymes alone had an annual worth
of 3.5 billion in 2015, which is a mere fraction of
the general white biotechnology products estimated to reach 450 billion in 2020 (Meyer et al.
2016). The vast majority of fungal processes are
performed with species that have not been
genetically engineered. Hence, the fungi have
either been domesticated through millennials
of human use, or their performance and yields
have been improved via classical genetic methods like mutagenesis. The importance of fungi
has sparked several multi-species genome
sequencing projects where one aim is to
uncover novel fungal genes involved in enzyme
secretion and secondary metabolite (SM) formation. The global fungal diversity currently
encompasses 144,000 classified species and
estimates reaching 3.8–6.0 million species (Taylor et al. 2014; Willis 2018), of which ~1500
species have been genome sequenced (de
Vries et al. 2018; Grigoriev et al. 2014). As
such, it is becoming increasingly clear that
fungi represent a vast reservoir of potentially
useful enzymes and secondary metabolites yet
to be discovered. As part of the sequencing
project of genus Aspergillus, Vesth et al. analyzed the genomes of 36 species of Aspergillus
section Nigri, predicting 40,424 unique genes,
including 17,903 carbohydrate-active enzymes
and an average of 70 putative secondary metabolite gene clusters per species (near 2700 total),
comprising 450 distinct compound classes
(Vesth et al. 2018). With the increasing number
of fungal genome sequences, many new genes
and potentially interesting products will be
uncovered in this post-genomic era.
However, many of these new products are
likely made by natural fungal producers that
are challenging to incorporate into industrial
1 Technical University of Denmark, Kgs. Lyngby, Denmark;
e-mail: jakren@dtu.dk; malfu@dtu.dk; zofja@bio.dtu.dk;
um@bio.dtu.dk
Genetics and Biotechnology, 3 rd Edition
The Mycota II
J.P. Benz, K. Schipper (Eds.)
© Springer Nature Switzerland AG 2020
Proteins and Secondary Metabolites in the Post-Genomic Era
JAKOB K. H. RENDSVIG
1
, MALGORZATA E. FUTYMA
1 , ZOFIA D. JARCZYNSKA
1
,
UFFE H. MORTENSEN
1
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
II. Expression Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 228
A. Construction of Simple Gene-Expression
Cassettes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
B. Introducing Gene-Expression Cassettes into
Fungal Hosts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
C. Bio-Block-Based Multi-GOI Expression
Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
III. Bio-Blocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
A. Gene of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
B. Selection Markers . . . . . . . . . . . . . . . . . . . . . . . . . 234
C. Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
D. Terminators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
E. Protein Tags and Linkers . . . . . . . . . . . . . . . . . . 242
IV. Heterologous Protein Production in
Filamentous Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
A. Engineering the Secretory Pathway . . . . . . . . 244
C. Building Blocks for Protein Synthesis . . . . . 248
D. Protease-Deficient Strains . . . . . . . . . . . . . . . . . 249
V. Heterologous Production of Secondary
Metabolites in Filamentous Fungi . . . . . . . . . . . 249
A. Challenges in Heterologous Secondary
Metabolite Production . . . . . . . . . . . . . . . . . . . . 250
B. Secondary Metabolite Discovery via
Different Gene-Expression Systems . . . . . . . . 252
VI. Concluding Remarks and Perspectives . . . . . . 254
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
I. Introduction
Fungi are well-established producers of foods,
food additives, industrial enzymes, and pharmaceutical drugs and contribute significantly
to human health and economy. Production of
industrial enzymes alone had an annual worth
of 3.5 billion in 2015, which is a mere fraction of
the general white biotechnology products estimated to reach 450 billion in 2020 (Meyer et al.
2016). The vast majority of fungal processes are
performed with species that have not been
genetically engineered. Hence, the fungi have
either been domesticated through millennials
of human use, or their performance and yields
have been improved via classical genetic methods like mutagenesis. The importance of fungi
has sparked several multi-species genome
sequencing projects where one aim is to
uncover novel fungal genes involved in enzyme
secretion and secondary metabolite (SM) formation. The global fungal diversity currently
encompasses 144,000 classified species and
estimates reaching 3.8–6.0 million species (Taylor et al. 2014; Willis 2018), of which ~1500
species have been genome sequenced (de
Vries et al. 2018; Grigoriev et al. 2014). As
such, it is becoming increasingly clear that
fungi represent a vast reservoir of potentially
useful enzymes and secondary metabolites yet
to be discovered. As part of the sequencing
project of genus Aspergillus, Vesth et al. analyzed the genomes of 36 species of Aspergillus
section Nigri, predicting 40,424 unique genes,
including 17,903 carbohydrate-active enzymes
and an average of 70 putative secondary metabolite gene clusters per species (near 2700 total),
comprising 450 distinct compound classes
(Vesth et al. 2018). With the increasing number
of fungal genome sequences, many new genes
and potentially interesting products will be
uncovered in this post-genomic era.
However, many of these new products are
likely made by natural fungal producers that
are challenging to incorporate into industrial
1 Technical University of Denmark, Kgs. Lyngby, Denmark;
e-mail: jakren@dtu.dk; malfu@dtu.dk; zofja@bio.dtu.dk;
um@bio.dtu.dk
Genetics and Biotechnology, 3 rd Edition
The Mycota II
J.P. Benz, K. Schipper (Eds.)
© Springer Nature Switzerland AG 2020