Superior promoters for fungal protein production

Abstract

The present invention relates to novel promoters for expression in filamentous fungi. The present invention further relates to expression constructs and expression vectors comprising the novel promoters as well as transgenic microorganisms comprising such expression constructs and expression vectors, as well as to methods for producing such expression constructs, expression vectors, and transgenic microorganisms and related uses. The present invention further relates to methods of production or expression of a nucleotide sequence of interest.

Description

[0001] Superior Promoters for Fungal Protein Production

[0002] Field of the Invention

[0003] The present invention relates to novel promoters for expression in filamentous fungi. The present invention further relates to expression constructs and expression vectors comprising the novel promoters, as well as transgenic microorganisms comprising such expression constructs and expression vectors, as well as to methods for producing such expression constructs, expression vectors, and transgenic microorganisms and related uses. The present invention further relates to methods of production or expression of a nucleotide sequence of interest.

[0004] Introduction

[0005] Biocatalytical and fermentative production of recombinant proteins and fine chemicals has been applied in industrial scale for a long time. In recent years, demand for products derived from biological processes is increasing. Various organisms have been used in such production including bacteria and fungi. Especially filamentous fungi, although difficult to transform and modify genetically have been used more and more as they have been proven to be highly efficient in biocatalytical and fermentative production.

[0006] Filamentous fungi have been shown to be excellent hosts for the production of a variety of proteins. Fungal strains, such as Aspergillus, Trichoderma, Penicillium and Thermothelomyces, have been applied in the industrial production of a wide range of enzymes, since they can secrete large amounts of protein into the fermentation broth. The protein-secreting capacity of these fungi makes them preferred hosts for the targeted production of specific enzymes or enzyme mixtures.

[0007] Wild type Thermothelomyces thermophilus (T. thermophilus) C1 (also known as Myceliophthora thermophila, previously described as Chrysosporium lucknowense) is a thermotolerant ascomycetous filamentous fungus, which has been described as being attractive for production of cellulases and other proteins on a commercial scale. Only recently, the strain has also been shown to be highly efficient for producing various fine chemicals, e.g. in WO 2020 / 161682 it is disclosed that T. thermophilus C1 is capable of producing cannabinoids and precursors thereof. WO 2000 / 20555 and US 2012 / 0005812 disclose transformation systems for T. thermophilus C1 and describe expressing and secreting heterologous proteins or polypeptides. Also disclosed is a process for producing large amounts of polypeptides or proteins in an economical manner. WO 2015 / 004241 discloses multiple proteases deficient filamentous fungal cells and methods useful for the production of heterologous proteins.

[0008] However, for strong expression of recombinant proteins in filamentous fungi, especially in T. thermophilus C1, only a limited number of suitable promoters are known in the art (e.g. Visser et al 2011, Industrial Biotechnology 7 (3), US2014 / 127788). There is a need for further regulatory elements functional in T. thermophilus C1 that allow robust and strong expression systems of recombinant proteins. Brief Summary of the Invention

[0009] In the art, between 1 and 1.5 kb of the intergenic region preceding, i.e., upstream of the start codon of any ORF would be considered to contain the promoter of said gene in filamentous fungi (see e.g., Brink et al. Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation. Microb Cell Fact. 2023 Aug 19;22(1):160; Blumhoff et al. Six novel constitutive promoters for metabolic engineering of Aspergillus niger. Appl Microbiol Biotechnol. 2013 Jan;97(1):259-67; Bando et al. Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture. Appl Microbiol Biotechnol. 2011 Nov;92(3):561-9; Polli F, Meijrink B, Bovenberg RAL, Driessen AJM. New promoters for strain engineering of Penicillium chrysogenum. Fungal Genet Biol. 2016 Apr; 89:62-71 including suppl. data). However, the inventors surprisingly found that longer (i.e., including at least 2kb or even the entire intergenic region) promoters based on the Thermothelomyces thermophilus C1 alp1 promoter are capable of directing strong and robust expression in filamentous fungi of the phylum Ascomycota, especially T. thermophilus C1 , at higher levels than a 1 kb or 1.5kb promoter tested at identical conditions.

[0010] In a first aspect, the invention provides an isolated promoter capable of conferring expression in a microorganism of the phylum Ascomycota comprising a nucleotide sequence of at least 2300 bp selected from the group consisting of:

[0011] (i) a nucleotide sequence of at least consecutive 2300 bp, wherein the nucleotide sequence comprises at least positions 1278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; and

[0012] (ii) a nucleotide sequence having at least 90% sequence identity over its entire length to a nucleotide sequence of (i).

[0013] In an embodiment, the isolated promoter comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 25 and 28-35.

[0014] In a second aspect, the invention provides an expression construct comprising an isolated promoter according to the first aspect of the invention operably linked to a heterologous nucleic acid encoding a protein of interest.

[0015] In a third aspect, the invention provides an expression vector comprising an isolated promoter according to the first aspect of the invention or the expression construct according to the second aspect of the invention.

[0016] In a fourth aspect, the invention provides a method for producing an expression construct, said method comprising operably linking an isolated promoter according to the first aspect of the invention to a heterologous nucleic acid encoding a protein of interest.

[0017] In a fifth aspect, the invention provides a process for producing a vector, said method comprising linking an isolated promoter according to the first aspect of the invention or an expression construct according to the second aspect of the invention to a vector.

[0018] In a sixth aspect, the invention provides a use of the isolated promoter of the first aspect of the invention for the production of an expression construct or an expression vector.

[0019] In a seventh aspect, the invention provides a use of the expression construct of the second aspect of the invention for the production of an expression vector.

[0020] In an eighth aspect, the invention provides a transgenic microorganism of the phylum Ascomycota comprising an expression construct according to the second aspect of the invention or an expression vector according to the third aspect of the invention.

[0021] In an embodiment of this eighth aspect of the invention, the expression construct or expression vector is integrated into the genome of said microorganism.

[0022] In a ninth aspect, the invention provides a method for producing a transgenic microorganism of the phylum Ascomycota, said method comprising introducing an isolated promoter according to the first aspect of the invention or an expression construct according to the second aspect of the invention or expression vector according to the third aspect of the invention into said microorganism.

[0023] In an embodiment of this ninth aspect of the invention, the protein of interest is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase.

[0024] In a tenth aspect, the invention provides a method for expression of a nucleotide sequence of interest in a recombinant microorganism of the phylum Ascomycota, comprising the step of cultivating the recombinant microorganism in a medium conducive for the expression of a heterologous nucleic acid encoding for a protein of interest, wherein the microorganism comprises a promoter according to the first aspect of the invention operably linked to the heterologous nucleic acid.

[0025] In an eleventh aspect, the invention provides a method for the production of a protein of interest in a recombinant microorganism of the phylum Ascomycota, comprising the steps of (i) cultivating a recombinant microorganism of the phylum Ascomycota in a medium conducive for the expression of the protein of interest, wherein the microorganism comprises a promoter according to the first aspect of the invention operably linked to a heterologous nucleic acid encoding said protein of interest, and

[0026] (ii) recovering said protein of interest.

[0027] Brief Description of the Figures

[0028] Figure 1 shows the relative phytase activity in % of the different phytase production strains after fermentation at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 48 hours, 72 hours or 96 hours. W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (1046 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 1046 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (1495 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 1495 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2517 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2517 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2717 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2717 bp_CC alp1 promoter) I W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2817 bp_CC)- phytase-Tcbhl (overexpression of phytase gene driven by 2817 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2917 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2917 bp_CC alp1 promoter) I \ L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (3017 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 3017 bp_CC alp1 promoter) I W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (3117 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 3117 bp_CC alp1 promoter) / W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (3317 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 3317 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (3575 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 3575 bp_CC al p1 promoter).

[0029] Figure 2 shows the relative alpha-amylase activity in % of the different alpha-amylase production strains after fermentation at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 96 hours. W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (1495 bp_CC)-alpha-amylase-Tcbh1 (overexpression of alphaamylase gene driven by 1495 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2517 bp_CC)-alpha-amylase-Tcbh1 (overexpression of alpha-amylase gene driven by 2517 bp_CC al p1 promoter) I W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2917 bp_CC)-alpha-amylase-Tcbh1 (overexpression of alpha-amylase gene driven by 2917 bp_CC alp1 promoter) I \ L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (3575 bp_CC)-alpha-amylase-Tcbh1 (overexpression of alpha-amylase gene driven by 3575 bp_CC alp1 promoter).

[0030] Figure 3 shows the relative glucanase activity in % of the different glucanase production strains after fermentation at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 96 hours. W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (1495 bp_CC)-glucanase-Tcbh1 (overexpression of glucanase gene driven by 1495 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2517 bp_CC)-glucanase-Tcbh1 (overexpression of glucanase gene driven by 2517 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2917 bp_CC)-glucanase-Tcbh1 (overexpression of glucanase gene driven by 2917 bp_CC alp1 promoter) I W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (3575 bp_CC)- glucanase-Tcbhl (overexpression of glucanase gene driven by 3575 bp_CC alp1 promoter).

[0031] Figure 4 shows the relative phytase activity in % of the different phytase production strains after fermentation at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 96 hours. W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (389 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by an alp1 promoter with a length of 391 bp as disclosed in Xing Qin et al: "Myceliophthora thermophila alp1 gene, SEQ ID 3", 7 June 2022, XP093218467 with improved Kozak sequence (389 bp_CC alp1 promoter)) L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel

[0032] Acbh1 ::Palp1 (1586 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by an alp1 promoter with a length of 1588 bp as disclosed in Emalfarb Mark et al: "Chrysosporium lucknowense protease Alp1 enzyme DNA sequence, SEQ ID 1", 19 June 2008, XP093218465 with improved Kozak sequence (1586 bp_CC alp1 promoter)) I

[0033] W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (1998 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 1998 bp_CC alp1 promoter) I \ L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2098 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2098 bp_CC alp1 promoter) I W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2198 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2198 bp_CC alp1 promoter) / W1 L#1OO.I Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2298 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2298 bp_CC alp1 promoter) I \ L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2398 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2398 bp_CC alp1 promoter) I W1 L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2517 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2517 bp_CC alp1 promoter).

[0034] Figure 5 shows the relative phytase activity in % of the different phytase production strains after fermentation at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 96 hours.

[0035] W1 L#100.l Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1::Palp1 (2517 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2517 bp_CC alp1 promoter) I \ L#100.1 Apyr5 Aalpl Achil Aku70B Apep4 Aprtl Aprt2 Aprt3 Aprt4 Alam2 Atrel Acbh1 ::Palp1 (2498 bp_CC)-phytase-Tcbh1 (overexpression of phytase gene driven by 2498 bp_CC alp1 promoter).

[0036] Detailed Description of the Invention

[0037] The entire intergenic region upstream of the alp1 ORF in T. thermophilus C1 is provided in SEQ ID NO: 25. SEQ ID NO: 35 differs from SEQ ID NO: 25 in that a) G at position 841 of SEQ ID NO: 25 is replaced with a C in SEQ ID NO: 35 (“G841C”), b) C at position 2528 of SEQ ID NO: 25 is replaced with a G in SEQ ID NO: 35 (“C2528G”), c) A at position 3576 of SEQ ID NO: 25 is replaced with a C in SEQ ID NO: 35 (“A3576C”), and d) G at position 3577 of SEQ ID NO: 25 is replaced with a C in SEQ ID NO: 35 (“G3577C”).

[0038] Changes G841C and C2428G each lead to the absence of a Bsal restriction site found in SEQ ID NO: 25 in SEQ ID NO: 35 (i.e., SEQ ID NO: 35 lacks two Bsal restriction sites found in SEQ ID NO: 25). Changes A3576C and G3577C change the Kozak sequence. Without being bound to any mechanism, it is believed that this change to the Kozak sequence may constitute an improvement in the translation of the ORF: CC at these positions is most commonly found in fungi.

[0039] In a first aspect, the invention provides an isolated promoter capable of conferring expression in a microorganism of the phylum Ascomycota comprising a nucleotide sequence of at least 2300 bp selected from the group consisting of:

[0040] (i) a nucleotide sequence of at least consecutive 2300 bp, wherein the nucleotide sequence comprises at least positions 1278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; and (ii) a nucleotide sequence having at least 90% sequence identity over its entire length to a nucleotide sequence of (i).

[0041] In an embodiment the microorganism of the phylum Ascomycota is selected from the list consisting of Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma. In one such embodiment, the microorganism of the phylum Ascomycota is selected from the list consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense). In an especially preferred embodiment, the microorganism is Thermothelomyces thermophilus.

[0042] In any embodiment of the first aspect, in (i), the nucleotide sequence of at least consecutive 2300 bp, wherein the nucleotide sequence comprises at least positions 1278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35 comprises at least 2400 bp (i.e. , comprises at least positions 1178 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 2500 bp (i.e., comprises at least positions 1078 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 2600 bp (i.e., comprises at least positions 978 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 2700 bp (i.e., comprises at least positions 878 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 2800 bp (i.e., comprises at least positions 778 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 2900 bp (i.e., comprises at least positions 678 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3000 bp (i.e., comprises at least positions 578 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3100 bp (i.e., comprises at least positions 478 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3200 bp (i.e., comprises at least positions 378 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3300 bp (i.e., comprises at least positions 278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3400 bp (i.e., comprises at least positions 178 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), at least 3500 bp (i.e., comprises at least positions 78 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35), or all of the nucleotide sequence set forth in SEQ ID NO: 25 or 35.

[0043] In a preferred embodiment, in (i), the nucleotide sequence of at least consecutive 2300 bp, wherein the nucleotide sequence comprises at least positions 1278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35, comprises 2519 bp (i.e. comprises positions 1059 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 28), comprises 2719 bp (i.e. comprises positions 859 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 29), comprises 2819 bp (i.e. comprises positions 759 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 30), comprises 2919 bp (i.e. comprises positions 659 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 31), comprises 3019 bp (i.e. comprises positions 559 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 32), comprises 3119 bp (i.e. comprises positions 459 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 33), or comprises 3319 bp (i.e. comprises positions 259 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; the latter is set forth in SEQ ID NO: 34). Accordingly, in one preferred embodiment, the isolated promoter comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 25 and 28-35.

[0044] In any embodiment of the first aspect, in (ii), a nucleotide sequence having at least 90% sequence identity over its entire length to a nucleotide sequence of (i) may have at least 90%, for example at least 91%, 92%, 93%, 94%, 95%, 96% or 97%, even more preferably at least 98% most preferably at least 99% sequence identity over its entire length to a nucleotide sequence of (i) or (ii).

[0045] Preferably, the expression derived from the promoters of the invention is at least 1 ,1 time, preferably 1,2 time, more preferably 1,3 time, more preferably at least 1 ,4 time higher than the expression derived from a 1 kb or a 1.5 kb fragment of the naturally occurring promoter of alp1 gene in T. thermophilus C1 (i.e. a fragment consisting of positions 2578 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or a fragment consisting of positions 2078 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25) or the expression derived from the promoter as set forth in SEQ ID NOs: 26 or 27. Preferably, the expression is increased by 20% or more, increased by 30% or more, increased by 40% or more, increased by 50% or more, for example by 100% or more, preferably by 200% or more compared to the expression derived from a 1 kb or a 1.5 kb fragment of the naturally occurring promoter of alp1 gene in T. thermophilus C1 (i.e. a fragment consisting of positions 2578 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or a fragment consisting of positions 2078 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25) or the expression derived from the promoter as set forth in SEQ ID NOs: 26 or 27. Preferably, the expression is 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more, for example 50 fold, relative to the expression derived from a 1 kb or a 1.5 kb fragment of the naturally occurring promoter of alp1 gene in T. thermophilus C1 (i.e. a fragment consisting of positions 2578 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or a fragment consisting of positions 2078 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25) or the expression derived from the promoter as set forth in SEQ ID NOs: 26 or 27.

[0046] In a second aspect, the invention provides an expression construct comprising an isolated promoter according to the first aspect of the invention operably linked to a heterologous nucleic acid encoding a protein of interest. The heterologous nucleic acid may comprise an ORF, and / or a regulatory sequence e.g. terminator or enhancer.

[0047] In a third aspect, the invention provides an expression vector comprising an isolated promoter according to the first aspect of the invention or the expression construct according to the second aspect of the invention. The expression vector may be selected from plasmids suitable for transformation of filamentous fungal cells, preferably Thermothelomyces thermophilus cells, and in particular plasmids suitable for expression of proteins in filamentous fungal cells, preferably Thermothelomyces thermophilus cells, e.g. plasmids which are capable of autonomous replication in other organisms, preferably in bacteria, in particular E. coli, and which can be prepared, e.g. digested, for genomic insertional transformation of filamentous fungal cells, preferably Thermothelomyces thermophilus cells.

[0048] In a fourth aspect, the invention provides a method for producing an expression construct, for example an expression construct according to the second aspect of the invention, said method comprising operably linking an isolated promoter according to the first aspect of the invention to a heterologous nucleic acid encoding a protein of interest. The heterologous nucleic acid may be a sequence to be expressed in a fungal host cell. The heterologous nucleic acid may further comprise an additional regulatory sequence such as an enhancer or terminator.

[0049] In a fifth aspect, the invention provides a process for producing a vector, said method comprising linking an isolated promoter according to the first aspect of the invention or an expression construct according to the second aspect of the invention to a vector.

[0050] In a sixth aspect, the invention provides a use of the isolated promoter of the first aspect of the invention for the production of an expression construct or an expression vector.

[0051] In a seventh aspect, the invention provides a use of the expression construct of the second aspect of the invention for the production of an expression vector. In an eighth aspect, the invention provides a transgenic microorganism of the phylum Ascomycota comprising an expression construct according to the second aspect of the invention or an expression vector according to the third aspect of the invention. In an embodiment, the phylum Ascomycota comprises Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Fili basidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma, more preferably Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense), most preferably Thermothelomyces thermophilus.

[0052] In one such embodiment of this eighth aspect of the invention, the expression construct or expression vector is integrated into the genome of said microorganism, which is preferably Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes or Trichoderma, more preferably Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica or Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense), most preferably Thermothelomyces thermophilus.

[0053] In a ninth aspect, the invention provides a method for producing a transgenic microorganism of the phylum Ascomycota, said method comprising introducing an isolated promoter according to the first aspect of the invention or an expression construct according to the second aspect of the invention or expression vector according to the third aspect of the invention into said microorganism. In an embodiment, the phylum Ascomycota comprises Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma, more preferably Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense), most preferably Thermothelomyces thermophilus.

[0054] In one such embodiment of this ninth aspect of the invention, the protein of interest is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase, preferably from the group consisting of aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphneoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase and beta-xylosidase.

[0055] In a tenth aspect, the invention provides a method for expression of a nucleotide sequence of interest in a recombinant microorganism of the phylum Ascomycota, comprising the step of cultivating the recombinant microorganism in a medium conducive for the expression of a heterologous nucleic acid encoding for ae protein of interest, wherein the microorganism comprises a promoter according to the first aspect of the invention operably linked to the heterologous nucleic acid. In an embodiment, the phylum Ascomycota comprises Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma, more preferably Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense), most preferably Thermothelomyces thermophilus.

[0056] In one such embodiment of this tenth aspect of the invention, the protein of interest is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase, preferably from the group consisting of aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphneoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase and beta-xylosidase.

[0057] In an eleventh aspect, the invention provides a method for the production of a protein of interest in a recombinant microorganism of the phylum Ascomycota, comprising the steps of

[0058] (i) cultivating a recombinant microorganism of the phylum Ascomycota in a medium conducive for the expression of the protein of interest, wherein the microorganism comprises a promoter according to the first aspect of the invention operably linked to a heterologous nucleic acid encoding said protein of interest, and

[0059] (ii) recovering said protein of interest. In an embodiment, the phylum Ascomycota comprises Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma, more preferably Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense), most preferably Thermothelomyces thermophilus.

[0060] In one such embodiment of this eleventh aspect of the invention, the protein of interest is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase, preferably from the group consisting of aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, nuclease, oxidase, pectinolytic enzyme, peroxidase, phosphodiesterase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase and beta-xylosidase.

[0061] In any of the embodiments of the eleventh aspect of the invention, the step of recovering the protein of interest comprises any suitable method for separating the recombinant polypeptide from the so-called “biomass” and ingredients of the medium. Suitable separation techniques known in the art include, but are not limited to, filtration, microfiltration, ultrafiltration, centrifugation, extraction, spray drying, evaporation, freeze drying and precipitation. The protein of interest may further be purified by a variety of procedures known in the art including, but not limited to, ammonium sulphate precipitation or other protein precipitation methods, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, size exclusion chromatography or electrophoretic procedures.

[0062] DEFINITIONS

[0063] It is to be understood that this invention is not limited to the particular methodology or protocols. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a vector" is a reference to one or more vectors and includes equivalents thereof known to those skilled in the art, and so forth. The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent, preferably 10 percent up or down (higher or lower). As used herein, the word "or" means any one member of a particular list and also includes any combination of members of that list. The words "comprise," "comprising," "include," "including," and "includes" when used in this specification and in the following claims are intended to specify the presence of one or more stated features, integers, components, or steps, but they do not preclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. The term “comprising” also includes the term “consisting of”.

[0064] For clarity, certain terms used in the specification are defined and used as follows:

[0065] Additional copies: When a sequence is said to comprise “one or more additional copies” of a sequence comprised therein, this means that on top of the original sequence being copied, one or more additional copies of the copied sequence are present. For example, if one additional copy is said to be present, this means that, including the original sequence being copied, there are two copies present in total (one original copy and one additional copy). Similarly, if ten additional copies are said to be present, this means that, including the original sequence being copied, there are eleven copies present in total (one original copy and ten additional copies).

[0066] Consecutive: The term “consecutive” as used herein when referring to base pairs or nucleotides means that the base pairs or nucleotides follow each other one after the other in order. That is, if a fragment of a nucleic acid sequence is said to contain “consecutive” base pairs or nucleotides, then the base pairs or nucleotides of the fragment occur one after the other in the same order as in the parent nucleic acid sequence of the fragment.

[0067] Coding region: As used herein the terms "coding region" or “open reading frame” or “ORF” when used in reference to a structural gene refers to the nucleotide sequences that encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5'-side by the nucleotide triplet "ATG" encoding the initiator methionine and on the 3'-side by one of the three triplets specifying stop codons (i.e., TAA, TAG, TGA). Genomic forms of a gene may contain introns and / or be flanked by sequences located on both the 5'- and 3'-end of the sequences. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5'-flanking region may contain regulatory sequences such as promoters and enhancers, which control or influence the transcription of the gene. The 3'-flanking region may contain sequences that direct the termination of transcription, post-transcriptional cleavage and polyadenylation.

[0068] Complementary: "Complementary" or "complementarity" refers to two nucleotide sequences, which comprise antiparallel nucleotide sequences capable of pairing with one another (by the base-pairing rules) upon formation of hydrogen bonds between the complementary base residues in the antiparallel nucleotide sequences. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases are not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acid molecules is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid molecule strands has significant effects on the efficiency and strength of hybridization between nucleic acid molecule strands. A "complement" of a nucleic acid sequence, as used herein, refers to a nucleotide sequence whose nucleic acid molecules show total complementarity to the nucleic acid molecules of the nucleic acid sequence of interest.

[0069] Cultivating: The terms “cultivating” or “cultivation” or “culturing” or “culture” are used interchangeably herein and mean the maintenance of cells (including, but not limited to, isolated cells, primary cells, cell lines, microorganisms such as bacteria and fungi) in conditions (including, but not limited to, nutrients, temperature, pressure, oxygenation, agitation) suitable for growth. Typically, such cells are cultivated in or on an appropriate “medium” or “growth medium” or “culture medium” that provides all necessary nutrients. A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of cells. The appropriate cultivating conditions and media are specific to the type of cell being cultivated. The skilled person will be aware of suitable conditions and media to choose for a given cell type. For certain member of the phylum Ascomycota, suitable cultivating conditions and media are described in WO 2001 / 79507 A2 and WO 2017 / 93451 A1.

[0070] Endogenous: An "endogenous" nucleotide sequence refers to a nucleotide sequence, which is present in the genome of the untransformed cell.

[0071] Enhanced expression: “enhance” or “increase” the expression of a nucleic acid molecule in a cell are used equivalently herein and mean that the level of expression of the nucleic acid molecule in a cell after applying a method of the present invention is higher than its expression in the cell before applying the method, or compared to a reference cell lacking a recombinant nucleic acid molecule of the invention. The term "enhanced” or “increased" as used herein are synonymous and means herein higher, preferably significantly higher expression of the nucleic acid molecule to be expressed. As used herein, an “enhancement” or “increase” of the level of an agent such as a protein, mRNA or RNA means that the level is increased relative to a substantially identical cell grown under substantially identical conditions, lacking a recombinant nucleic acid molecule of the invention, the recombinant construct or recombinant vector of the invention. As used herein, “enhancement” or “increase” of the level of an agent, such as for example a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNA expressed by a gene of interest and / or of the protein product encoded by it (the “protein of interest”), means that the level is preferably increased by 20% or more, increased by 30% or more, increased by 40% or more, increased 50% or more, for example 100% or more, preferably 200% or more, more preferably 5 fold or more, even more preferably 10 fold or more, most preferably 20 fold or more for example 50 fold relative to a cell or organism lacking a recombinant nucleic acid molecule of the invention. The enhancement or increase can be determined by methods with which the skilled worker is familiar. Thus, the enhancement or increase of the nucleic acid or protein quantity can be determined for example by an immunological detection of the protein. Moreover, techniques such as protein assay, fluorescence, Northern hybridization, nuclease protection assay, reverse transcription (quantitative RT-PCR), ELISA (enzyme-linked immunosorbent assay), Western blotting, radioimmunoassay (RIA) or other immunoassays and fluorescence-activated cell analysis (FACS) can be employed to measure a specific protein or RNA in a cell. Depending on the type of the induced protein product, its activity or the effect on the phenotype of the cell may also be determined. Methods for determining the protein quantity are known to the skilled worker. Examples, which may be mentioned, include: the micro-Biuret method (Goa J (1953) Scand J Clin Lab Invest 5:218-222), the Folin-Ciocalteau method (Lowry OH et al. (1951) J Biol Chem 193:265-275) or measuring the absorption of CBB G-250 (Bradford MM (1976) Analyt Biochem 72:248-254). Expression: "Expression" refers to the biosynthesis of a gene product, preferably to the transcription and / or translation of a nucleotide sequence, for example an endogenous gene or a heterologous gene, in a cell. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and - optionally - the subsequent translation of mRNA into one or more polypeptides. In other cases, expression may refer only to the transcription of the DNA harbouring an RNA molecule.

[0072] Expression construct: "Expression construct" as used herein means a DNA sequence capable of directing expression of a particular nucleotide sequence in a cell, comprising a promoter functional in said cell into which it will be introduced, operatively linked to the nucleotide sequence of interest which is - optionally - operatively linked to termination signals. If translation is required, it also typically comprises sequences required for proper translation of the nucleotide sequence. The expression construct comprising the nucleotide sequence of interest may be chimeric, meaning that one or more of its components is heterologous with respect to one or more of its other components. The expression construct may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression construct is heterologous with respect to the host, i.e., the particular DNA sequence of the expression construct does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.

[0073] Foreign: The term "foreign" refers to any nucleic acid molecule (e.g., gene sequence), which is introduced into the genome of a cell by experimental manipulations and may include sequences found in that cell so long as the introduced sequence contains some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) and is therefore distinct relative to the naturally-occurring sequence.

[0074] Fragment: The term “fragment” as used herein when referring to nucleic acid sequences means a piece of consecutive nucleotides that, compared to the parent sequence, is shortened on one or both ends.

[0075] Functional linkage: The term "functional linkage" or "functionally linked" are to be understood as meaning, for example, the sequential arrangement of a regulatory element (e.g. a promoter) with a nucleic acid sequence to be expressed and, if appropriate, further regulatory elements (such as e.g., a terminator) in such a way that each of the regulatory elements can fulfil its intended function to allow, modify, facilitate or otherwise influence expression of said nucleic acid sequence. As a synonym the wording “operable linkage” or “operably linked” may be used. The expression may result depending on the arrangement of the nucleic acid sequences in relation to sense or antisense RNA. To this end, direct linkage in the chemical sense is not necessarily required. Genetic control sequences such as, for example, enhancer sequences, can also exert their function on the target sequence from positions which are further away, or indeed from other DNA molecules. Preferred arrangements are those in which the nucleic acid sequence to be expressed recombinantly is positioned behind the sequence acting as promoter, so that the two sequences are linked covalently to each other. The distance between the promoter sequence and the nucleic acid sequence to be expressed recombinantly is preferably less than 200 base pairs, especially preferably less than 100 base pairs, very especially preferably less than 50 base pairs, most preferably 0 base pairs. Functional linkage, and an expression construct, can be generated by means of customary recombination and cloning techniques as described (e.g., in Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY); Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing Assoc, and Wiley Interscience). However, further sequences, which, for example, act as a linker with specific cleavage sites for restriction enzymes, or as a signal peptide, may also be positioned between the two sequences. The insertion of sequences may also lead to the expression of fusion proteins. Preferably, the expression construct, consisting of a linkage of a promoter and nucleic acid sequence to be expressed, can exist in a vector-integrated form and be inserted into the genome, for example by transformation.

[0076] Gene: The term "gene" refers to a region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner. A gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (downstream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e. , introns) between individual coding regions (i.e. , exons). The term "structural gene" as used herein is intended to mean a DNA sequence that is transcribed into mRNA, which is then in turn translated into a sequence of amino acids characteristic of a specific polypeptide or protein of interest.

[0077] Genome and genomic DNA: The terms “genome” or “genomic DNA” is referring to the heritable genetic information of a host organism. Preferably, the terms genome or genomic DNA is referring to the chromosomal DNA of the nucleus.

[0078] Heterologous: The term "heterologous” with respect to a nucleic acid molecule or DNA refers to a nucleic acid molecule, which is operably linked to, or is manipulated to become operably linked to, a second nucleic acid molecule, e.g. a promoter, to which it is not operably linked in nature, e.g. in the genome of a WT cell, or to which it is operably linked at a different location or position in nature, e.g. in the genome of a WT cell. Preferably the term "heterologous”, with respect to a nucleic acid molecule or DNA, e.g. promoter, refers to a nucleic acid molecule which is operably linked to, or is manipulated to become operably linked to, a second nucleic acid molecule, e.g. an ORF, to which it is not operably linked in nature. A heterologous expression construct comprising a nucleic acid molecule and one or more regulatory nucleic acid molecules (such as a promoter or a transcription termination signal) linked thereto may be a construct resulting from experimental manipulations in which either a) said nucleic acid molecule, or b) said regulatory nucleic acid molecule or c) both (i.e. (a) and (b)) is not located in its natural (native) genetic environment or has been modified by experimental manipulations. Examples of modifications include a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. “Natural genetic environment” refers to the natural chromosomal locus in the organism of origin, or to the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the sequence of the nucleic acid molecule is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least at one side and has a sequence of at least 50 bp, preferably at least 500 bp, especially preferably at least 1,000 bp, very especially preferably at least 5,000 bp, in length. A naturally occurring expression construct - for example the naturally occurring combination of a promoter with the corresponding gene - becomes a transgenic expression construct when it is modified by non-natural, synthetic, “artificial” methods such as, for example, mutagenesis. Such methods have been described (US 5,565,350; WO 2000 / 15815). For example, a protein encoding a nucleic acid molecule operably linked to a promoter, which is not the native promoter of this molecule, is considered to be heterologous with respect to the promoter. Preferably, heterologous DNA is not endogenous to or not naturally associated with the cell into which it is introduced, but has been obtained from another cell or has been synthesized. “Heterologous DNA” also includes an endogenous DNA sequence that contains some modification; non- naturally occurring, multiple copies of an endogenous DNA sequence; or a DNA sequence which is not naturally associated with another DNA sequence physically linked thereto.

[0079] “Identity”: “Identity” when used in respect to the comparison of two or more nucleic acid sequences means that the sequences share a certain degree of sequence similarity, the sequences being partially identical. The percent sequence identity (% sequence identity) between two nucleic acid sequences is determined from a pairwise global sequence alignment created by using program “Needle” (as implemented in the European Molecular Biology Open Software Suite (EMBOSS), version 6.3.1.2 or later (Trends in Genetics (2000) 16 (6), p. 276- 277) with applying a gap open penalty of 10, a gap extension penalty of 0.5, and matrix EDNAFULL (which is the “Needle” EMBOSS version of matrix DNAFULL), by the following calculation: % sequence identity = (Identical Residues x 100) / (Length of Alignment showing both sequences over their complete lengths - Total Number of Gaps in Alignment), which is also reported as “Longest dentity” in the output of “Needle” from EMBOSS when parameter nobrief” is applied. A Gap in the alignment is a position in the alignment, in which one of the two sequences has no residue, and which is typically represented by a symbol in the alignment. Program “Needle” EMBOSS has implemented the algorithm of Needleman & Wunsch (J. Mol. Biol. (1979) 48, p. 443-453) for aligning two sequences.

[0080] Example:

[0081] Seq A = AAGATACTG

[0082] Seq B = GATCTGA

[0083] Producing a pairwise global alignment which is showing both sequences over their complete lengths results in:

[0084] Seq A : AAGATACTG-

[0085] I I I I I I

[0086] Seq B : — GAT-CTGA

[0087] The percent-identity then is = (Identical Residues x 100) I (Length of Alignment showing both sequences over their complete lengths - Total Number of Gaps in Alignment) = ( 6 x 100 ) / ( 10 - 4 ) = 600 / 6 = 100 %

[0088] The term “introducing”, “introduction” and the like with respect to the introduction of a donor DNA molecule in the target site of a target DNA means any introduction of the sequence of the donor DNA molecule into the target region for example by the physical integration of the donor DNA molecule or a part thereof into the target region or the introduction of the sequence of the donor DNA molecule or a part thereof into the target region wherein the donor DNA is used as template for a polymerase.

[0089] Isogenic: organisms (e.g., fungi), which are genetically identical, except that they may differ by the presence or absence of a heterologous DNA sequence.

[0090] Isolated: The term "isolated" as used herein means that a material has been removed by the hand of man and exists apart from its original, native environment and is therefore not a product of nature. An isolated material or molecule (such as a DNA molecule or enzyme) may exist in a purified form or may exist in a non-native environment such as, for example, in a transgenic host cell. For example, a naturally occurring polynucleotide or polypeptide present in a living cell is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides can be part of a vector and / or such polynucleotides or polypeptides could be part of a composition and would be isolated in that such a vector or composition is not part of its original environment. Preferably, the term "isolated" when used in relation to a nucleic acid molecule, as in "an isolated nucleic acid sequence" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source. Isolated nucleic acid molecule is nucleic acid molecule present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acid molecules are nucleic acid molecules such as DNA and RNA, which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighbouring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs, which encode a multitude of proteins. However, an isolated nucleic acid sequence comprising for example SEQ ID NO: 25 includes, by way of example, such nucleic acid sequences in cells which ordinarily contain SEQ ID NO: 25 where the nucleic acid sequence is in a chromosomal or extrachromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid sequence may be present in single-stranded or double-stranded form. When an isolated nucleic acid sequence is to be utilized to express a protein, the nucleic acid sequence will contain at a minimum at least a portion of the sense or coding strand (i.e., the nucleic acid sequence may be single-stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the nucleic acid sequence may be double-stranded).

[0091] Isolated promoter sequences as used herein have a length of max. 5000 nucleotides and can be differentiated in length from much longer isolated genome fragments comprising promoter sequences together with genes or gene fragments.

[0092] Minimal Promoter: The term “minimal promoter” refers to promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.

[0093] Naturally occurring: The term “naturally occurring” as used herein refers to cells, organisms, genes, nucleic acids, nucleic acid sequences, etc. that identically occur in nature, i.e., that are not created or influenced by the hand of man. As soon as such a naturally occurring cell, organism, gene, nucleic acid, nucleic acid sequence, etc. is changed by man, e.g. by mutation, fusion, deletion, insertion, transformation, or other means, it is no longer naturally occurring but “modified” or “engineered”. Non-coding: The term "non-coding" refers to sequences of nucleic acid molecules that do not encode part or all of an expressed protein. Non-coding sequences include but are not limited to functional RNAs, introns, enhancers, promoter regions, 3' untranslated regions, and 5' untranslated regions.

[0094] Nucleic acids and nucleotides: The terms "Nucleic Acids" and "Nucleotides" refer to naturally occurring or synthetic or artificial nucleic acid or nucleotides. The terms “nucleic acids” and "nucleotides” comprise deoxyribonucleotides or ribonucleotides or any nucleotide analogue and polymers or hybrids thereof in either single- or double-stranded, sense or antisense form. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The term "nucleic acid" is used inter-changeably herein with "gene", "cDNA, "mRNA", "oligonucleotide," and "polynucleotide". Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and / or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, substitution of 5-bromo-uracil, and the like; and 2'-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN. Short hairpin RNAs (shRNAs) also can comprise non-natural elements such as non-natural bases, e.g., inosine and xanthine, nonnatural sugars, e.g., 2'-methoxy ribose, or non-natural phosphodiester linkages, e.g., methylphosphonates, phosphorothioates and peptides.

[0095] Nucleic acid sequence: The phrase "nucleic acid sequence" refers to a single or doublestranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'- to the 3'-end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. "Nucleic acid sequence" also refers to a consecutive list of abbreviations, letters, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a relatively short nucleic acid, usually less than 100 nucleotides in length. Often a nucleic acid probe is from about 50 nucleotides in length to about 10 nucleotides in length. A "target region" of a nucleic acid is a portion of a nucleic acid that is identified to be of interest. A "coding region" of a nucleic acid is the portion of the nucleic acid, which is transcribed and translated in a sequence-specific manner to produce into a particular polypeptide or protein of interest when placed under the control of appropriate regulatory sequences. The coding region is said to encode such a polypeptide or protein of interest. Oligonucleotide: The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof, as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. An oligonucleotide preferably includes two or more nucleotide monomers covalently coupled to each other by linkages (e.g., phosphodiesters) or substitute linkages.

[0096] Overhang: An "overhang" is a relatively short single-stranded nucleotide sequence on the 5'- or 3'-hydroxyl end of a double-stranded oligonucleotide molecule (also referred to as an "extension," "protruding end," or "sticky end").

[0097] Phylum Ascomycota: The term “phylum Ascomycota” as used herein includes, but is not limited to, Acremonium, Aspergillus, Agaricus, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia, Thermoascus, Thermothelomyces, Thielavia, Tolypocladium, Trametes and Trichoderma. In particularly preferred embodiments the phylum Ascomycota includes, but is not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Neurospora crassa, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Thermothelomyces heterothallica and Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila and as Chrysosporium lucknowense). Among these, Thermothelomyces thermophilus is especially preferred.

[0098] Polypeptide: The terms "polypeptide", "peptide", "oligopeptide", "polypeptide", "gene product", "expression product" and "protein" are used interchangeably herein to refer to a polymer or oligomer of consecutive amino acid residues.

[0099] “Precise” with respect to the introduction of a donor DNA molecule in target region means that the sequence of the donor DNA molecule is introduced into the target region without any InDeis, duplications or other mutations as compared to the unaltered DNA sequence of the target region that are not comprised in the donor DNA molecule sequence.

[0100] Promoter: The terms "promoter", or "promoter sequence" are equivalents and as used herein, refer to a DNA sequence, which, when operably linked to a nucleotide sequence of interest, is capable of controlling the transcription of the nucleotide sequence of interest into RNA. A promoter is located 5' (i.e., upstream), proximal to the start codon of a nucleotide sequence of interest, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Said promoter comprises for example the at least 3.5 kb, for example 3 kb or 2 kb proximal to the transcription start site. The promoter may for example be heterologous or homologous to the respective cell. A polynucleotide sequence is "heterologous to" an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence or an allele from a different ecotype or variety). Suitable promoters can be derived from genes of the host cells where expression should occur. Activity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of a cell and detecting the expression of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene).

[0101] Purified: As used herein, the term "purified" refers to molecules, either nucleic or amino acid sequences that are removed from their natural environment, isolated, or separated. "Substantially purified" molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. A purified nucleic acid sequence may be an isolated nucleic acid sequence.

[0102] Recombinant: The term "recombinant" with respect to nucleic acid molecules refers to nucleic acid molecules produced by recombinant DNA techniques. Recombinant nucleic acid molecules may also comprise molecules that as such do not exist in nature but are modified, changed, mutated or otherwise manipulated by man. Preferably, a "recombinant nucleic acid molecule" is a non-naturally occurring nucleic acid molecule that differs in sequence from a naturally occurring nucleic acid molecule by at least one nucleic acid. A “recombinant nucleic acid molecule” may also comprise a “recombinant construct” which comprises, preferably operably linked, a sequence of nucleic acid molecules not naturally occurring in that order. Preferred methods for producing said recombinant nucleic acid molecule may comprise cloning techniques, directed or non-directed mutagenesis, synthesis, or recombination techniques.

[0103] Recovery: The term “recovery” or “recovering” refers to separating the protein of interest from the recombinant microorganism used to produce it. The recovery process of any fermentative or biocatalytically produced peptide, protein or fine chemical comprises any suitable method for separating the recombinant polypeptide from the so-called “biomass” and ingredients of the culture medium. Suitable separation techniques known in the art include, but are not limited to, filtration, microfiltration, ultrafiltration, centrifugation, extraction, spray drying, evaporation, freeze drying and precipitation. The recombinant polypeptide may further be purified by a variety of procedures known in the art including, but not limited to, ammonium sulphate precipitation or other protein precipitation methods, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, size exclusion chromatography or electrophoretic procedures.

[0104] Significant increase or decrease: An increase or decrease, for example in enzymatic activity or in gene expression, that is larger than the margin of error inherent in the measurement technique, preferably an increase or decrease by about 1,1 -fold or greater, for example 1,2-fold or greater, 1,3-fold or greater, 1,4-fold or greater, 1,5-fold or greater, 1,6-fold or greater, 1,7-fold or greater, 1 ,8-fold or greater, 1 ,9-fold or greater, or 2-fold or greater of the activity of the control enzyme or expression in the control cell, more preferably an increase or decrease by about 2,5- fold or greater, 3-fold or greater, 3,5-fold or greater, 4-fold or greater, 4,5-fold or greater, or 5- fold or greater, and most preferably an increase or decrease by about 10-fold or greater.

[0105] “Target site” as used herein means the position in the genome at which a double strand break or one or a pair of single strand breaks (nicks) are induced using recombinant technologies such as Zn-finger, TALEN, restriction enzymes, homing endonucleases, RNA-guided nucleases, RNA-guided nickases such as CRISPR / Cas nucleases or nickases and the like.

[0106] Transgene: The term "transgene" as used herein refers to any nucleic acid sequence, which is introduced into the genome of a cell by experimental manipulations. A transgene may be an "endogenous DNA sequence," or a "heterologous DNA sequence" (i.e. , "foreign DNA"). The term "endogenous DNA sequence" refers to a nucleotide sequence, which is naturally found in the cell into which it is introduced so long as it does not contain some modification (e.g., a point mutation, the presence of a selectable marker gene, etc.) relative to the naturally occurring sequence.

[0107] Transgenic: The term transgenic when referring to an microorganism means transformed, preferably stably transformed, with a recombinant DNA molecule that preferably comprises a suitable promoter operatively linked to a DNA sequence of interest.

[0108] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a genomic integrated vector, or "integrated vector", which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In the present specification, "plasmid" and "vector" are used interchangeably unless otherwise clear from the context. Expression vectors designed to produce RNAs as described herein in vitro or in vivo may contain sequences recognized by any RNA polymerase, including mitochondrial RNA polymerase, RNA pol I, RNA pol II, and RNA pol III. These vectors can be used to transcribe the desired RNA molecule in the cell according to this invention.

[0109] Wild-type: The term "wild-type", "natural" or "natural origin" means with respect to an organism, polypeptide, or nucleic acid sequence, that said organism is naturally occurring or available in at least one naturally occurring organism which is not changed, mutated, or otherwise manipulated by man.

[0110] Examples

[0111] Example 1

[0112] Transformation of Thermothelomyces thermophilus

[0113] Several methods for the transformation of T. thermophilus protoplasts are described in the literature (WO 00 / 20555, US 2012 / 0005812, Verdoes et al. (2007) Industrial Biotechnology 3(1): 48-57).

[0114] Protoplasts of T. thermophilus strain were prepared by inoculating a 25 ml preculture of a standard fungal growth media with 0.7-1x 105spores / ml in a 100 ml shake flask for 24 h at 37°C and 250 rpm. The main culture was prepared by inoculating 100 ml of a standard fungal growth media with 20 ml of the preculture in a 500 ml shake flask for 24 h at 37°C and 250 rpm. The mycelium was harvested by filtration through a sterile cell strainer (VWR) and washed with 100 ml 2000 mosmol / L NaCI / CaCh (0.6 M NaCI and 0.27 M CaCh*H2O). 1 g of the washed mycelium was transferred into a 100 ml flask. The mycelium was mixed with 150 mg VinoTaste Pro solution (2.5 mg / ml in 2000 mosmol / L NaCI / CaCh) and 10 mg yatalase solution (0.625 mg / ml in 2000 mosmol / L NaCI / CaCh) and 10 ml of 2000 mosmol / L NaCI / CaCh. The mycelium suspension was incubated at 30°C and 70 rpm for 50-70 min until protoplasts are visible under the microscope. Harvesting of protoplasts was done by filtration through a sterile cell strainer into a sterile 50 ml tube. After the addition of 25 ml ice-cold STC solution (1.2 M sorbitol, 50 mM CaCh, 35 mM NaCI, 10 mM Tris / HCI pH 7.5) to the flow through, the protoplasts were harvested by centrifugation (1200 x g, 10 min, 4°C). The protoplasts were washed again in 50 ml STC and resuspended in 0.5-1.2 ml STC.

[0115] For transformation, 5-10 pg of linearized DNA, 1 pl 0.5 M aurintricarboxylic acid (ATA) and 100 pl of protoplast suspension were mixed and incubated for 30-40 min on ice. Then 1.7 ml of PEG solution (60% PEG4000 [polyethylenglycol], 50 mM CaCI2, 35 mM NaCI, 10 mM Tris / HCI pH

[0116] 7.5) was added and mixed gently. After incubation for 30 min at 4°C, the tube was filled with 11 ml STC solution, centrifuged (900 x g, 10 min, 4°C), and the supernatant discarded. The pellet was resuspended in the remaining STC and plated on selective media plates as known in the art. After incubation of the plates for 3-6 days at 37°C, transformants were picked and restreaked on selective media.

[0117] Selective media plates

[0118] If the amdS gene is used as selection marker, enriched minimal medium with uridine and uracil and with acetamide is used to select positive transformants (sucrose is only added in case protoplasts are plated): Glucose 10 g / L Sucrose 230 g / L Mg2SO4*7H2O 0.49 g / L KCI 0.52 g / L KH2PO41.52 g / L CUSO4*5H2O 1.6 mg / L FeSO4*7H2O 5 mg / L ZnSO4*7H2O 22 mg / L MnSO4*H2O 4.3 mg / L COCI2*6H2O 1.6 mg / L Na2MoO4*2H2O 1.5 mg / L H3BO3 11 mg / L CsCI 2.52 g / L EDTA 50 mg / L Penicilline 50000 U / L

[0119] Streptomycin 50 mg / L

[0120] Uracil 1.12 g / L

[0121] Uridine 2.44 g / L

[0122] Acetamide 0.6 g / L

[0123] Agar 16 g / L set pH to 6.5

[0124] If selection of clones with lost acetamidase functionality is carried out, enriched minimal medium with uridine and uracil and with fluoroacetamide is used:

[0125] Glucose 10 g / L

[0126] Sucrose 230 g / L Mg2SO4*7H2O 0.49 g / L KCI 0.52 g / L

[0127] KH2PO41.52 g / L

[0128] CUSO4*5H2O 1.6 mg / L

[0129] FeSO4*7H2O 5 mg / L

[0130] ZnSO4*7H2O 22 mg / L

[0131] MnSO4*H2O 4.3 mg / L

[0132] COCI2*6H2O 1.6 mg / L Na2MoO4*2H2O 1.5 mg / L H3BO3 11 mg / L

[0133] CsCI 2.52 g / L

[0134] EDTA 50 mg / L

[0135] Penicilline 50000 U / L

[0136] Streptomycin 50 mg / L Uracil 1.12 g / L Uridine 2.44 g / L

[0137] Fluoroacetamide 5 g / L

[0138] Urea 0.3 g / L

[0139] Agar 16 g / L set pH to 6.5

[0140] If the pyr5 gene is used as selection marker, enriched minimal medium without uridine and uracil is used to select positive transformants (sucrose is only added in case protoplasts are plated):

[0141] Glucose 10 g / L

[0142] Sucrose 230 g / L

[0143] Mg2SO4*7H2O 0.49 g / L

[0144] KCI 0.52 g / L

[0145] KH2PO41.52 g / L

[0146] NaNO36 g / L

[0147] CUSO4*5H2O 1.6 mg / L

[0148] FeSO4*7H2O 5 mg / L

[0149] ZnSO4*7H2O 22 mg / L

[0150] MnSO4*H2O 4.3 mg / L

[0151] COCI2*6H2O 1.6 mg / L

[0152] Na2MoO4*2H2O 1.5 mg / L

[0153] H3BO3 11 mg / L EDTA 50 mg / L

[0154] Penicilline 50000 U / L

[0155] Streptomycin 50 mg / L

[0156] Casamino acids 1 g / L

[0157] Agar 16 g / L set pH to 6.5

[0158] Example 2

[0159] Generation of phytase expression plasmids with different alp1 promoters

[0160] Expression construct with alp1 promoter (389 bp_CC)

[0161] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 36) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3133 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0162] Expression construct with alp1 promoter (1046 bp_CC)

[0163] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 26) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2560 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0164] Expression construct with alp1 promoter (1495 bp_CC)

[0165] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 27) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2559 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0166] Expression construct with alp1 promoter (1586 bp_CC)

[0167] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 37) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3132 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0168] Expression construct with alp1 promoter (1998 bp_CC)

[0169] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 38) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3131 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0170] Expression construct with alp1 promoter (2098 bp_CC)

[0171] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 39) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3130 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0172] Expression construct with alp1 promoter (2198 bp_CC)

[0173] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 40) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3129 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0174] Expression construct with alp1 promoter (2298 bp_CC)

[0175] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 41) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3128 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0176] Expression construct with alp1 promoter (2398 bp_CC)

[0177] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 42) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3127 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0178] Expression construct with alp1 promoter (2498 bp_CC)

[0179] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 43) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT3126 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0180] Expression construct with alp1 promoter (2517 bp_CC)

[0181] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 28) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2556 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0182] Expression construct with alp1 promoter (2717 bp_CC)

[0183] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 29) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2596 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0184] Expression construct with alp1 promoter (2817 bp_CC)

[0185] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 30) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2738 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0186] Expression construct with alp1 promoter (2917 bp_CC)

[0187] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 31) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2595 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0188] Expression construct with alp1 promoter (3017 bp_CC)

[0189] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 32) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2645 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0190] Expression construct with alp1 promoter (3117 bp_CC)

[0191] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 33) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2594 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0192] Expression construct with alp1 promoter (3317 bp_CC)

[0193] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 34) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2593 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0194] Expression construct with alp1 promoter (3575 bp_CC)

[0195] A synthetic gene (SEQ ID NO: 1) encoding a synthetic phytase from bacterial origin (disclosed in WO 2012 / 143862; as phytase PhV-99; SEQ ID NO: 2) was used for the construction of a phytase expression plasmids. For the secretion of the phytase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the phytase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 35) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the phytase. Using standard cloning techniques, the expression plasmid MT2558 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the phytase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the phytase expression cassette was later used for transformation.

[0196] Example 3

[0197] Generation of alpha-amylase expression plasmids with different alp1 promoters

[0198] Expression construct with alp1 promoter (1495 bp_CC)

[0199] A synthetic gene (SEQ ID NO: 3) encoding the alpha-amylase Termamyl from Bacillus licheniformis (disclosed in US 5753460; SEQ ID NO: 4) was used for the construction of an alpha-amylase expression plasmids. For the secretion of the alpha-amylase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the alpha-amylase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 27) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the alpha-amylase. Using standard cloning techniques, the expression plasmid MT3031 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the alpha-amylase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the alpha-amylase expression cassette was later used for transformation.

[0200] Expression construct with alp1 promoter (2517 bp_CC)

[0201] A synthetic gene (SEQ ID NO: 3) encoding the alpha-amylase Termamyl from Bacillus licheniformis (disclosed in US 5753460; SEQ ID NO: 4) was used for the construction of an alpha-amylase expression plasmids. For the secretion of the alpha-amylase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the alpha-amylase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 28) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the alpha-amylase. Using standard cloning techniques, the expression plasmid MT3032 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the alpha-amylase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the alpha-amylase expression cassette was later used for transformation.

[0202] Expression construct with alp1 promoter (2917 bp_CC)

[0203] A synthetic gene (SEQ ID NO: 3) encoding the alpha-amylase Termamyl from Bacillus licheniformis (disclosed in US 5753460; SEQ ID NO: 4) was used for the construction of an alpha-amylase expression plasmids. For the secretion of the alpha-amylase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the alpha-amylase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 31) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the alpha-amylase. Using standard cloning techniques, the expression plasmid MT3034 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the alpha-amylase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the alpha-amylase expression cassette was later used for transformation.

[0204] Expression construct with alp1 promoter (3575 bp_CC)

[0205] A synthetic gene (SEQ ID NO: 3) encoding the alpha-amylase Termamyl from Bacillus licheniformis (disclosed in US 5753460; SEQ ID NO: 4) was used for the construction of an alpha-amylase expression plasmids. For the secretion of the alpha-amylase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the alpha-amylase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 35) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the alpha-amylase. Using standard cloning techniques, the expression plasmid MT3033 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the alpha-amylase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the alpha-amylase expression cassette was later used for transformation.

[0206] Example 4

[0207] Generation of glucanase expression plasmids with different alp1 promoters

[0208] Expression construct with alp1 promoter (1495 bp_CC)

[0209] A synthetic gene (SEQ ID NO: 5) encoding a glucanase CEA from Rasamsonia emersonii (SEQ ID NO: 6) was used for the construction of a glucanase expression plasmids. For the secretion of the glucanase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the glucanase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 27) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the glucanase. Using standard cloning techniques, the expression plasmid MT3016 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the glucanase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the glucanase expression cassette was later used for transformation.

[0210] Expression construct with alp1 promoter (2517 bp_CC)

[0211] A synthetic gene (SEQ ID NO: 5) encoding a glucanase CEA from Rasamsonia emersonii (SEQ ID NO: 6) was used for the construction of a glucanase expression plasmids. For the secretion of the glucanase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the glucanase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 28) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the glucanase. Using standard cloning techniques, the expression plasmid MT3017 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the glucanase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the glucanase expression cassette was later used for transformation.

[0212] Expression construct with alp1 promoter (2917 bp_CC)

[0213] A synthetic gene (SEQ ID NO: 5) encoding a glucanase CEA from Rasamsonia emersonii (SEQ ID NO: 6) was used for the construction of a glucanase expression plasmids. For the secretion of the glucanase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the glucanase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 31) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the glucanase. Using standard cloning techniques, the expression plasmid MT3019 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the glucanase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the glucanase expression cassette was later used for transformation.

[0214] Expression construct with alp1 promoter (3575 bp_CC)

[0215] A synthetic gene (SEQ ID NO: 5) encoding a glucanase CEA from Rasamsonia emersonii (SEQ ID NO: 6) was used for the construction of a glucanase expression plasmids. For the secretion of the glucanase, a signal sequence encoding for a signal peptide derived from T. thermophilus (SEQ ID NO: 7) was added to the mature sequence of the glucanase. A promoter sequence derived from the upstream region of the Alp1 encoding gene (SEQ ID NO: 35) and a terminator sequence amplified from the downstream region of the Cbh1 encoding gene from T. thermophilus (SEQ ID NO: 8) were used as regulatory elements to drive the expression of the glucanase. Using standard cloning techniques, the expression plasmid MT3018 was constructed based on the E. coli standard cloning vector MT940 (SEQ ID NO: 9). Plasmid MT940 consists of the pMB1 origin of replication, kanamycin resistance, pyr5 gene, upstream and downstream regions of the Cbh1 encoding gene from T. thermophilus for homologous recombination, and lacZ for blue / white screening. The alp1 promoter sequence, the glucanase gene including a signal sequence and the cbh1 terminator sequence were cloned in reverse direction between bases 3858 and 4523. The plasmid was digested with Swal to remove the vector backbone and the fragment containing the glucanase expression cassette was later used for transformation.

[0216] Example 5

[0217] Generation of the Thermothelomyces thermophilus strain W1 L#100.l \pyr5 \alp1 \chi1 \ku70B \pep4 \prt1 \prt2 \prt3 \prt4 \lam2 \tre1 \cbh1::amdS

[0218] The T. thermophilus ku70 homologous gene was knocked out in strain W1 L#100.l alpl chU pyr5 (construction described in detail in WO2010 / 107303). The applied ku70 knock-out construct contained the amdS (acetamidase) gene expression cassette from Aspergillus nidulans, which was flanked by identical C1 cbh1 (cellobiohydrolase 1) gene fragments thereby creating a repeat sequence. The cbh1 -repeat flanked amdS cassette was cloned in between ku70 flanks to create the knock-out construct pdel-ku70B (SEQ ID NO: 44). One ku70 flank (5’) comprised a region ranging from about 0.5 kb upstream of the ku70 ATG start codon to about 0.5 kb downstream of the ATG start codon, while the other ku70 flank (3’) comprised a region ranging from about 0.8 kb upstream of the ku70 stop-codon to about 0.4 kb downstream of the stop-codon. After transformation of W1 L#1OO.IAa / p7Ac / 7 / 7Apyr5 and homologous recombination of this ku70 knock-out cassette using the ku70 flanks, the central ± 0.7 kb of the ku70 gene was replaced by the amdS cassette, thereby inactivating ku70 function. Spores of this (intermediate) strain were subsequently plated onto agar medium containing fluoro-acetamide to select for amdS removal. A few spores among thousands used the cbh1 repeats that flank the amdS cassette to recombine and as such excised the amdS cassette from the genome. This is a natural frequency spontaneous mutation event. After amdS excision, one copy of the cbh1 repeat will be left in the disrupted ku70 locus. The resulting strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B was co-transformed as described in example 1 with the two isolated deletion fragments from plasmids MT185 (SEQ ID NO: 10) and MT186 (SEQ ID NO: 11) to delete gene pep4. Enriched minimal medium for amdS selection was used for incubation. After re-streaking on enriched minimal medium with fluoroacetamide for counterselection, the transformants were analysed by PCR for the correct integration of the deletion cassettes in the targeted locus, for the disappearance of the intact gene, and for removal of the amdS marker. The resulting strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 was modified in consecutive transformation rounds, and the following genes were deleted in the identical manner; gene prt1 with the two isolated deletion fragments from plasmids MT 111 (SEQ ID NO: 12) and MT 112 (SEQ ID NO: 13), after removal of the amdS marker strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April was obtained; gene pri2 with the two isolated deletion fragments from plasmids MT368 (SEQ ID NO: 14) and MT453 (SEQ ID NO: 15), after removal of the amdS marker strain W1 L#100.l Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 was obtained; gene pri3 with the two isolated deletion fragments from plasmids MT392 (SEQ ID NO: 16) and MT393 (SEQ ID NO: 17), after removal of the amdS marker strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 was obtained; gene prt4 with the two isolated deletion fragments from plasmids MT386 (SEQ ID NO: 18) and MT649 (SEQ ID NO: 19), after removal of the amdS marker strain

[0219] W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 was obtained; gene lam2 with the two isolated deletion fragments from plasmids MT706 (SEQ ID NO: 20) and MT707 (SEQ ID NO: 21), after removal of the amdS marker strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 was obtained; gene tre1 with the two isolated deletion fragments from plasmids MT710 (SEQ ID NO: 22) and MT711 (SEQ ID NO: 23), after removal of the amdS marker strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 Atrel was obtained; gene cbh1 was replaced by an amdS marker with the isolated deletion fragment from plasmid MT2178 (SEQ ID NO: 24), and strain W1 L#100.l Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 Atrel Acbh1::amdS was obtained.

[0220] Example 6

[0221] Generation of T. thermophilus strains with phytase, alpha-amylase or glucanase expression constructs integrated at cbh1 locus

[0222] For the expression of a phytase, the T. thermophilus host strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 Atrel Acbh1::amdS (construction described in example 5) from the C1 lineage, a strain with uracil auxotrophy, reduced protease activity, and impaired non-homologous end joining (NHEJ) repair system, was transformed as described in example 1 with the Swal-digested and isolated phytase expression constructs (cf. example 2) from plasmids MT2556, MT2558, MT2559, MT2560, MT2593, MT2594, MT2595, MT2596, MT2645, MT2738, MT3126, MT3127, MT3128, MT3129, MT3130, MT3131, MT3132 or MT3133.

[0223] For the expression of an alpha-amylase, the T. thermophilus host strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 Atrel Acbh1::amdS (construction described in example 5) from the C1 lineage, a strain with uracil auxotrophy, reduced protease activity, and impaired non-homologous end joining (NHEJ) repair system, was transformed as described in example 1 with the Swal-digested and isolated alpha-amylase expression constructs (cf. example 3) from plasmids MT3031 , MT3032, MT3033 or MT3034.

[0224] For the expression of a glucanase, the T. thermophilus host strain W1 L#100.1 Apyr5 Aalpl AchU Aku70B Apep4 April Apri2 Apri3 Apri4 Alam2 Atrel Acbh1::amdS (construction described in example 5) from the C1 lineage, a strain with uracil auxotrophy, reduced protease activity, and impaired non-homologous end joining (NHEJ) repair system, was transformed as described in example 1 with the Swal-digested and isolated glucanase expression constructs (cf. example 4) from plasmids MT3016, MT3017, MT3018 or MT3019. The transformants were incubated for 3-6 days at 37°C on enriched minimal medium for pyr5 selection to select for restored uracil prototrophy by complementing the pyr5 deletion with the pyr5 marker as known in the art. Colonies were re-streaked and checked for the integration of the phytase, alpha-amylase or glucanase expression cassette using PCR with primer pairs specific for the phytase, alpha-amylase or glucanase expression cassette and the cbh1 locus as known in the art. Transformants tested positive for the phytase, alpha-amylase or glucanase expression construct at the cbh1 locus were selected for further characterization.

[0225] Example 7

[0226] Phytase activity assay

[0227] The phytase activity is determined in microtiter plates. The phytase containing supernatant is diluted in reaction buffer (250 mM Na-acetate, 1 mM CaCh, 0.01 % Tween 20, pH 5.5). 10 pl of the enzyme solution are incubated with 140 pl substrate solution (6 mM Na-phytate (Sigma P3168) in reaction buffer) for 1 h at 37°C. The reaction is quenched by adding 150 pl of trichloroacetic acid solution (15% w / w). To detect the liberated phosphate, 20 pl of the quenched reaction solution are treated with 280 pl of freshly made-up color reagent (60 mM L- ascorbic acid (Sigma A7506), 2.2 mM ammonium molybdate tetrahydrate, 325 mM H2SO4), and incubated for 20 min at 37°C, and the absorption at 820 nm is subsequently determined. For the blank value, the substrate buffer on its own is incubated at 37°C and the 10 pl of enzyme sample are only added after quenching with trichloroacetic acid. The colour reaction is performed analogously to the remaining measurements. The amount of liberated phosphate is determined via a calibration curve of the colour reaction with a phosphate solution of known concentration.

[0228] Example 8

[0229] Alpha-amylase activity assay

[0230] The amylase activity was determined by a method employing the substrate ethylidene-4- nitrophenyl-a-D-maltoheptaoside (EPS) (Roche CostumBiotech 10880078103). D- maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage an alpha-glucosidase liberates a para-nitrophenol (PNP) molecule which has a yellow colour and thus can be measured by visible spectrophotometry at 405nm. The slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.

[0231] Example 9

[0232] Glucanase activity assay

[0233] The glucanase activity is determined in microtiter plates using the DNS method. The glucanase containing supernatant is diluted in reaction buffer (100 mM Na-acetate, 0.005 % Tween 20, pH 4.5). As substrate, low viscosity carboxymethyl cellulose (CMC) was used which was dissolved at a concentration of 4% in reaction buffer with stirring while being heated up. Using a PCR Cycler 35 pl of the diluted enzyme solution is mixed with 35 pL of the substrate solution 30min at 40°C. The reaction is quenched by the addition of 105 pl of DNS solution (16 g / L NaOH, 10 g / L dinitro salicylic acid, 300 g / L Na-K-tartrate tetra hydrate). After 20 min of heating using 80°C, the samples were cooled to 4°C and 150 pL of the solution is transferred to flat-bottom microtiter plates. The resulting dark orange colour was detected at 540 nm. By subtracting a suitable blank and using a series of glucose solutions with known concentration the enzyme activity was determined and is reported as U / mL of enzyme solution with U is defined as pmol released reducing ends per min of reaction time.

[0234] Example 10

[0235] Analysis of phytase, alpha-amylase or glucanase production in small-scale cultivation Generated mutant strains were fermented in small-scale cultivation and the supernatants were analysed. T. thermophilus strains grown on agar were inoculated in 1 ml cultivation medium as shown in Table 1 in a 96-deepwell microtiter plate. The strains were fermented at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 72 hours. 300 pl of the 72-hour- preculture were transferred in 700 pl cultivation medium as shown in Table 2 in a 96-deepwell microtiter plate. The strains were fermented at 37°C on a microtiter plate shaker at 900 rpm and 80% humidity for 48 hours, 72 hours or 96 hours.

[0236] Cell-free supernatants were harvested at the end of cultivation and subjected to a phytase activity assay (cf. example 7), an alpha-amylase activity assay (cf. example 8) or a glucanase activity assay (cf. example 9).

[0237] Table 1 : Cultivation medium

[0238] Glucose 10 g / L

[0239] Mg2SO4*7H2O 0.49 g / L

[0240] K2SO41.21 g / L

[0241] CaSO4*2H2O 0.47 g / L

[0242] KH2PO41 .75 g / L

[0243] (NH4)2SO44.63 g / L

[0244] CUSO4*5H2O 1 .46 mg / L

[0245] FeSO4*7H2O 4.56 mg / L

[0246] ZnSO4*7H2O 20.05 mg / L

[0247] MnSO4*H2O 3.92 mg / L Na2MoO4*2H2O 1.37 mg / L EDTA 50 mg / L Biotin 0.006 mg / L

[0248] Penicilline 50000 U / L

[0249] Streptomycin 50 mg / L

[0250] Casamino acids 1 g / L

[0251] MES 21.33 g / L set pH to 6.0

[0252] Table 2: Cultivation medium

[0253] Glucose 14.29 g / L

[0254] Mg2SO4*7H2O 0.65 g / L

[0255] K2SO41 .63 g / L

[0256] CaSO4*2H2O 0.63 g / L

[0257] KH2PO42.35 g / L

[0258] (NH4)2SO49.31 g / L

[0259] CUSO4*5H2O 2.09 mg / L

[0260] FeSO4*7H2O 6.51 mg / L

[0261] ZnSO4*7H2O 28.64 mg / L

[0262] MnSO4*H2O 5.60 mg / L

[0263] Na2MoO4*2H2O 1.96 mg / L

[0264] EDTA 71.43 mg / L

[0265] Biotin 0.009 mg / L

[0266] Penicilline 71429 U / L

[0267] Streptomycin 71.43 mg / L MES 60.93 g / L set pH to 6.75

[0268] The results are shown in Figs. 1 ,4 and 5 for phytase and in figures 2 and 3 for alpha amylase and glucanase repsectively:

[0269] The production of phytase was significantly higher when the expression of the phytase gene was driven by an alp1 promoter with a length of 2300 bp (2298 bp_CC alp1 promoter) or longer (see column F, G and H in figure 4 for 2300, 2400, 2517 bp) compared to the state-of-the-art alp1 promoters in column A and B with a length of only 389bp (389 bp_CC alp1 promoter) or 1588 bp (1586 bp_CC alp1 promoter).

[0270] It was again higher when the expression of the phytase gene was driven by an alp1 promoter with a length of 2400 bp (2398 bp_CC alp1 promoter) or longer as column G and H in Figure 4 and column H and I in figure 5 show.

[0271] The production of phytase, alpa-amylase or glucanase was higher when the expression of the phytase, alpha-amylase or glucanase gene was driven by the 2517 bp_CC alp1 promoter compared to the 1495 bp_CC alp1 promoter and again higher when the expression of the phytase, alpha-amylase or glucanase gene was driven by the 2917 bp_CC alp1 promoter (see figures 1 , 2 and 3).

Claims

Claims1. An isolated promoter capable of conferring expression in a microorganism of the phylum Ascomycota comprising a nucleotide sequence of at least 2300 bp selected from the group consisting of:(i) a nucleotide sequence of at least consecutive 2300 bp, wherein the nucleotide sequence comprises at least positions 1278 to 3577 of the nucleotide sequence set forth in SEQ ID NO: 25 or 35; and(ii) a nucleotide sequence having at least 90% sequence identity over its entire length to a nucleotide sequence of (i).

2. The isolated promoter of claim 1, wherein the promoter comprises a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 25 and 28-35.

3. An expression construct comprising an isolated promoter according to claim 1 or claim 2 operably linked to a heterologous nucleic acid encoding a protein of interest.

4. An expression vector comprising an isolated promoter according to claim 1 or claim 2 or the expression construct according to claim 3.

5. A method for producing an expression construct, said method comprising operably linking an isolated promoter according to claim 1 or claim 2 to a heterologous nucleic acid encoding a protein of interest.

6. A process for producing a vector, said method comprising linking an isolated promoter according to claim 1 or claim 2 or an expression construct according to claim 3 to a vector.

7. Use of the isolated promoter of claim 1 or claim 2 for the production of an expression construct or an expression vector.

8. Use of the expression construct of claim 3 for the production of an expression vector.

9. A transgenic microorganism of the phylum Ascomycota comprising an expression construct according to claim 3 or an expression vector according to claim 4.

10. The transgenic microorganism according to claim 9 wherein the expression construct or expression vector is integrated into the genome of said microorganism.

11. A method for producing a transgenic microorganism of the phylum Ascomycota, said method comprising introducing an isolated promoter according to claim 1 or claim 2 or an expression construct according to claim 3 or expression vector according to claim 4 into said microorganism.

12. The expression construct according to claim 3, the expression vector according to claim 4 or the microorganism according to claim 9 or claim 10, wherein the protein of interest is selected from the group consisting of hydrolase, isomerase, ligase, lyase, oxidoreductase and transferase.

13. A method for expression of a nucleotide sequence of interest in a recombinant microorganism of the phylum Ascomycota, comprising the step of cultivating the recombinant microorganism in a medium conducive for the expression of a heterologous nucleic acid encoding for a protein of interest, wherein the microorganism comprises a promoter according to claim 1 or claim 2 operably linked to the heterologous nucleic acid.

14. A method for the production of a protein of interest in a recombinant microorganism of the phylum Ascomycota, comprising the steps of(i) cultivating a recombinant microorganism of the phylum Ascomycota in a medium conducive for the expression of the protein of interest, wherein the microorganism comprises a promoter according to claim 1 or claim 2 operably linked to a heterologous nucleic acid encoding said protein of interest, and(ii) recovering said protein of interest.

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