Fungal expression system for hypoxic conditions

Abstract

This document discloses a nucleic acid containing a system for expressing a sequence of interest in fungi, comprising the following operablely linked components: (a) a promoter induced by hypoxia conditions; (b) the sequence of interest; and (c) an upstream Kozak region and / or a transcription terminator, wherein the upstream Kozak region and / or the transcription terminator is not intrinsic to the promoter. Such expression cassettes are capable of providing high expression of the sequence of interest under hypoxia conditions, which may be dominant, for example, in later stages of fungal fermentation.

Description

[0001] Cross-reference with related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 596662, filed November 7, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list electronically submitted in XML format, which is incorporated herein by reference in its entirety. The XML sequence list was created on November 6, 2024, and is named BPC-018WO_SL.xml, with a size of 110,538 bytes. Background Technology

[0005] Yeast and other fungi are single-celled eukaryotic organisms that have been used in food production processes for thousands of years. In recent decades, technologies in the biotechnology field have enabled fungi to be used in bio-industrial processes beyond their traditional roles in baking, brewing, and winemaking.

[0006] Generally, food production and bioindustrial processes utilizing fungi involve the production of recombinant peptides, either as products of interest or for catalytic production of products of interest. To maximize the yield of recombinant peptides, it is desirable to allow the fungi to grow to a specific biomass and then proceed to the production stage, where the fungi shift from increasing biomass to protein production.

[0007] There is a need in this field for expression cassettes that can improve the production of recombinant peptides during the production stage. Summary of the Invention

[0008] This disclosure provides fungal expression cassettes and expression systems induced by hypoxia conditions, which are therefore suitable for the preferential expression of sequences of interest during the production phase of fungal cultures, for example, for industrial applications.

[0009] In various embodiments, the fungal expression system comprises an expression cassette containing a hypoxia-inducible promoter and a Kozak region and / or transcription terminator operatively linked to a sequence of interest, wherein the Kozak region and / or transcription terminator is not native to the promoter. Surprisingly, specific combinations of the hypoxia-inducible promoter described herein with non-native Kozak regions and / or transcription terminators have been found to achieve hypoxia-inducible expression at levels comparable to the well-known yeast constitutive promoter TEF1p.

[0010] Examples of the expression boxes disclosed herein are described in Section 6.2 and in numbered embodiments 1 to 221.

[0011] The expression cassettes of this disclosure can be included in various recombinant nucleic acids. Examples of nucleic acids containing the expression cassettes of this disclosure are described in Section 6.3 and in embodiments numbered 222 to 225.

[0012] Nucleic acids containing expression cassettes can be engineered into fungal cells, such as yeast cells, using various techniques. Examples of fungal cells containing said nucleic acids are described in Section 6.4 and in embodiments numbered 226 to 231.

[0013] Engineered cells containing nucleic acids with expression cassettes can be used to express sequences of interest. These sequences of interest can be transcribed to produce RNA of interest or translated to produce protein of interest. The use of engineered cells to express sequences of interest is described in Section 6.5.3 and in embodiments numbered 232 to 235.

[0014] In some embodiments where the sequence of interest is expressed to produce the protein of interest, the protein of interest can be used to produce the product. This production can be carried out in vivo in cells that produce the protein of interest, or in vitro after the protein of interest has been produced in cells and purified. Production can involve the catalysis of a substrate to produce a product or precursor, which can be produced by the activity of other enzymes and / or by chemical catalysis. The use of the protein of interest in product production is described in Section 6.5.4 and in embodiments numbered 236 through 244. Attached Figure Description

[0015] Figure 1 The overall configuration of the expression cassette of this disclosure is schematically depicted, comprising a hypoxia-inducible promoter including a non-transcriptional region, a transcription start site, and a 5' untranslated region (UTR). The expression cassette also includes a Kozak region (which includes the Kozak upstream region in the 5' UTR and nucleotides +1 to +4 of the sequence of interest), the sequence of interest, and a transcription terminator. The depicted components are not drawn to scale.

[0016] Figure 2 The GFP expression (relative to the negative control) of GFP ORF operatively linked to the DAN1p, DAN4p, TIR4p, TIR3p, TIR1p, tHEM13p, HES1p, HEM13p, ANB1p or AAC3p promoter and PGK1t terminator as described in Example 1 is shown. The GFP expression ratio under anaerobic and aerobic conditions is also shown.

[0017] Figure 3The GFP expression (relative to the negative control) of GFP ORF operatively linked to the DAN1p, DAN1pm1, DAN1pm5 or DAN1pm6 promoter and the PGK1t or SPG5t terminator as described in Example 2 is shown, along with the GFP expression ratio under anaerobic and aerobic conditions.

[0018] Figure 4 The GFP expression (relative to the negative control) of GFP ORF operatively linked to the DAN1pm1, DAN1pm5, or DAN1pm6 promoters with Kozak upstream regions TCTGAATA, TCTTATAACC, or TCTCAACC and the SPG5t terminator is shown in Example 3, and the GFP expression ratio under anaerobic and aerobic conditions is also presented.

[0019] Figure 5 The expression of GFP ORF operatively linked to the DAN1p promoter and PGK1t, CPS1t, VPS13t, HIS5t, PRM9t, ADH1t, IDP1t, PDC6t, GAT2t, LSC2t, SPG5t, UBX6t, SPO1t, or PRM5t terminator as described in Example 4 is shown in yeast (relative to the negative control).

[0020] Figure 6 The GFP expression (relative to the negative control) of GFP ORF operatively linked to the DAN1pm1 promoter and PGK1t, CPS1t, VPS13t, HIS5t, PRM9t, ADH1t, IDP1t, PDC6t, GAT2t, LSC2t, SPG5t, UBX6t, SPO1t, or PRM5t terminators as described in Example 5 is shown. The GFP expression ratio under anaerobic and aerobic conditions is also shown.

[0021] Figure 7 The expression of GFP ORF in yeast (relative to the negative control) is shown as described in Example 5, operatively linked to the DAN1pm1 promoter and PGK1t, CPS1t, VPS13t, HIS5t, PRM9t, ADH1t, IDP1t, PDC6t, GAT2t, LSC2t, SPG5t, UBX6t, SPO1t, or PRM5t terminator (X-axis) or operatively linked to the DAN1p promoter and the same terminator (Y-axis).

[0022] Figure 8The expression of GFP ORF operatively linked to the ANB1p, HEM13p, AAC3p, DAN1pm1, DAN1pm5, and DAN1pm6 promoters and the PGK1t or SPG5t terminator in yeast is shown (relative to the negative control) as described in Example 6.

[0023] Figure 9 The GFP expression (relative to the negative control) of GFPORF operably linked with different combinations of promoters and terminators as described in Example 10 and the GFP expression ratio under anaerobic and aerobic conditions are shown.

[0024] Figure 10 It shows brewing yeast ( S. cerevisiae A diagram of the isobutanol pathway starting from glucose in ( ). Figure 10 Adapted from Generosos et al., 2017, FEMS Yeast Res 17:1-10, DOI: 10.1093 / femsyr / fox029.

[0025] Figure 11 This illustrates, as described in Example 12, the transformation of *Saccharomyces cerevisiae* (a type of yeast) with different expression constructs under aerobic and anaerobic conditions. S. cerevisiae Accumulation of 2,3-dihydroxyisovalerate (DIV) in strains. Detailed Implementation

[0026] 6.1. Definition

[0027] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and embodiments, the words “having” and “comprising” or variations thereof shall be understood to imply inclusion of the stated integers or groups of integers, but not to exclude any other integers or groups of integers. All publications and other references mentioned herein are incorporated herein by reference in their entirety. Although numerous documents are cited herein, such citations do not constitute an acknowledgment that any of these documents constitutes part of common general knowledge in the art.

[0028] Aerobic The term “aerobic” as used in relation to culture media and other aqueous solutions in this article refers to culture media and other aqueous solutions containing more than 5 ppm (5 mg / L) of dissolved oxygen.

[0029] Anaerobic The term “anaerobic” as used in connection with culture media and other aqueous solutions in this article refers to culture media and other aqueous solutions containing 2 ppm (2 mg / L) or less of dissolved oxygen.

[0030] EC XXXX A polypeptide described as having an Enzyme Commission category “EC XXXX” is considered to have activity belonging to that EC category, and may have activity belonging to one or more other EC categories. Sometimes, the phrase “e.g., EC XXXX” is used to indicate a polypeptide having activity belonging to that EC category and optionally one or more other EC categories.

[0031] Expression Box As used herein, the term "expression cassette" refers to a nucleic acid sequence that contains a sequence of interest (e.g., a coding sequence for an RNA of interest or an open reading frame encoding a protein of interest) and has regulatory elements that allow said sequence of interest to be transcribed (and optionally translated) in a host cell (e.g., a fungal cell). Expression cassettes can be integrated into the genome or chromosome (e.g., within the host cell genome) or extrachromosomal (e.g., within plasmids, transposons, or other vectors).

[0032] Expression system As used herein, the term "expression system" refers to a cellular (e.g., recombinant fungal cell) or in vitro reaction mixture containing a machine (e.g., RNA polymerase, ribosome, etc.) that allows the sequence of interest to be transcribed from the expression cassette and optionally translated under suitable conditions (e.g., hypoxia).

[0033] Heterogeneous The term "heterologous" as used herein in association with nucleic acid or polypeptide sequences in an organism refers to a nucleic acid or polypeptide having a nucleotide or amino acid sequence that is not present in the native nucleic acid or polypeptide of the organism (or, in the case of nucleic acids, at least not at that particular location in the genome of the organism). For example, in some cases, a heterologous nucleic acid is heterologous to the genome because it is not present in the native genome. In other cases, the heterologous nucleic acid is heterologous because it is typically present at a different location in the genome of the organism. With respect to the components of the expression cassette of this disclosure, the term "heterologous" refers to a component that is not typically associated with another component, such as a promoter that may be heterologous to the sequence of interest transcribed in the expression cassette.

[0034] hypoxia The term “hypoxia” as used in this article in connection with culture media and other aqueous solutions refers to culture media and other aqueous solutions containing 2 ppm (2 mg / L) to 5 ppm (5 mg / L) of dissolved oxygen.

[0035] Kozak DistrictThe “Kozak region” used in this article refers to a DNA sequence spanning the region 9 nucleotides upstream of the ATG start codon (“-9”) and 1 nucleotide downstream of the ATG start codon (“+4”).

[0036] Kozak sequence The Kozak sequence is a nucleic acid motif that acts as a protein translation initiation site in eukaryotic mRNA transcripts. Kozak sequences regulate the specificity and efficiency of translation initiation. They also mediate the recruitment and assembly of ribosomes on messenger RNA (mRNA) transcripts. It is also known that Kozak sequences are involved in recognizing the correct AUG start codon to initiate translation.

[0037] The shared Kozak sequence varies across species, but it is typically contained within approximately 5 to 8 nucleotides upstream and downstream of the AUG start codon. Several conserved positional effects exist for the nucleotides within the shared Kozak sequence, which can influence the overall strength of translation. If the +4, -1, -2, and -3 positions of the Kozak sequence match the shared Kozak sequence of the stated species relative to the A nucleotide in the AUG start codon (referred to as the +1 position), it is classified as having strong mRNA translation efficiency. If only one of the -3 and +4 positions of the Kozak sequence matches the shared Kozak sequence of the stated species, it is classified as having adequate mRNA translation efficiency. If neither the -3 nor +4 positions of the Kozak sequence match the shared Kozak sequence of the stated species, it is classified as having weak mRNA translation efficiency.

[0038] Kozak upstream area As used herein, the “Kozak upstream region” refers to the portion of the Kozak region located at the 5' position of the ATG start codon in non-recombinant (e.g., wild-type) organisms, corresponding to a DNA sequence spanning nine nucleotides upstream of the ATG start codon (“-9”). In non-recombinant organisms, the nucleotide immediately preceding the 5' position of the ATG start codon is referred to as having a “-1” position. In the expression cassette of this disclosure, a short (20 bp or less, e.g., 12 bp) utility sequence may be included between the Kozak upstream region and the ATG start codon, such as containing restriction sites or other sites useful in recombinant DNA technology.

[0039] hypoxia The term “hypoxia” as used in this article in connection with culture media and other aqueous solutions refers to culture media and other aqueous solutions containing less than 5 ppm (5 mg / L) of dissolved oxygen, and therefore covers both anoxic and anaerobic culture media and other aqueous solutions.

[0040] hypoxia-inducible promotersThe term "hypoxia-inducible promoter" is used herein to refer to a promoter that can be induced by hypoxia (e.g., hypoxia or anaerobic) conditions, and / or whose expression under hypoxia (e.g., hypoxia or anaerobic) conditions is at least 1.25 times that under aerobic conditions (in some embodiments, at least 1.5 times or at least 2 times). Sometimes, for convenience, hypoxia-inducible promoters are referred to herein as "anaerobic promoters," although such promoters may also be induced under hypoxia conditions.

[0041] Non-original For the purposes of the expression cassettes disclosed herein, the term “non-native” and its grammatical variations as used herein refer to components of the expression cassette that are not typically associated with each other in non-recombinant organisms, such as (a) components aggregated from (i) different genes or (ii) different organisms and / or (b) components engineered to include variant sequences.

[0042] promoter The term "promoter" as used in this article refers to a nucleic acid sequence that can interact with RNA polymerase to initiate transcription of an operable ligation sequence.

[0043] Proteins of interest The term "protein of interest" as used in this article refers to a peptide, polypeptide, or protein encoded by a sequence of interest. Therefore, the nucleic acid sequence encoding the protein of interest is a subset of the sequence of interest.

[0044] Reorganization In this document, the term "recombinant" as used in relation to nucleic acids or peptides refers to nucleic acids or peptides that are the product of gene editing or genetic engineering, producing sequences (e.g., structural, coding, or non-coding sequences) that are distinguishable from endogenous nucleic acids or peptides present in natural systems. The term "recombinant" as used in relation to expression cassettes refers to non-naturally occurring expression cassettes, such as those created by modifying natural Kozak and / or terminator sequences and / or by combining originally separated sequence segments. Similarly, the term "recombinant" peptide refers to a peptide that is not naturally occurring or expressed by non-naturally occurring nucleic acids.

[0045] RNA of interest In this paper, "RNA of interest" refers to an RNA molecule transcribed from a sequence of interest. In other words, the sequence transcribed to produce the RNA of interest is a subset of the sequence of interest. RNA of interest includes mRNAs from which translation of proteins of interest is desired. In this paper, mRNA may be referred to as "coding RNA". RNA of interest also includes RNAs that are not expected and / or do not need to be translated, such as RNAs that perform regulatory, structural, or similar functions. RNAs that are not expected and / or do not need to be translated may be referred to as "non-coding RNA" in this paper.

[0046] Sequence identity "Sequence identity" in relation to the nucleotide or amino acid sequence of a nucleic acid or polypeptide molecule refers to the overall relevance between two such sequences. For example, the percentage of sequence identity (nucleotide or amino acid sequence identity) between two sequences can be calculated as follows: The two sequences are aligned for optimal comparison purposes (e.g., vacancies can be introduced in one or both of the first and second nucleic acid or amino acid sequences to achieve optimal alignment). The nucleotides or amino acids at the corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, the molecule is identical at that position. The percentage of identity between two sequences is a function of the number of common positions in the sequences, taking into account the number of vacancies introduced to achieve optimal alignment and the length of each vacancies. Once the nucleotide or amino acid sequence alignment is generated, the percentage of sequence identity can be determined manually. Queries of nucleotide or amino acid sequence alignments with reference nucleotide or amino acid sequences can be generated using the computer program ClustalW (version 1.83, default parameters), which allows alignment across the entire length of a nucleic acid or protein sequence (global alignment). ClustalW calculates and aligns the best match between the query and one or more reference sequences to determine identity, similarity, and differences. One or more residues can be inserted in the query sequence, reference sequence, or both to maximize sequence alignment. For rapid alignment of nucleotide sequences, the following default parameters are used: word length: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; gap penalty: 5. For rapid alignment of amino acid sequences, the following parameters are used: word length: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. Unless otherwise specified, the percentage of sequence identity between the reference nucleotide or amino acid sequence (e.g., the sequence with the defined SEQ ID NO disclosed herein) and the query nucleotide or amino acid sequence is calculated over the entire length of the reference sequence.

[0047] Sequences of interest As used herein, "sequence of interest" refers to a nucleic acid sequence operatively linked to a promoter for transcription. The term "sequence of interest" includes sequences encoding a "protein of interest" and sequences encoding an "RNA of interest." In some embodiments, in the expression cassette containing a hypoxia-inducible promoter of this disclosure, the sequence of interest is heterologous to the hypoxia-inducible promoter.

[0048] TerminationAs used in this article, a “terminator” or “transcription terminator” is a nucleotide sequence that, when placed downstream of a sequence of interest (e.g., an open reading frame (ORF)) during transcription, causes the termination of transcription of the nucleotide sequence containing that sequence of interest. Eukaryotic terminators typically contain a highly conserved AAUAA region, a GU-rich region, and a polyadenylation signal sequence (a nucleotide included in the mRNA transcript). These regions of the terminator are contained within the 3' untranslated region (UTR) of the mRNA transcript.

[0049] 6.2. Expression Box

[0050] This disclosure provides hypoxia-inducible expression cassettes suitable for expression in fungi such as yeast. The expression cassette typically includes a promoter induced by hypoxia (anaerobic or hypoxic) conditions, an upstream Kozak region, a transcription terminator, and a sequence of interest. Figure 1 Examples of configurations for these components are shown in the diagram. Other configurations, such as the configuration where the Kozak upstream region is not included in the expression cassette (this may be suitable when the sequence of interest encodes an RNA of interest that does not require translation).

[0051] In some embodiments, the hypoxia-inducible expression cassette comprises the following operably linked components: a hypoxia-inducible promoter; a sequence of interest; and an upstream Kozak region and / or a transcription terminator; wherein the upstream Kozak region and / or transcription terminator is non-native to the promoter. In some embodiments, the hypoxia-inducible expression cassette comprises the following components operably linked in a 5' to 3' orientation: a hypoxia-inducible promoter; an upstream Kozak region; a sequence of interest; and a transcription terminator; wherein the upstream Kozak region and the transcription terminator are non-native to the hypoxia-inducible promoter. In some embodiments, the hypoxia-inducible expression cassette comprises the following components operably linked in a 5' to 3' orientation: a hypoxia-inducible promoter; an upstream Kozak region; and a sequence of interest; wherein the upstream Kozak region is non-native to the hypoxia-inducible promoter. In some embodiments, the hypoxia-inducible expression cassette includes the following components operably linked in a 5' to 3' orientation: a hypoxia-inducible promoter; a sequence of interest; and a transcription terminator; wherein the transcription terminator is non-native to the hypoxia-inducible promoter.

[0052] In some embodiments, the expression of the sequence of interest under hypoxic conditions is improved relative to an expression cassette where the Kozak upstream region and / or transcription terminator is intrinsic to the promoter, compared to aerobic conditions. In some embodiments, the improved expression of the sequence of interest is due to an increased absolute expression level under hypoxic conditions (e.g., anaerobic or hypoxic conditions). In some embodiments, the improved expression of the sequence of interest is due to an increased ratio of expression under hypoxic conditions to expression under aerobic conditions. In some embodiments, the improved expression of the sequence of interest is due to both (a) an increased ratio of expression under hypoxic conditions to expression under aerobic conditions and (b) an increased absolute expression level under hypoxic conditions. In some embodiments, the improved expression of the sequence of interest is due to (a) an increased ratio of expression under hypoxic conditions to expression under aerobic conditions, even though the absolute expression level is decreased or unchanged under hypoxic conditions. In some embodiments, the expression of the sequence of interest is decreased or unchanged under aerobic conditions relative to a comparable expression cassette having a Kozak upstream region and / or transcription terminator intrinsic to the hypoxia-inducible promoter. In some cases, it is desirable to have reduced (e.g., as little as possible) expression under aerobic conditions, and the expression cassette of this disclosure can achieve this by pairing a heterologous terminator sequence and / or an upstream Kozak region with a hypoxia-inducible promoter.

[0053] In some embodiments, the expression cassette of this disclosure provides at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times the expression of the sequence of interest under hypoxic conditions (e.g., hypoxia or anaerobic conditions) compared to aerobic conditions. In some embodiments, the expression of the sequence of interest provided by the expression cassette of this disclosure under hypoxic conditions (e.g., hypoxia or anaerobic conditions) compared to aerobic conditions is less than 20, 25, 30, 35, 40, 45, 50, 60, 70, or 80 times the expression of the sequence of interest provided by the expression cassette of this disclosure under hypoxic conditions (e.g., hypoxia or anaerobic conditions) compared to aerobic conditions. In some embodiments, the expression of the sequence of interest provided by the expression cassette of this disclosure under hypoxic conditions (e.g., hypoxia or anaerobic conditions) compared to aerobic conditions is 5 to 40 times, 20 to 40 times, 5 to 80 times, or 20 to 80 times the expression of the sequence of interest provided by the expression cassette of this disclosure under hypoxic conditions (e.g., hypoxia or anaerobic conditions) compared to aerobic conditions. In some embodiments, the expression cassette of this disclosure provides at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times the expression of the sequence of interest under anaerobic conditions compared to aerobic conditions. In some embodiments, the expression cassette of this disclosure provides less than 20, 25, 30, 35, 40, 45, 50, 60, 70, or 80 times the expression of the sequence of interest under anaerobic conditions compared to aerobic conditions. In some embodiments, the expression cassette of this disclosure provides 5 to 40, 20 to 40, 5 to 80, or 20 to 80 times the expression of the sequence of interest under anaerobic conditions compared to aerobic conditions. In some embodiments, the expression cassette of this disclosure is present within cells, and when the cells are under anaerobic conditions, the expression of the sequence of interest in the cells is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 times, or 5 to 40 times, 20 to 40 times, 5 to 80 times, or 20 to 80 times higher than the expression of the sequence of interest in the cells under aerobic conditions. In some embodiments, the expression cassette of this disclosure is present within cells in a culture, and when the culture is under anaerobic conditions (e.g., when the culture medium is anaerobic), the expression of the sequence of interest produced by the cultured cells is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times higher, or 5 to 40, 20 to 40, 5 to 80, or 20 to 80 times higher, than when the culture is under aerobic conditions (e.g., when the culture medium is aerobic). In some embodiments, the anaerobic to aerobic expression ratio of the expression cassette of this disclosure is measured as described in the examples below (Section 8). In some embodiments, the anaerobic to aerobic expression ratio of the expression cassette of this disclosure is measured as described in Section 8.1 (Example 1).In some embodiments, the anaerobic to aerobic expression ratio of the expression cassette of this disclosure is measured using GFP as a reporter gene (i.e., the sequence of interest is GFP). In some embodiments, the sequence of interest encodes GFP, and expression under hypoxic conditions compared to aerobic conditions is measured by observing GFP fluorescence. In some embodiments, expression under aerobic conditions is measured after culturing in a medium with at least 5 ppm dissolved oxygen for 24 hours. In some embodiments, expression under aerobic conditions is measured after culturing in a medium with 10 ppm dissolved oxygen for 24 hours. In some embodiments, expression under anaerobic conditions is measured after culturing in a medium with less than 2 ppm dissolved oxygen for 24 hours. In some embodiments, expression under anaerobic conditions is measured after culturing in a medium with 1 ppm dissolved oxygen for 24 hours.

[0054] In some implementations, the improvement in expression of the sequence of interest includes an increase in transcription of the sequence of interest.

[0055] In some embodiments, the sequence of interest encodes a polypeptide. In some other embodiments, the expression of the polypeptide is improved under hypoxic conditions compared to normoxic conditions, with respect to an expression cassette that is intrinsic to the sequence of interest, relative to the promoter and Kozak upstream region and / or the transcription terminator.

[0056] In some implementations, the sequence of interest is non-native to the promoter.

[0057] Examples of hypoxia-inducible promoters that can be incorporated into the expression cassette of this disclosure are described in Section 6.2.1.

[0058] Instances of the Kozak upstream region that can be incorporated into the expression box of this disclosure are described in Section 6.2.2.

[0059] Examples of transcription terminators that can be incorporated into the expression cassettes of this disclosure are described in Section 6.2.3.

[0060] Instances of sequences of interest that can be incorporated into the expression box of this disclosure are described in Section 6.2.4.

[0061] 6.2.1. Hypoxia-inducible promoters

[0062] Promoters that can be used in the expression cassettes of this disclosure include any promoter capable of being operatively linked to a sequence of interest, such that the sequence of interest can be transcribed in a fungal species (e.g., yeast) upon exposure to hypoxic conditions (e.g., during the production phase of a cell culture). The hypoxia-inducible promoter may have a wild-type or variant sequence, may be at its native locus or at a non-native locus, and may be native or heterologous for fungal species engineered to include the expression cassettes of this disclosure.

[0063] In some implementations, hypoxia-inducible promoters include Saccharomyces cerevisiae (Saccharomyces cerevisiae). S. cerevisiae The DAN1 promoter (DAN1p; SEQ ID NO: 1) or a variant thereof. DAN1 encodes a cell wall protein whose expression is induced during anaerobic growth and strongly repressed during aerobic growth.

[0064] Variants of DAN1p include promoters comprising a nucleotide sequence having 80% or higher sequence identity with SEQ ID NO: 1, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. Variants of DAN1p include those reported by Nevoigt et al., 2006, Biotechnol. Bioeng. https: / / doi.org / 10.1002 / bit.21129. Exemplary DAN1p variants include DAN1pm1 (SEQ ID NO: 2), DAN1pm5 (SEQ ID NO: 3), and DAN1pm6 (SEQ ID NO: 4).

[0065] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with TIR1p (SEQ ID NO: 6), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. TIR1 is a cold shock-induced protein, found in *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae ) Components of the cell wall. TIR1 expression is required for anaerobic growth.

[0066] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with TIR3p (SEQ ID NO: 7), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. TIR3 is a sacchariformis yeast (Sacchariformis spp.) S. cerevisiae ) Components of the cell wall. TIR3 expression is required for anaerobic growth.

[0067] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with TIR4p (SEQ ID NO: 8), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. TIR4 is a sacchariformis yeast (Sacchariformis spp.) S. cerevisiae ) Components of the cell wall. TIR4 expression is required for anaerobic growth.

[0068] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with tHEM13p (SEQ ID NO: 9), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, or a promoter containing a nucleotide sequence having 80% or higher sequence identity with HEM13p (SEQ ID NO: 10), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. HEM13 in Saccharomyces cerevisiae ( S. cerevisiaeIn heme biosynthesis, it plays a role by catalyzing the aerobic oxidative decarboxylation of the propionic acid groups of rings A and B of coproporphyrinogen III to produce vinyl groups in protoporphyrinogen IX (e.g., EC 1.3.3.3). Although not bound by theory, its expression under hypoxic conditions may contribute to the provision of heme cofactors for flavohemoglobin, which has nitric oxide reductase, FAD reductase, and nitric oxide dioxygenase activities and is responsive to nitrite stress. tHEM13p of SEQ ID NO: 9 is a truncated fragment of HEM13p with the upstream activating sequence removed in an attempt to improve anaerobic specificity and remove the aerobic induction observed from HEM13p (Castro-Prego et al., 2010, FEMS YeastRes 10:309–321, DOI: 10.1111 / j.1567-1364.2010.00616).

[0069] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with HES1p (SEQ ID NO: 11), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. HES1 is a sacchariformis yeast (Sacchariformis spp.) S. cerevisiae It is an oxidosterol-binding protein homolog that participates in non-vesicular lipid transfer between membranes and may play a role in ergosterol synthesis and / or transport.

[0070] In some embodiments, the hypoxia-inducible promoter includes a promoter comprising a nucleotide sequence having 80% or higher sequence identity with AAC3p (SEQ ID NO: 12), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. AAC3 is a sacchariformis yeast (Sacchariformis spp.) S. cerevisiae ADP:ATP reverse transporter is a protein that mediates the importation of ADP into the mitochondrial matrix for ATP synthesis and the export of ATP to the cytoplasm.

[0071] In some embodiments, the hypoxia-inducible promoter comprises a promoter containing a nucleotide sequence having 80% or higher sequence identity with ANB1p (SEQ ID NO: 13), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. ANB1 is a type of Saccharomyces cerevisiae (Saccharomyces cerevisiae). S. cerevisiae Translational factors promote translation elongation and termination, especially after ribosome delay, and may be involved in stress responses and cell wall maintenance pathways.

[0072] In some embodiments, the hypoxia-inducible promoter includes a component containing *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae Hypoxia-inducible promoters of yeast other than Kluyveromyces (e.g., Kluyveromyces) Kluyveromyces (The hypoxia-inducible promoter) has a promoter with 80% or higher sequence identity, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity of the nucleotide sequence.

[0073] 6.2.2. Kozak sequence and Kozak upstream region

[0074] This disclosure provides an expression cassette comprising a hypoxia-inducible promoter, in embodiments of which the hypoxia-inducible promoter is operatively linked to a Kozak upstream region. In some embodiments, the Kozak upstream region differs from a native Kozak upstream region associated with the hypoxia-inducible promoter by at least one nucleotide. In some embodiments, the Kozak upstream region is non-native to the sequence of interest or the promoter. In some embodiments, the non-native Kozak upstream region results in higher expression of the sequence of interest than that observed with a Kozak upstream region intrinsic to the sequence of interest.

[0075] Suitable for use in yeast (e.g., yeast ( Saccharomyces Exemplary coding sequences of the Kozak region expressing the sequence of interest are illustrated in Table 1 below, where A is adenine, G is guanine, C is cytosine, T is thymine, R is purine (A or G), Y is pyrimidine (C or T), N is any nucleotide, W is weak (A or T), S is strong (G or C), M is amino (A or C), K is ketone (G or T), B is not A (G, C, or T), H is not G (A, C, or T), D is not C (A, G, or T), and V is not T (A, G, or C).

[0076]

[0077] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of NNNNNANN (-9 to -1 of C1 or C2), optionally including an A at position -4.

[0078] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of NNNNAANN (-9 to -1 of C3).

[0079] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of TCTNNNANN (-9 to -1 of C4), optionally including an A at position -4.

[0080] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of TCTNNAANN (C5 -9 to -1).

[0081] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of NNNWAMAMA (C6 -9 to -1), optionally including an A at position -4.

[0082] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of NNNNAANN (C7 -9 to -1).

[0083] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of TCTGAATA (K1 -9 to -1), optionally including an A at position -3 and / or an A at position -4.

[0084] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of TCTATAACC (K2 from -9 to -1), optionally including an A at position -3 and / or an A at position -4.

[0085] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of TCTCCAACC (K3 from -9 to -1), optionally including an A at position -3 and / or an A at position -4.

[0086] Suitable for use in yeasts (e.g., Kluyveromyces) KluyveromycesExemplary coding sequences of the Kozak region expressing the sequence of interest are illustrated in Table 2 below, where A is adenine, G is guanine, C is cytosine, T is thymine, R is purine (A or G), Y is pyrimidine (C or T), N is any nucleotide, W is weak (A or T), S is strong (G or C), M is amino (A or C), K is ketone (G or T), B is not A (G, C, or T), H is not G (A, C, or T), D is not C (A, G, or T), and V is not T (A, G, or C).

[0087]

[0088] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides of NNNNNNAAG (-9 to -1 of Klu1), optionally including an A at position -4.

[0089] In some implementations, the Kozak upstream region contains at least seven, at least eight, or nine nucleotides NNNNNNAAA (-9 to -1 of Klu2), optionally including an A at position -4.

[0090] In some embodiments, the expression cassette of this disclosure includes a Kozak upstream region comprising or consisting of any of the Kozak upstream region sequences set forth in Table 8 or Table 9. In some embodiments, the expression cassette includes a Kozak upstream region comprising any of the sequences designated herein as DK1 to DK1365 or TK1 to TK262. In some embodiments, the six nucleotides immediately upstream of the ATG start codon of the sequence of interest consist of any of the Kozak upstream region sequences designated herein as DK1 to DK1365 or TK1 to TK262.

[0091] In some embodiments, the expression box includes a Dan1p promoter or a Dan1p promoter variant operatively connected in the 5' to 3' direction, a Kozak upstream region contained in any of the sequences designated herein as DK1 to DK1365, and a sequence of interest, wherein the Kozak upstream region sequence is heterologous to the Dan1p promoter or Dan1p promoter variant, and wherein the Kozak upstream region sequence is immediately upstream of the sequence of interest.

[0092] In some embodiments, the expression box includes a Tir1p promoter operably connected in the 5' to 3' direction, a Kozak upstream region containing any of the sequences designated herein as TK1 to TK262, and a sequence of interest, wherein the Kozak upstream region sequence is heterogeneous with respect to the Tir1p promoter, and wherein the Kozak upstream region sequence is immediately upstream of the sequence of interest.

[0093] In some embodiments, an expression cassette having a Kozak upstream region sequence that is heterologous with respect to the hypoxia-inducible promoter and / or transcription terminator as disclosed herein results in the expression cassette providing one or more of the following effects relative to an expression cassette having a Kozak upstream region that is intrinsic with respect to the hypoxia-inducible promoter and otherwise identical: (a) an absolute increase in expression of the sequence of interest under induction conditions; (b) a decrease in absolute expression of the sequence of interest under aerobic conditions; (c) an increase in the expression ratio of the sequence of interest under hypoxic conditions (e.g., anaerobic or hypoxic conditions) versus aerobic conditions; or any combination of (a), (b), and (c). In some embodiments, an expression cassette with a heterologous Kozak upstream region exhibits an expression rate at least 1, 1.5, 2, 3, 4, 5, 10, 15, or 20 times higher under hypoxic conditions (e.g., anaerobic or hypoxic conditions) compared to an expression cassette having a transcription terminator that is intrinsic to the hypoxia-inducible promoter and is otherwise identical, or 1 to 2, 2 to 10, 5 to 20, or 10 to 20 times higher under aerobic conditions. In some embodiments, the expression is determined by mRNA levels. In some embodiments, the expression is determined by protein levels. In some embodiments, expression under anaerobic conditions is determined as described in the examples below (Section 8). In some embodiments, the anaerobic to aerobic expression ratio of the expression cassette of this disclosure is measured as described in Section 8.1 (Example 1). In some embodiments, the anaerobic to aerobic expression ratio of the expression cassette of this disclosure is measured using GFP as a reporter gene (i.e., the sequence of interest is GFP). In some embodiments, the expression under aerobic conditions is measured after culturing in a medium with at least 5 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under aerobic conditions is measured after culturing in a medium with 10 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under anaerobic conditions is measured after culturing in a medium with less than 2 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under anaerobic conditions is measured after culturing in a medium with 1 ppm dissolved oxygen for 24 hours.

[0094] 6.2.3. Transcription terminators

[0095] Transcription terminators that can be used in the expression cassettes of this disclosure include any terminator capable of being operatively linked to a sequence of interest. In some embodiments, the transcription terminator is non-intrinsic to the sequence of interest. In some embodiments, the transcription terminator is heterologous to all other sequences operatively linked to it. In some embodiments, the transcription terminator is heterologous to the promoter (e.g., a hypoxia-inducible promoter) operatively linked to the transcription terminator. In some embodiments, non-intrinsic transcription terminators result in higher expression of the sequence of interest than that observed with transcription terminators endogenous to the sequence of interest or the promoter.

[0096] In some embodiments, the expression cassette of this disclosure includes a hypoxia-inducible promoter and a transcription terminator heterologous to the hypoxia-inducible promoter, wherein the transcription terminator causes the expression cassette to exhibit a higher expression ratio under hypoxia (e.g., anaerobic or hypoxic conditions) versus aerobic conditions compared to an expression cassette having a transcription terminator intrinsic to the hypoxia-inducible promoter and otherwise identical. In some embodiments, an expression cassette including a transcription terminator sequence heterologous to the operatively linked hypoxia-inducible promoter and / or sequence of interest as disclosed herein causes the expression cassette to provide one or more of the following effects relative to an expression cassette having a transcription terminator intrinsic to the hypoxia-inducible promoter and / or sequence of interest and otherwise identical: (a) increased absolute expression of the sequence of interest under hypoxia conditions; (b) decreased absolute expression of the sequence of interest under aerobic conditions; (c) increased expression ratio of the sequence of interest under hypoxia versus aerobic conditions; or any combination of (a), (b), and (c). In some embodiments, compared to expression cassettes having a transcription terminator that is original to the hypoxia-inducible promoter and otherwise identical, expression cassettes having a hypoxia-to-aerobic expression ratio that is at least 1, 1.5, 2, 3, 4, 5, 10, 15, or 20 times higher, or 1 to 2, 2 to 10, 5 to 20, or 10 to 20 times higher. In some embodiments, the expression is determined by mRNA levels. In some embodiments, the expression is determined by protein levels. In some embodiments, expression under anaerobic conditions is determined as described in the examples below (Section 8). In some embodiments, the anaerobic-to-aerobic expression ratio of the expression cassettes of this disclosure is measured as described in Section 8.1 (Example 1). In some embodiments, the anaerobic-to-aerobic expression ratio of the expression cassettes of this disclosure is measured using GFP as a reporter gene (i.e., the sequence of interest is GFP). In some embodiments, the expression under aerobic conditions is measured after culturing in a medium with at least 5 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under aerobic conditions is measured after culturing in a medium with 10 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under anaerobic conditions is measured after culturing in a medium with less than 2 ppm dissolved oxygen for 24 hours. In some embodiments, the expression under anaerobic conditions is measured after culturing in a medium with 1 ppm dissolved oxygen for 24 hours.

[0097] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with PGK1t (SEQ ID NO: 14), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0098] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with SPG5t (SEQ ID NO: 15), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0099] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with UBX6t (SEQ ID NO: 16), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0100] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with SpO1t (SEQ ID NO: 17), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0101] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with PRM5t (SEQ ID NO: 18), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0102] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with IDP1t (SEQ ID NO: 19), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0103] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with LSC2t (SEQ ID NO: 20), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0104] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with ADH1t (SEQ ID NO: 21), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0105] In some embodiments, the transcription terminator includes a terminator comprising a nucleotide sequence having 80% or higher sequence identity with CYC1t (SEQ ID NO: 47), such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0106] 6.2.4. Sequences of Interest

[0107] The expression cassette disclosed herein can be used to express any sequence of interest that is desired to be expressed in fungi (e.g., yeast) under hypoxic conditions. Sequences of interest include both coding proteins and non-coding sequences of interest.

[0108] 6.2.4.1. Proteins of Interest

[0109] In some implementations, the protein of interest is any or all proteins encoded by a coding region operatively linked to an endogenous hypoxia-inducible promoter.

[0110] In other embodiments, the protein of interest is a protein encoded by a coding region that is not naturally operably linked to a hypoxia-inducible promoter.

[0111] In some implementations, the nucleotide at position +4 of the coding region of the protein of interest is T. Therefore, in some implementations, the first nucleotide in the codon immediately following the start codon is T.

[0112] In some implementations, the nucleotide at position +4 of the coding region of the protein of interest is A. Therefore, in some implementations, the first nucleotide in the codon immediately following the start codon is A.

[0113] In some implementations, the nucleotide at position +4 of the coding region of the protein of interest is G. Therefore, in some implementations, the first nucleotide in the codon immediately following the start codon is G.

[0114] The expression cassette with +4 bits as T is particularly suitable for use in yeast ( Saccharomyces Expression in Kluyveromyces (Kluyveromyces). The expression cassette with +4 bits set to A is particularly suitable for expression in Kluyveromyces (Kluyveromyces). Kluyveromyces This is expressed in ().

[0115] In some implementations, the sequence of interest is polycistronic and contains two or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) coding regions. When the sequence of interest contains more than one coding region, each coding region may, but does not necessarily, be operatively connected to a unique Kozak upstream region. Therefore, the translation efficiency of multiple coding regions may differ, at least in part, due to the differences in the Kozak upstream regions.

[0116] In some embodiments, the proteins of interest include those described below in sections 6.2.4.1.1 to 6.2.4.1.6.

[0117] In some embodiments, the expression cassettes of this disclosure contain sequences of interest encoding an enzyme of interest that is part of a multi-enzyme pathway for the production of a specific product of interest (e.g., isobutanol, butanol, mevalonic acid, fatty acids, or isoprene-like compounds). In some embodiments, the activity of a single sequence of interest (e.g., the sequence of interest encoding the first enzyme in the multi-enzyme pathway) may be critical or a bottleneck in the production of the product of interest, and tight oxygen sensitivity control of the single sequence of interest may be particularly beneficial. In some embodiments, the expression of other enzymes in the pathway may not need to be so tightly controlled and can be optimized to increase absolute expression levels (whether under hypoxia-inducible control or other controls) to provide optimal production of the product during the production phase. In some embodiments, only a portion (e.g., just one) of the enzymes in the multi-enzyme pathway is operatively linked within the expression cassettes of this disclosure, while sequences of interest encoding other enzymes in the pathway are expressed from expression cassettes in which oxygen regulation levels are not so tightly controlled or are absent (e.g., under constitutive promoters or other non-hypoxia-inducible promoters (e.g., promoters native to other enzymes)). In some respects, the selection of appropriate combinations of hypoxia-inducible promoters, Kozak sequences, and terminator sequences allows for the tunable regulation of the expression of various components of multi-enzyme pathways.

[0118] In some embodiments, the production of the multi-enzyme pathway end product is effectively “turned off” during the aerobic growth phase because one enzyme in the multi-enzyme pathway is under strict control of the hypoxia-inducible expression cassette disclosed herein, even if other enzymes in the pathway may be expressed during the aerobic growth phase.

[0119] 6.2.4.1.1. Isobutanol / butanol pathway enzymes

[0120] In some embodiments, sequences of interest include sequences encoding enzymes active in the conversion of glucose to butanol or isobutanol. Butanol and isobutanol are of great interest as biofuels.

[0121] In some embodiments, the sequence of interest includes a sequence encoding a component of the acetolactate synthase complex (e.g., EC2.2.1.6). In an exemplary embodiment, the sequence of interest includes a sequence encoding a component of the acetolactate synthase complex comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 22, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, or having 80% or higher sequence identity with SEQ ID NO: 23, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. In an exemplary embodiment, the sequence of interest includes a sequence encoding acetolactate synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 60, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0122] In some embodiments, the sequence of interest includes a sequence encoding a ketocolate reductase (e.g., EC1.1.1.86). In an exemplary embodiment, the sequence of interest includes a sequence encoding a ketocolate reductase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 24, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. In an exemplary embodiment, the sequence of interest includes a sequence encoding a ketool acid reductase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 62, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0123] In some embodiments, the sequence of interest includes a sequence encoding a dihydroxy acid dehydratase (e.g., EC 4.2.1.9). In an exemplary embodiment, the sequence of interest includes a sequence encoding a dihydroxy acid dehydratase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 25, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0124] In some embodiments, the sequence of interest includes a sequence encoding an aromatic amino acid decarboxylase (e.g., EC4.1.1.28). In exemplary embodiments, the sequence of interest includes a sequence encoding an aromatic amino acid decarboxylase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 26, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity. The aromatic amino acid decarboxylase may also catalyze one or more reactions identified by EC4.1.1.43, EC 4.1.1.74, EC 4.1.1.80, and EC 4.1.1.72.

[0125] In some embodiments, the sequence of interest includes a sequence encoding an alcohol dehydrogenase (e.g., EC 1.1.1.1). In an exemplary embodiment, the sequence of interest includes a sequence encoding an alcohol dehydrogenase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 27, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0126] 6.2.4.1.2. Mevalonate pathway enzymes

[0127] In some embodiments, the sequence of interest includes a sequence encoding a mevalonate pathway enzyme. The mevalonate pathway converts acetyl-CoA into isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are precursors to isoprene-like compounds, including compounds of interest as biofuels.

[0128] In some embodiments, the sequence of interest includes a sequence encoding acetyl-CoA thiolytic enzyme (e.g., EC2.3.1.9). In an exemplary embodiment, the sequence of interest includes a sequence encoding acetyl-CoA thiolytic enzyme containing an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 28, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0129] In some embodiments, the sequence of interest includes a sequence encoding hydroxymethylglutaryl-CoA (HMG-CoA) synthase (e.g., EC 2.3.3.10). In an exemplary embodiment, the sequence of interest includes a sequence encoding HMG-CoA synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 29, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0130] In some embodiments, sequences of interest include those encoding hydroxymethylglutaryl-CoA (HMG-CoA) reductase (e.g., EC 1.1.1.34). In an exemplary embodiment, the sequence of interest includes a sequence encoding HMG-CoA reductase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 30, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, or having 80% or higher sequence identity with SEQ ID NO: 31, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0131] In some embodiments, sequences of interest include those encoding pyruvate decarboxylases (e.g., EC 4.1.1.43; EC 4.1.1.72; EC 4.1.1.74). In an exemplary embodiment, the sequence of interest includes a sequence encoding pyruvate decarboxylase, comprising 80% or higher sequence identity with SEQ ID NO: 51, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity; 80% or higher sequence identity with SEQ ID NO: 52, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity; and / or with SEQ ID NO: 52. NO:53 has an amino acid sequence with 80% or higher sequence identity, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0132] In some embodiments, the sequence of interest includes a sequence encoding mevalonate kinase (e.g., EC 2.7.1.36). In an exemplary embodiment, the sequence of interest includes a sequence encoding mevalonate kinase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 32, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0133] In some embodiments, the sequence of interest includes a sequence encoding mevalonate kinase (e.g., EC 2.7.4.2). In an exemplary embodiment, the sequence of interest includes a sequence encoding mevalonate kinase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 33, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0134] In some embodiments, the sequence of interest includes a sequence encoding mevalonate pyrophosphate decarboxylase (e.g., EC4.1.1.33). In an exemplary embodiment, the sequence of interest includes a sequence encoding mevalonate pyrophosphate decarboxylase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 34, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0135] In some embodiments, the sequence of interest includes a sequence encoding geranyl diphosphate synthase (e.g., EC 2.5.1.1). In an exemplary embodiment, the sequence of interest includes a sequence encoding a geranyl diphosphate synthase subunit comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 35, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, and / or having 80% or higher sequence identity with SEQ ID NO: 48, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0136] In some embodiments, the sequence of interest includes a sequence encoding farnesyl diphosphate synthase (e.g., EC2.5.1.10). In an exemplary embodiment, the sequence of interest includes a sequence encoding farnesyl diphosphate synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 35, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0137] 6.2.4.1.3. Fatty acid / polyketide pathway enzymes

[0138] In some embodiments, sequences of interest include sequences encoding fatty acid / polyketide pathway enzymes, which are involved in the synthesis of fatty acids and / or polyketides from simpler precursor molecules.

[0139] In some embodiments, the sequence of interest includes a sequence encoding acetyl-CoA carboxylase (e.g., EC 6.4.1.2). In an exemplary embodiment, the sequence of interest includes a sequence encoding acetyl-CoA carboxylase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 36, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0140] In some embodiments, the sequence of interest includes a sequence encoding malonyl-CoA synthase (e.g., EC6.2.1.14). In an exemplary embodiment, the sequence of interest includes a sequence encoding malonyl-CoA synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 37, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0141] In some embodiments, the sequence of interest includes a sequence encoding a subunit or regulatory unit of a fatty acid synthase (e.g., EC 2.3.1.85). In an exemplary embodiment, the sequence of interest includes a sequence encoding a subunit or regulatory unit of a fatty acid synthase, comprising 80% or higher sequence identity with SEQ ID NO: 38, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, and 80% or higher sequence identity with SEQ ID NO: 39, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity, and / or with SEQ ID NO: 39. NO:40 has an amino acid sequence with 80% or higher sequence identity, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0142] In some embodiments, the sequence of interest includes a sequence encoding lovastatin nonaketone synthase (e.g., EC2.3.1.161). In an exemplary embodiment, the sequence of interest includes a sequence encoding lovastatin nonaketone synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 41, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0143] In some embodiments, the sequence of interest includes a sequence encoding an acyl-CoA hydrolase / thioesterase (e.g., EC3.1.2.20). In an exemplary embodiment, the sequence of interest includes a sequence encoding an acyl-CoA hydrolase / thioesterase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 42, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0144] 6.2.4.1.4. Nonribosomal peptides

[0145] In some embodiments, the sequence of interest includes sequences encoding non-ribosomal peptides, which are peptides not synthesized by ribosomes.

[0146] In some embodiments, the sequence of interest includes a sequence encoding L-cysteine-D-valine synthase (e.g., EC6.3.2.26). In an exemplary embodiment, the sequence of interest includes a sequence encoding L-cysteine-D-valine synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 43, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0147] In some embodiments, the sequence of interest includes a sequence encoding isopentiprine N synthase (e.g., EC 1.21.3.1). In an exemplary embodiment, the sequence of interest includes a sequence encoding isopentiprine N synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 44, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0148] In some embodiments, the sequence of interest includes a sequence encoding a nonribosomal peptide synthase (e.g., EC 6.3.2). In an exemplary embodiment, the sequence of interest includes a sequence encoding a nonribosomal peptide synthase comprising an amino acid sequence having 80% or higher sequence identity with SEQ ID NO: 45, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0149] 6.2.4.1.5. Entner-Doudoroff pathway enzymes

[0150] In some embodiments, the sequence of interest includes the sequence encoding an enzyme that phosphorylates the Entner-Doudoroff pathway. The Entner-Doudoroff pathway converts glucose into pyruvate and glyceraldehyde-3-phosphate, with 2-keto-3-deoxy-6-phosphoglucate (KDGP) as an intermediate.

[0151] In some embodiments, the sequence of interest includes the sequence encoding an enzyme in the non-phosphorylated Entner-Doudoroff pathway. The non-phosphorylated Entner-Doudoroff pathway converts glucose into pyruvate and glyceraldehyde, with 2-keto-3-deoxygluconic acid (KDG) as an intermediate.

[0152] In some embodiments, the sequence of interest includes a sequence encoding gluconate dehydratase (e.g., EC 4.2.1.39). In an exemplary embodiment, the sequence of interest includes a sequence encoding gluconate dehydratase that has 80% or higher sequence identity with SEQ ID NO: 46, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0153] 6.2.4.1.6. Isoprene-like pathway enzymes

[0154] In some embodiments, the sequences of interest include those encoding enzymes involved in isoprene synthesis. Isoprene is an organic compound consisting of one or more isoprene units arranged in a specific pattern. Isoprene has a wide range of applications, including polymers, pigments, fuels, and lubricants.

[0155] In some embodiments, the sequence of interest includes a sequence encoding a dimethylallyltransferase (e.g., EC2.5.1.1). In an exemplary embodiment, the sequence of interest includes a sequence encoding a farnesyl pyrophosphate synthase having dimethylallyltransferase activity, which has 80% or higher sequence identity with SEQ ID NO: 54, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0156] In some embodiments, the sequence of interest includes a sequence encoding a dimethylallyl cis-transferase (e.g., EC2.5.1.28).

[0157] In some embodiments, sequences of interest include those encoding gerany-1,2-bisphosphate phosphatases (e.g., EC3.1.7.11 and / or EC 3.6.1.).

[0158] In some embodiments, sequences of interest include those encoding nerolithyl bisphosphatase (e.g., EC3.1.7.13 and / or EC 3.6.1.).

[0159] In some embodiments, sequences of interest include sequences encoding isoprene synthases (e.g., EC 4.2.3.27 and / or EC 1.17.7.4).

[0160] In some embodiments, sequences of interest include sequences encoding limonene synthase (e.g., EC 4.2.3.16 and / or EC 4.2.3.20).

[0161] In some embodiments, the sequence of interest includes a sequence encoding myrcene synthase (e.g., EC 4.2.3.15). In an exemplary embodiment, the sequence of interest includes a sequence encoding myrcene synthase that has 80% or higher sequence identity with SEQ ID NO: 55, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0162] In some embodiments, sequences of interest include sequences encoding farnesene synthases (e.g., EC 4.2.3.47 and / or EC 4.2.3.46).

[0163] In some embodiments, the sequences of interest include those encoding linalool synthase (e.g., EC 4.2.3.25 and / or EC 4.2.3.25).

[0164] In some embodiments, the sequence of interest includes a sequence encoding pinene synthase (e.g., EC 4.2.3.14).

[0165] In some embodiments, sequences of interest include sequences encoding ocimene synthases (e.g., EC 4.2.3.B69 and / or EC 4.2.3.B40).

[0166] In some embodiments, sequences of interest include sequences encoding 1,8-cineole synthase (e.g., EC 4.2.3.108).

[0167] In some embodiments, the sequence of interest includes a sequence encoding phytoene synthase / lycopene cyclase (e.g., EC 2.5.1.32). In an exemplary embodiment, the sequence of interest includes a sequence encoding phytoene synthase / lycopene cyclase that has 80% or higher sequence identity with SEQ ID NO: 49, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0168] In some embodiments, the sequence of interest includes a sequence encoding lycopene cyclase (e.g., EC 5.5.1.19).

[0169] In some embodiments, the sequence of interest includes a sequence encoding a phytoene desaturase (e.g., EC1.3.99.31). In an exemplary embodiment, the sequence of interest includes a sequence encoding a phytoene desaturase that has 80% or higher sequence identity with SEQ ID NO: 50, such as 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, or 100% sequence identity.

[0170] In some embodiments, the sequence of interest includes a sequence encoding geraniol geraniol diphosphate synthase (e.g., EC2.5.1.29).

[0171] 6.2.4.2. Non-coding sequences of interest

[0172] The expression cassette disclosed herein can be used to produce any sequence of interest that does not encode a protein or has a reduced potential to be translated into a protein. By placing the non-coding or non-translating sequence of interest under the regulation of a hypoxia-inducible promoter, such sequences of interest can be transcribed only at the desired time or under the desired conditions.

[0173] For example, expression cassettes can be used to produce RNA molecules that function in the cell (e.g., ribosomal RNA, tRNA, ribozymes) or RNA molecules that can disrupt one or more of the cell's activities (e.g., RNA, such as microRNA (miRNA) or small interfering RNA (siRNA) for RNA interference (RNAi) applications; or antisense RNA (asRNA)). RNAs that disrupt one or more of the cell's activities may be referred to herein as "regulatory RNAs".

[0174] The expression of regulatory RNAs can be used to silence genes by interfering with the translation or stability of messenger RNAs. By operatively linking the expression of a sequence of interest capable of transcribing regulatory RNAs to the regulatory regions disclosed herein, the silencing effect of regulatory RNAs can be conditioned on the induction of expression, thus applicable to genes essential for growth. Furthermore, generating expression vectors for regulatory RNAs may be simpler than gene knockout methods. RNA-mediated regulatory mechanisms and their potential applications in synthetic biology and metabolic engineering have been described (e.g., Na et al., 2013, Nat. Biotechnol. 31:170-174).

[0175] In some implementations, the sequence of interest is transcribed into microRNA (miRNA).

[0176] In some implementations, the sequence of interest is transcribed into small interfering RNA (siRNA).

[0177] In some implementations, the sequence of interest is transcribed into antisense RNA (asRNA).

[0178] In some implementations, the sequence of interest is transcribed into a single guide RNA (sgRNA) for use in CRISPR-based technologies (e.g., CRISPR interference (CRISPRi); CRISPR gene editing).

[0179] 6.3. Nucleic acids containing expression cassettes

[0180] In implementation, this disclosure relates to nucleic acids comprising expression cassettes as described in Section 6.2.

[0181] In some embodiments, the nucleic acid containing the expression cassette may be in the form of a vector. Cloning and expression vectors have been extensively described in the literature, for example in Sambrook et al., 2001, *Molecular Cloning: A Laboratory Manual* (CSHL Press) and Ausubel et al., 2002, *Short Protocols in Molecular Biology* (Current Protocols), the contents of which relate to expression vectors are expressly incorporated herein by reference. Other exemplary expression vectors suitable for fungal host cells are described in van den Hondel et al., 1991, Bennett and Lasure (eds.), *More Gene Manipulations in Fungi*, Academic Press, pp. 396-428. These vectors can then be introduced into or replicated in suitable host cells, including, for example, fungal cells (especially in the case of expression vectors) or bacterial cells (especially in the case of cloning vectors). Any vector can be used, as long as it is replicable and viable in the cell in which it is introduced.

[0182] In addition to the expression cassette, the vector may also contain one or more nucleic acid sequences that allow or enhance the ability of the construct to be introduced into a host cell, selected upon introduction into the cell, or replicated independently of the host cell's genome. Vectors intended for introduction into fungal cells may also include sequences that allow the vector to integrate into the host cell's genome to replace endogenous genes, coding regions, and / or regulatory regions, or both or more, in the recombinant fungal genome. Many suitable vectors are known to those skilled in the art, and a large number of them are commercially available. An exemplary vector suitable for expression in yeast is the yeast expression plasmid pRS416.

[0183] In some embodiments, the vector contains a selectable marker or reporter gene that can be used to identify cells in which the selectable marker / reporter gene (and an expression cassette present on the same vector) is retained in non-integrating nucleic acids and / or integrated into the genome of a recombinant fungus. Common selectable markers include genes encoding antifungal resistance, fluorescent markers, enzymes that catalyze the formation of readily detectable products, and enzymes or cofactors required for cell survival or growth.

[0184] Nucleic acids also include the host cell genome containing the expression cassette. Section 6.4.1 describes the engineering of fungal cell genomes to incorporate expression cassettes.

[0185] The host cell genome containing the expression cassette may also be characterized by one or more gene modifications, such as deletion (complete or partial), insertion, substitution, replacement, or other modifications, of coding and / or regulatory regions. In embodiments, host cell gene modifications may enable recombinant fungi to increase the amount or productivity of substrates available for the catalytic activity of peptides encoded by the sequence of interest, to more efficiently utilize the product of the catalytic activity of peptides encoded by the sequence of interest, or to thrive more effectively under hypoxic conditions, as well as other properties beneficial to the expression of the sequence of interest, which will be apparent to those skilled in the art who benefit from this disclosure.

[0186] Nucleic acids also include the yeast artificial chromosome (YAC), which contains an expression cassette, two yeast telomere sequences, a yeast centromere sequence, and a yeast autonomous replication sequence (ARS).

[0187] Within the expression cassette, the sequence of interest encoding the amino acid sequence of the heterologous polypeptide can be codon-optimized for recombinant fungi. Therefore, in some embodiments, the nucleotide sequence may contain one or more codons that can lead to faster translation and / or fewer translation errors compared to the native nucleotide sequence encoding the amino acid sequence of the heterologous polypeptide, thereby reducing the likelihood of transcript secondary structure formation or providing other advantages in polypeptide expression in recombinant fungi. In some embodiments, the codon-optimized nucleotide sequence can be generated using an Integrated DNA Technology (IDT) algorithm (www.idtdna.com / pages / tools / codon-optimization-tool).

[0188] When optimizing the first and second codons of the nucleotide sequence, care should be taken to avoid changing the nucleotide at any of the +1 to +4 positions in the Kozak region, particularly at position +4, to a non-common nucleotide of the Kozak sequence of the recombinant fungus. In some embodiments, the first and second codons of the nucleotide sequence are engineered to change the nucleotide at positions +1 to +4 in the Kozak region to a common nucleotide of the Kozak sequence of the recombinant fungus.

[0189] Nucleic acids can contain more than one expression cassette. For example, a plasmid or fungal genome can contain two or more expression cassettes. A fungal genome can contain multiple expression cassettes engineered therein as described in Section 6.4.1.

[0190] 6.4. Fungal cells containing nucleic acids

[0191] In embodiments, this disclosure relates to recombinant fungi comprising expression cassettes of this disclosure (e.g., as described in sections 6.2 and 6.3). Any parent fungus can be engineered to form the nucleic acid-containing recombinant fungus disclosed herein.

[0192] In some embodiments, the fungus is yeast.

[0193] In some embodiments, the yeast belongs to the genus *Saccharomyces* (Saccharomyces). Saccharomyces In this embodiment, the yeast belongs to the Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ) species, such as the brewer's yeast strain BY4741 ( Saccharomyces cerevisiae ).

[0194] In some embodiments, the yeast belongs to the genus Pichia pastoris (Pichia pastoris). Pichia ).

[0195] In some embodiments, the yeast belongs to the genus *Hansenula* (…). Hansenula ).

[0196] In some embodiments, the yeast belongs to the genus *Yersinia* (…). Yarrowia ).

[0197] In some embodiments, the yeast belongs to the genus Kluyveromyces (Kluyveromyces). Kluyveromyces ).

[0198] In some embodiments, the yeast belongs to the genus *Tetranychus* (…). Pachysolen ).

[0199] In some embodiments, the yeast belongs to the genus *Rhodotorula* (Yeast). Rhodotorula ).

[0200] In some embodiments, the yeast belongs to the genus *Zygosaccharomyces* (…). Zygosaccharomyces ).

[0201] In some embodiments, the yeast belongs to the genus *Schizosaccharomyces* (…). Schizosaccharomyces ).

[0202] In some embodiments, the yeast belongs to the genus *Cyclophorus* (…). Torulaspora ).

[0203] In some embodiments, the yeast belongs to the genus *Debaliyces* (…). Debaryomyces ).

[0204] In some embodiments, the yeast belongs to the genus *Pseudomonas* (…). Williopsis ).

[0205] In some embodiments, the yeast belongs to the genus *Dracaena* (…). Dekkera ).

[0206] In some embodiments, the yeast belongs to the genus *Klerkia* (…). Kloeckera ).

[0207] In some embodiments, the yeast belongs to the genus *Metschia* (…). Metschnikowia ).

[0208] In some embodiments, the yeast belongs to the genus *Issa yeast* (…). Issatchenkia ).

[0209] In some embodiments, the yeast belongs to the genus Candida (Candida). Candida ).

[0210] 6.4.1. Engineering Methods

[0211] Fungal cells can be engineered using techniques known in the art to incorporate the expression cassettes of this disclosure. For example, a vector containing an expression cassette, as described in Section 6.3, can be introduced into fungal cells using techniques known in the art.

[0212] For example, fungal cells can be transformed in a manner known per se through processes involving protoplast formation, protoplast transformation, and cell wall regeneration. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153: 163; Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920; and Gietz et al., 2007, Nat Protoc. 2(1):31-34.

[0213] The vector can be maintained in a free state and therefore contains sequences for autonomous replication, such as autosomal replication sequences.

[0214] Nucleic acids containing expression cassettes can also be integrated into the fungal genome in one or more copies. As is well known in the art, integration into the fungal genome can occur randomly via non-homologous recombination or at selected locations via homologous recombination.

[0215] In some embodiments, nucleic acids containing only a portion of the expression cassette are introduced into fungal cells, and a chromosome-integrated expression cassette is formed in situ through recombination of the nucleic acids with the fungal cell genome using a technique sometimes referred to as "gene transformation." In this gene transformation method, nucleic acids containing sequences of one or more components of the expression cassette and corresponding to the genomic region to be replaced are constructed in vitro and then transformed into fungi. Through homologous recombination, the constructed nucleic acid sequences replace the endogenous genomic region. It may be desirable that the constructed nucleic acid sequences also encode markers that can be used to select fungal cell lines containing the expression cassette components. For example, the components of the expression cassette can be introduced onto non-replicating or temperature-sensitive plasmids by binding to a selection marker. Selection for plasmid integration is achieved by selecting the marker under conditions that disallow plasmid replication. Selection of a second recombination event leading to gene substitution is achieved by examining the loss of the selection marker and the acquisition of the expression cassette components in the colony. Alternatively, the nucleic acid sequences of the expression cassette components may contain insertions, substitutions, and / or deletions of one or more nucleotides corresponding to the genomic region.

[0216] Furthermore, in some embodiments, recombinant fungi can be prepared by homologous recombination, such that one or more of the hypoxia-inducible promoter, the Kozak upstream region, and / or the transcription terminator can replace the corresponding regulatory element of the endogenous or previously incorporated sequence of interest in the fungal genome.

[0217] Homologous recombination can be achieved using plasmids constructed to continuously contain 5′ and 3′ regions flanking the genomic sequence to be replaced. These continuous 5′ and 3′ regions can be introduced into fungal cells, for example, on a temperature-sensitive plasmid, at an allowable temperature that allows the plasmid to establish itself in the cells. The cells are then transferred to a non-allowable temperature to select cells that will integrate the plasmid into the chromosome at one of the homologous flanking regions. Selection for plasmid integration is achieved by selecting the second optional marker. After integration, recombination events at the second homologous flanking region are stimulated by transferring the cells to an allowable temperature for several generations without selection. The cells are plated to obtain individual colonies, and the loss of both selection markers in the colonies is examined.

[0218] In some embodiments, recombinant fungi can be prepared by homologous recombination, such that one or more of the hypoxia-inducible promoter, the Kozak upstream region, and / or the sequence of interest can replace the corresponding element that is intrinsic to the fungal genome or a previously incorporated transcription terminator.

[0219] In some embodiments, recombinant fungi can be prepared by homologous recombination, such that one or more of the Kozak upstream region, the sequence of interest, and / or the transcription terminator can replace the corresponding element that is intrinsic to the fungal genome or a previously incorporated hypoxia-inducible promoter.

[0220] In some embodiments, recombinant fungi can be prepared by homologous recombination, such that one or more of the hypoxia-inducible promoter, the sequence of interest, and / or the transcription terminator can replace the corresponding element that is intrinsic to the fungal genome or the previously incorporated Kozak upstream region.

[0221] More generally, recombinant fungi can be prepared by homologous recombination, in which one, two, or three of the fungal endogenous hypoxia-inducible promoter, Kozak upstream region, sequence of interest, and / or transcription terminator are retained, and the other one, two, or three are replaced by elements that are non-native to the retained elements.

[0222] Recombinant fungal cells can be constructed by introducing, replacing, or removing one or more nucleotides from a genomic sequence to produce the expression cassettes of this disclosure. Such modifications can be achieved by site-directed mutagenesis or PCR-generated mutations, according to methods known in the art.

[0223] Optionally or additionally, gene editing technologies can be used to generate heterologous expression cassettes in the fungal genome.

[0224] Various genome editing technologies, including but not limited to homologous recombination, CRISPR-Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), can be used to delete or disrupt genes in parental fungi. Genome editing technologies can be used to operatively link sequences of interest in parental fungi (optionally having one or two intrinsic regulatory elements selected from hypoxia-inducible promoters, Kozak upstream regions, and transcription terminators) to one or more non-intrinsic regulatory elements, thereby producing the expression cassettes of this disclosure. Optionally or additionally, genome editing technologies can be used to alter the nucleotide sequences of sequences of interest and / or one or more regulatory elements in parental fungi to produce the expression cassettes of this disclosure.

[0225] In some implementations, CRISPR-Cas9 editing utilizes nucleic acid-guided endonucleases that locate their target DNA by binding to guide RNA (e.g., Cas9) and Cpf1 or guide DNA (e.g., NgAgo) (which recruits CRISPR endonucleases to target genomic regions), wherein the endonuclease can produce single-strand or double-strand breaks in the DNA. This targeted DNA break becomes a substrate for DNA repair and can be recombinated with a provided editing template to modify, delete, or replace the genomic region. For example, a gene encoding a nucleic acid-guided endonuclease (from *Streptococcus pyogenes* for this purpose) is used. S. pyogenes A gene encoding Cas9 (or the Cas9 nuclease) or a codon-optimized gene operably linked to an active promoter and an active terminator in yeast cells produces a yeast Cas9 expression cassette. Similarly, those skilled in the art can readily identify one or more target sites specific to a genomic region. For example, to construct a DNA construct encoding a gRNA targeting a target site within a genomic region, a variable targeting domain (VT) may contain a nucleotide at the 5' of the protospacer region adjacent to the motif (TGG) of the target site, said nucleotide being coupled with the gene encoding *Streptococcus pyogenes* (…). S. pyogenes DNA is fused to the Cas9 endonuclease recognition domain (CER) of Cas9. The combination of DNA encoding the VT domain and DNA encoding the CER domain produces DNA encoding gRNA. Therefore, a yeast expression cassette of said gRNA can be generated by operatively linking the DNA encoding said gRNA to a promoter and a terminator that are active in yeast cells.

[0226] In some embodiments, DNA breaks induced by the endonuclease can be repaired / replaced using an input sequence. For example, to precisely repair DNA breaks generated by the Cas9 expression cassette and gRNA expression cassette described above, a nucleotide editing template can be provided, allowing the cell's DNA repair mechanism to utilize the editing template. For example, approximately 500 bp from the 5' end and approximately 500 bp from the 3' end of the target genomic region, along with an input sequence (e.g., one, two, three, or four components of the expression cassette), can provide an editing template that is used by the yeast host's mechanisms to repair the DNA breaks using the input sequence.

[0227] The Cas9 expression cassette, gRNA expression cassette, and editing template can be co-delivered to fungal cells using various methods, such as protoplast fusion, electroporation, naturally competent cells, or induced competent cells. The transformed cells can be screened by PCR amplification of the target locus using forward and reverse primers. These primers can amplify wild-type loci or edited modified loci. The amplicon can be sequenced using sequencing primers to identify the edited colonies.

[0228] Before, after, or simultaneously with the engineering of recombinant fungi to incorporate into the expression cassette of this disclosure, the recombinant fungi containing the expression cassette of this disclosure may be engineered to have additional features (e.g., expression of peptides required for the intended purpose of incorporating the expression cassette of this invention, or elimination or reduction of peptides not required for that purpose).

[0229] 6.5. Instructions for Use

[0230] In embodiments, this disclosure relates to a method of using fungi as described in Section 6.4 to express sequences of interest. Typically, the method includes culturing fungal cells (e.g., yeast cells) under growth conditions in a medium containing 2 ppm to 20 ppm oxygen; and culturing the fungi in a medium containing less than 2 ppm oxygen to induce the expression of the sequence of interest.

[0231] 6.5.1. Culture medium

[0232] Recombinant fungi can be cultured in liquid media under appropriate conditions. Media suitable for fungal growth and / or expression of sequences of interest in fungi are known to those skilled in the art. The media may contain D-glucose, glycerol, D-galacturonic acid, L-galacturonic acid, D-tagaguronic acid, D-adarosenic acid, methylglucuronic acid, D-glucuronic acid, D-fructuronic acid, D-mannonic acid, gluconolactone, and / or D-gluconic acid. In some embodiments, the media contains glycerol and glucose. In some embodiments, glucose is added to the media at the end of the logarithmic phase of cell growth (also known as the growth phase). In some embodiments, glucose is added to the media at the beginning of the stationary phase of cell growth (also known as the production phase).

[0233] 6.5.2. Cultivation Conditions

[0234] In some implementations, the recombinant cells undergo fermentation. Fermentation conditions include batch, fed-batch, and continuous fermentation. Classical batch fermentation is a closed system in which the composition of the culture medium is not artificially altered during fermentation. In fed-batch fermentation, the substrate is added incrementally as fermentation progresses. In both classical batch and fed-batch fermentation, the products remain in the bioreactor until the process is complete. Batch and fed-batch fermentation are common and well-known in the art. In continuous fermentation, a prescribed culture medium is continuously added to the bioreactor while an equal volume of product-containing medium is removed. Continuous fermentation aims to maintain steady-state growth conditions. Methods for regulating nutrients and growth factors in continuous fermentation processes, as well as techniques for maximizing product formation rates, are well-known in the field of industrial microbiology.

[0235] The fermentation process can be carried out at the optimal temperature for the growth of recombinant fungi. In embodiments, fermentation is carried out at temperatures ranging from about 20°C to about 45°C, from about 25°C to about 40°C, or from about 30°C to about 37°C. In embodiments, fermentation is carried out at pH values ​​ranging from 4 to 8, from 5 to 7, or from 5.5 to 6.5. In embodiments, fermentation is carried out over time periods ranging from about 8 to 240 hours, from about 12 hours to about 168 hours, from about 16 hours to about 144 hours, from about 20 hours to about 120 hours, from about 24 hours to about 72 hours, or from about 36 to about 48 hours.

[0236] In this embodiment, the recombinant fungi are cultured to increase their biomass during the growth phase and maintain it during the production phase. During the growth phase, the culture medium is aerobic, for example, having a dissolved oxygen concentration of 5 ppm to 20 ppm, which can be achieved or maintained, if desired, by continuously or batchively adding oxygen, air, or other oxygen-containing gas mixtures to the medium. The transition from the growth phase to the production phase can be achieved by allowing the fungi to consume the oxygen in the culture medium without replenishment through the addition of oxygen, air, or other oxygen-containing gas mixtures. In this embodiment, oxygen in the culture medium can be deprived by adding nitrogen or other gas mixtures containing little or no oxygen.

[0237] In some implementations, yeast cultures (e.g., Saccharomyces cerevisiae) Saccharomyces cerevisiae The growth phase of the culture continues until the yeast reaches a certain biomass concentration or cell density threshold. This threshold may depend on the type of culture medium and culture conditions (e.g., oxygen content, temperature, and agitation). In some embodiments, the aerobic growth phase continues until the yeast in the culture reaches a biomass concentration of at least about 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, or 20 mg / ml of dry yeast mass. In some embodiments, after the yeast in the culture reaches a biomass concentration of at least about 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, or 20 mg / ml of dry yeast mass, the production phase is initiated by establishing or allowing the establishment of anoxic or anaerobic conditions. In some embodiments, the aerobic growth phase continues until the yeast in the culture reaches at least about 5 mg / ml of dry yeast mass. 10 6 6 cells / ml 10 6 7 cells / ml 10 6 8 cells / ml, 8 10 6 9 cells / ml, 9 10 6 cells / ml, 1 10 7 cells / ml, 2 10 7 3 cells / ml, 3 107 4 cells / ml 10 7 5 cells / ml, 5 10 7 6 cells / ml 10 7 7 cells / ml 10 7 8 cells / ml, 8 10 7 9 cells / ml, 9 10 7 cells / ml, 1 10 8 cells / ml, 2 10 8 cells / ml or 3 10 8 A cell density of [number] cells / ml. In some embodiments, the yeast in the culture reaches at least about 5 [cells / ml]. 10 6 6 cells / ml 10 6 7 cells / ml 10 6 8 cells / ml, 8 10 6 9 cells / ml, 9 10 6 cells / ml, 1 10 7 cells / ml, 2 10 7 3 cells / ml, 3 10 7 4 cells / ml 10 7 5 cells / ml, 5 10 7 6 cells / ml 10 7 7 cells / ml 10 7 8 cells / ml, 8 10 7 9 cells / ml, 9 10 7 cells / ml, 1 10 8 cells / ml, 2 10 8 cells / ml or 3 10 8After achieving a cell density of [number] cells / ml, the production phase is initiated by establishing or allowing the establishment of hypoxic or anaerobic conditions. In some embodiments, the aerobic growth phase continues until the yeast in the culture reaches at least approximately 5 [cells / ml]. 10 6 CFU / ml, 6 10 6 CFU / ml, 7 10 6 CFU / ml, 8 10 6 CFU / ml, 9 10 6 CFU / ml, 1 10 7 CFU / ml, 2 10 7 CFU / ml, 3 10 7 CFU / ml, 4 10 7 CFU / ml, 5 10 7 CFU / ml, 6 10 7 CFU / ml, 7 10 7 CFU / ml, 8 10 7 CFU / ml, 9 10 7 CFU / ml, 1 10 8 CFU / ml, 2 10 8 CFU / ml or 3 10 8 Cell density of CFU / ml. In some embodiments, the yeast in the culture reaches at least about 5... 10 6 CFU / ml, 6 10 6 CFU / ml, 7 10 6 CFU / ml, 8 10 6 CFU / ml, 9 10 6 CFU / ml, 1 10 7 CFU / ml, 2 10 7 CFU / ml, 3 10 7 CFU / ml, 4 10 7 CFU / ml, 5 10 7 CFU / ml, 6 10 7 CFU / ml, 7 10 7 CFU / ml, 8 10 7 CFU / ml, 9 10 7 CFU / ml, 1 10 8 CFU / ml, 2 10 8 CFU / ml or 3 10 8 After achieving a cell density of CFU / ml, the production phase can begin by establishing or allowing the establishment of hypoxic or anaerobic conditions.

[0238] The hypoxia-inducible promoter of the expression cassette described herein allows expression of sequences of interest during the production phase after fungal biomass has grown to a selected extent. At sufficiently high fungal biomass, the fungal oxygen consumption is typically high enough that hypoxia predominates in the culture, thereby inducing expression of the sequence of interest operatively linked to the hypoxia-inducible promoter without the need for an inducer molecule. Depriving the culture medium of oxygen by adding nitrogen or other gas mixtures with little or no oxygen can aid in the transition to and / or maintenance of hypoxia.

[0239] 6.5.3. Methods for expressing sequences of interest

[0240] This disclosure also relates to a method for generating sequences of interest in fungi, such as yeast cells containing nucleic acids comprising the expression cassettes described herein. Fungal cells can be grown in a culture medium to a desired cell concentration. After reaching the desired cell concentration, the cells can be cultured under anaerobic conditions to induce the expression of the sequences of interest.

[0241] In some implementations, the sequence of interest is transcribed to produce the RNA of interest. The RNA of interest includes coding RNA and non-coding RNA.

[0242] In some embodiments, the RNAs of interest are microRNAs (miRNAs) and small interfering RNAs (siRNAs). Both miRNAs and siRNAs can be extracted and purified from recombinant fungi using known techniques. Purified miRNAs and / or siRNAs can be used for RNAi technology in eukaryotic cells and / or in vitro studies. In some embodiments, miRNAs and siRNAs can be used for RNAi technology in fungal cells that express them.

[0243] In some implementations, the RNA of interest is antisense RNA (asRNA), which can be extracted, purified, and used in antisense RNA technology in cells other than recombinant fungi to express them. Additionally, asRNAs can be selectively paired with and neutralized from transcripts of interest in the recombinant fungi expressing them, and / or the asRNA can be used for in vitro studies.

[0244] In some implementations, the RNA of interest is a single guide RNA (sgRNA), which can be generated for use, for example, after extraction and purification, in CRISPR-based technologies (e.g., CRISPR interference (CRISPRi); CRISPR gene editing).

[0245] In implementations where the sequence of interest is transcribed to produce non-coding RNA, the expression cassette may omit the Kozak region or the Kozak upstream region.

[0246] In some embodiments, the sequence of interest is translated to produce the protein of interest. In some embodiments, the protein of interest is extracted and purified from the cells of the recombinant fungus. In some embodiments, the protein of interest is retained within the cells of the recombinant fungus.

[0247] Proteins of interest can include both enzymes and non-catalytic proteins. Non-catalytic proteins include structural proteins, proteins configured to transport other molecules (e.g., serum albumin), signaling peptides, antibodies and their antigen-binding fragments, and transcriptional regulators. Enzymes include peptides that catalyze any reaction, through which a precursor can be converted into a product or a second precursor of the product can be produced by the action of another enzyme.

[0248] Proteins of interest as enzymes can be part of existing pathways in the cell, for example, by increasing the amount of endogenous proteins from the parent fungus and / or recombinant fungus at desired points on the cell growth curve, or by having activity levels different from the corresponding endogenous proteins in the cell. Proteins of interest as enzymes can also be part of pathways in the recombinant fungus that are not present in the parent fungus.

[0249] In the implementation, the proteins of interest include those described in Section 6.2.4.1.

[0250] 6.5.4. Methods for producing the product

[0251] This disclosure also relates to a method for producing the product. The method includes introducing a nucleic acid comprising an expression cassette described herein into fungal cells, wherein the sequence of interest encodes a protein of interest, the protein catalyzing a reaction in a pathway between the precursor and the product. Similar to the method of expressing the sequence of interest to produce the protein of interest, the fungus can be grown to a desired cell concentration under aerobic conditions. After reaching the desired cell concentration, the fungus can be cultured in a medium under anaerobic conditions to induce the expression of the protein of interest.

[0252] Following expression, the protein of interest can catalyze the reaction. In some embodiments, catalysis occurs in vitro. The protein of interest can be extracted and purified from cells using known techniques. The purified protein of interest can be used in solution, after immobilization on a substrate, or under other conditions to catalyze a reaction upon contact with a substrate containing the protein of interest.

[0253] In some embodiments, catalysis occurs in vivo, for example, in the cells of recombinant fungi. The substrate can be added to the cells by incorporating it into the culture medium, and / or the cells can produce the substrate via metabolic pathways that may exist in the parent microorganism or be engineered into the recombinant fungus.

[0254] Whether catalysis occurs in vitro or in vivo, the protein of interest can catalyze a reaction that can directly convert a substrate into a product, making the substrate a direct precursor of the product, and / or convert a substrate into an intermediate, which can be further converted into the product by the action of one or more other enzymes.

[0255] In embodiments, the product of interest includes products generated directly from the catalytic activity of the protein of interest as described in Section 6.2.4.1 or from the generated intermediates.

[0256] 7. Numbering and Implementation Methods

[0257] Although various specific embodiments have been described and illustrated, it should be understood that various changes can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments set forth below.

[0258] 1. An expression box comprising the following operatively connected components: (a) Hypoxia-inducible promoters; (b) The sequence of interest; and (c) Kozak upstream region and / or transcription terminator; The Kozak upstream region and / or transcription terminator therein are non-native to the promoter.

[0259] 2. The expression cassette according to embodiment 1, wherein the expression of the sequence of interest under hypoxic conditions is improved relative to an expression cassette in which the Kozak upstream region and / or transcription terminator is intrinsic to the promoter, compared to aerobic conditions.

[0260] 3. The expression cassette according to embodiment 2, wherein the improvement in expression of the sequence of interest includes an increase in transcription of the sequence of interest.

[0261] 4. An expression cassette according to any one of embodiments 1 to 3, wherein the sequence of interest encodes a polypeptide.

[0262] 5. The expression cassette according to embodiment 4, wherein the expression of the polypeptide is improved relative to an expression cassette in which the Kozak upstream region and / or transcription terminator is intrinsic to the promoter.

[0263] 6. An expression box according to any one of embodiments 1 to 5, wherein the sequence of interest is non-originating with respect to the promoter.

[0264] 7. The expression box according to any one of embodiments 1 to 6, wherein the low oxygen condition is an anoxic condition.

[0265] 8. The expression cassette according to any one of embodiments 1 to 6, wherein the low oxygen condition is an anaerobic condition.

[0266] 9. An expression cassette according to any one of embodiments 1 to 6, wherein the expression level of the sequence of interest under hypoxia conditions is at least 5 times that under aerobic conditions compared to aerobic conditions.

[0267] 10. An expression cassette according to any one of embodiments 1 to 7, wherein the expression level of the sequence of interest under hypoxia conditions is at least 10 times that under aerobic conditions compared to aerobic conditions.

[0268] 11. The expression cassette according to any one of embodiments 1 to 10, wherein the expression level of the sequence of interest under hypoxia conditions is at least 20 times that under aerobic conditions compared to aerobic conditions.

[0269] 12. The expression cassette according to any one of embodiments 1 to 11, wherein the expression level of the sequence of interest under hypoxia conditions is no more than 80 times that under aerobic conditions compared to aerobic conditions.

[0270] 13. The expression cassette according to any one of embodiments 1 to 12, wherein the expression level of the sequence of interest under hypoxia conditions is no more than 40 times that under aerobic conditions compared to aerobic conditions.

[0271] 14. The expression cassette according to any one of embodiments 9 to 13, wherein the expression under hypoxic conditions compared with aerobic conditions is measured according to any of the methods described in Section 8 (Examples).

[0272] 15. The expression cassette according to any one of embodiments 9 to 13, wherein the expression under hypoxic conditions compared to aerobic conditions is measured according to the method described in Section 8.1 (Example 1).

[0273] 16. An expression cassette according to any one of embodiments 9 to 13, wherein the sequence of interest encodes GFP, and expression under hypoxic conditions compared to aerobic conditions is measured by observing GFP fluorescence.

[0274] 17. The expression cassette according to any one of embodiments 9 to 16, wherein measuring expression under hypoxic conditions compared to aerobic conditions comprises measuring the expression of the sequence of interest after culturing a cell culture containing cells containing the expression cassette under anaerobic conditions for 24 hours.

[0275] 18. The expression cassette according to embodiment 17, wherein the anaerobic conditions include dissolved oxygen in the cell culture being less than 2 ppm.

[0276] 19. The expression cassette according to any one of embodiments 9 to 18, wherein measuring expression under hypoxic conditions compared to aerobic conditions comprises measuring the expression of the sequence of interest after culturing a cell culture containing cells containing the expression cassette under aerobic conditions for 24 hours.

[0277] 20. The expression cassette according to embodiment 19, wherein the aerobic conditions include dissolved oxygen in the cell culture exceeding 10 ppm.

[0278] 21. The expression cassette according to embodiment 20, wherein the aerobic conditions include dissolved oxygen in the cell culture being 10 ppm to 20 ppm.

[0279] 22. An expression cassette according to any one of embodiments 1 to 21, wherein the expression cassette provides a higher ratio of hypoxic to aerobic expression compared to an expression cassette having an upstream Kozak region that is intrinsic to the hypoxia-inducible promoter and is otherwise identical.

[0280] 23. The expression box according to embodiment 22, wherein the higher ratio is a ratio that is 1 to 2 times higher.

[0281] 24. An expression cassette according to any one of embodiments 1 to 23, wherein the expression cassette provides a higher ratio of hypoxic expression to aerobic expression compared to an expression cassette having a transcription terminator that is intrinsic to the hypoxia-inducible promoter and is otherwise identical.

[0282] 25. The expression box according to embodiment 24, wherein the higher ratio is a ratio that is 1 to 2 times higher.

[0283] 26. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 1.

[0284] 27. An expression cassette according to any one of embodiments 1 to 26, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 1.

[0285] 28. An expression cassette according to any one of embodiments 1 to 27, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 1.

[0286] 29. An expression cassette according to any one of embodiments 1 to 28, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 1.

[0287] 30. An expression cassette according to any one of embodiments 1 to 29, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 1.

[0288] 31. An expression cassette according to any one of embodiments 1 to 30, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 2.

[0289] 32. An expression cassette according to any one of embodiments 1 to 31, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 2.

[0290] 33. An expression cassette according to any one of embodiments 1 to 32, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 2.

[0291] 34. An expression cassette according to any one of embodiments 1 to 33, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 2.

[0292] 35. An expression cassette according to any one of embodiments 1 to 34, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 2.

[0293] 36. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 3.

[0294] 37. An expression cassette according to any one of embodiments 1 to 25 and 36, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 3.

[0295] 38. An expression cassette according to any one of embodiments 1 to 25 and 36 to 37, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 3.

[0296] 39. An expression cassette according to any one of embodiments 1 to 25 and 36 to 38, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 3.

[0297] 40. An expression cassette according to any one of embodiments 1 to 25 and 36 to 39, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 3.

[0298] 41. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 4.

[0299] 42. An expression cassette according to any one of embodiments 1 to 25 and 41, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 4.

[0300] 43. An expression cassette according to any one of embodiments 1 to 25 and 41 to 42, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 4.

[0301] 44. An expression cassette according to any one of embodiments 1 to 25 and 41 to 43, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 4.

[0302] 45. An expression cassette according to any one of embodiments 1 to 25 and 41 to 44, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 4.

[0303] 46. ​​An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 5.

[0304] 47. An expression cassette according to any one of embodiments 1 to 25 and 46, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 5.

[0305] 48. An expression cassette according to any one of embodiments 1 to 25 and 46 to 47, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 5.

[0306] 49. An expression cassette according to any one of embodiments 1 to 25 and 46 to 48, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 5.

[0307] 50. An expression cassette according to any one of embodiments 1 to 25 and 46 to 49, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 5.

[0308] 51. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 6.

[0309] 52. An expression cassette according to any one of embodiments 1 to 25 and 51, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 6.

[0310] 53. An expression cassette according to any one of embodiments 1 to 25 and 51 to 52, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 6.

[0311] 54. An expression cassette according to any one of embodiments 1 to 25 and 51 to 53, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 6.

[0312] 55. An expression cassette according to any one of embodiments 1 to 25 and 51 to 54, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 6.

[0313] 56. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 7.

[0314] 57. An expression cassette according to any one of embodiments 1 to 25 and 56, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 7.

[0315] 58. An expression cassette according to any one of embodiments 1 to 25 and 56 to 57, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 7.

[0316] 59. An expression cassette according to any one of embodiments 1 to 25 and 56 to 58, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 7.

[0317] 60. An expression cassette according to any one of embodiments 1 to 25 and 56 to 59, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 7.

[0318] 61. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 8.

[0319] 62. An expression cassette according to any one of embodiments 1 to 25 and 61, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 8.

[0320] 63. An expression cassette according to any one of embodiments 1 to 25 and 61 to 62, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 8.

[0321] 64. An expression cassette according to any one of embodiments 1 to 25 and 61 to 63, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 8.

[0322] 65. An expression cassette according to any one of embodiments 1 to 25 and 61 to 64, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 8.

[0323] 66. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 9.

[0324] 67. An expression cassette according to any one of embodiments 1 to 25 and 66, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 9.

[0325] 68. An expression cassette according to any one of embodiments 1 to 25 and 66 to 67, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 9.

[0326] 69. An expression cassette according to any one of embodiments 1 to 25 and 66 to 68, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 9.

[0327] 70. An expression cassette according to any one of embodiments 1 to 25 and 66 to 69, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 9.

[0328] 71. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 10.

[0329] 72. An expression cassette according to any one of embodiments 1 to 25 and 71, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 10.

[0330] 73. An expression cassette according to any one of embodiments 1 to 25 and 71 to 72, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 10.

[0331] 74. An expression cassette according to any one of embodiments 1 to 25 and 71 to 73, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 10.

[0332] 75. The expression cassette according to any one of embodiments 1 to 25 and 71 to 74, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 10.

[0333] 76. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 11.

[0334] 77. An expression cassette according to any one of embodiments 1 to 25 and 76, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 11.

[0335] 78. An expression cassette according to any one of embodiments 1 to 25 and 76 to 77, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 11.

[0336] 79. An expression cassette according to any one of embodiments 1 to 25 and 76 to 78, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 11.

[0337] 80. An expression cassette according to any one of embodiments 1 to 25 and 76 to 79, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 11.

[0338] 81. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 12.

[0339] 82. An expression cassette according to any one of embodiments 1 to 25 and 81, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 12.

[0340] 83. An expression cassette according to any one of embodiments 1 to 25 and 81 to 82, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 12.

[0341] 84. An expression cassette according to any one of embodiments 1 to 25 and 81 to 83, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 12.

[0342] 85. An expression cassette according to any one of embodiments 1 to 25 and 81 to 84, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 12.

[0343] 86. An expression cassette according to any one of embodiments 1 to 25, wherein the promoter comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 13.

[0344] 87. An expression cassette according to any one of embodiments 1 to 25 and 86, wherein the promoter comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 13.

[0345] 88. An expression cassette according to any one of embodiments 1 to 25 and 86 to 87, wherein the promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 13.

[0346] 89. An expression cassette according to any one of embodiments 1 to 25 and 86 to 88, wherein the promoter comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 13.

[0347] 90. An expression cassette according to any one of embodiments 1 to 25 and 86 to 89, wherein the promoter comprises a nucleotide sequence having 100% identity with SEQ ID NO: 13.

[0348] 91. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of TCTGAATA (K1 -9 to -1), optionally including an A at position -3 and / or an A at position -4.

[0349] 92. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of TCTATAACC (K2 -9 to -1), optionally including A at position -3 and / or A at position -4.

[0350] 93. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of TCTCCAACC (K3 -9 to -1), optionally including A at position -3 and / or A at position -4.

[0351] 94. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of NNNNNANN (-9 to -1 of C1 or C2), optionally including an A at position -4.

[0352] 95. An expression cassette according to any one of embodiments 1 to 90, wherein the upstream region of Kozak comprises at least seven, at least eight, or nine nucleotides of NNNNAANN (-9 to -1 of C3).

[0353] 96. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of TCTNNNANN (-9 to -1 of C4), optionally including an A at position -4.

[0354] 97. The expression cassette according to any one of embodiments 1 to 90, wherein the upstream region of Kozak contains at least seven, at least eight, or nine nucleotides of TCTNNAANN (C5 -9 to -1).

[0355] 98. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of NNNWAMAMA (C6 -9 to -1), optionally including an A at position -4.

[0356] 99. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of NNNNAANN (C7 -9 to -1).

[0357] 100. An expression cassette according to any one of embodiments 1 to 90, wherein the upstream region of Kozak comprises at least seven, at least eight, or nine nucleotides of NNNNNNAAG (Klu1 -9 to -1), optionally including an A at position -4.

[0358] 101. An expression cassette according to any one of embodiments 1 to 90, wherein the upstream region of Kozak comprises at least seven, at least eight, or nine nucleotides of NNNNNNAAA (-9 to -1 of Klu2), optionally including an A at position -4.

[0359] 102. An expression cassette according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises at least seven, at least eight, or nine nucleotides of NNNGCCACC (F1 -9 to -1), optionally including an A at position -4.

[0360] 103. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises any one of sequences DK1 to DK1365.

[0361] 104. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises any one of sequences TK1 to TK262.

[0362] 105. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises CGAGAC (DK1).

[0363] 106. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises ACAAAA (DK2).

[0364] 107. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises GCTTAG (DK3).

[0365] 108. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises CCAGCT (DK4).

[0366] 109. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises AATCAGA (DK5).

[0367] 110. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises AACGA (DK6).

[0368] 111. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises CTCGGG (DK7).

[0369] 112. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises GCTTAA (DK8).

[0370] 113. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises CGCATT (DK9).

[0371] 114. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises AAGAAG (DK10).

[0372] 115. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region includes CCTATA (TK1).

[0373] 116. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises CAAGAG (TK2).

[0374] 117. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises AACAGT (TK3).

[0375] 118. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises GTTAGC (TK4).

[0376] 119. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises TTAAAA (TK5).

[0377] 120. An expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises ATGCAT (TK6).

[0378] 121. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region includes CCAACT (TK7).

[0379] 122. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region includes ATCATA (TK8).

[0380] 123. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region comprises GTAATA (TK9).

[0381] 124. The expression box according to any one of embodiments 1 to 90, wherein the Kozak upstream region includes TATCAG (TK10).

[0382] 125. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 14.

[0383] 126. An expression cassette according to any one of embodiments 1 to 125, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 14.

[0384] 127. An expression cassette according to any one of embodiments 1 to 126, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 14.

[0385] 128. An expression cassette according to any one of embodiments 1 to 127, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 14.

[0386] 129. An expression cassette according to any one of embodiments 1 to 128, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 14.

[0387] 130. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 15.

[0388] 131. An expression cassette according to any one of embodiments 1 to 124 and 130, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 15.

[0389] 132. An expression cassette according to any one of embodiments 1 to 124 and 130 to 131, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 15.

[0390] 133. An expression cassette according to any one of embodiments 1 to 124 and 130 to 132, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 15.

[0391] 134. An expression cassette according to any one of embodiments 1 to 124 and 130 to 133, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 15.

[0392] 135. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 16.

[0393] 136. An expression cassette according to any one of embodiments 1 to 124 and 135, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 16.

[0394] 137. An expression cassette according to any one of embodiments 1 to 124 and 135 to 136, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 16.

[0395] 138. An expression cassette according to any one of embodiments 1 to 124 and 135 to 137, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 16.

[0396] 139. An expression cassette according to any one of embodiments 1 to 124 and 135 to 138, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 16.

[0397] 140. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 17.

[0398] 141. An expression cassette according to any one of embodiments 1 to 124 and 140, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 17.

[0399] 142. An expression cassette according to any one of embodiments 1 to 124 and 140 to 141, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 17.

[0400] 143. An expression cassette according to any one of embodiments 1 to 124 and 140 to 142, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 17.

[0401] 144. An expression cassette according to any one of embodiments 1 to 124 and 140 to 143, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 17.

[0402] 145. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 18.

[0403] 146. An expression cassette according to any one of embodiments 1 to 124 and 145, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 18.

[0404] 147. An expression cassette according to any one of embodiments 1 to 124 and 145 to 146, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 18.

[0405] 148. An expression cassette according to any one of embodiments 1 to 124 and 145 to 147, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 18.

[0406] 149. An expression cassette according to any one of embodiments 1 to 124 and 145 to 148, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 18.

[0407] 150. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 19.

[0408] 151. An expression cassette according to any one of embodiments 1 to 124 and 150, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 19.

[0409] 152. An expression cassette according to any one of embodiments 1 to 124 and 150 to 151, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 19.

[0410] 153. An expression cassette according to any one of embodiments 1 to 124 and 150 to 152, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 19.

[0411] 154. An expression cassette according to any one of embodiments 1 to 124 and 150 to 153, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 19.

[0412] 155. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 20.

[0413] 156. An expression cassette according to any one of embodiments 1 to 124 and 155, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 20.

[0414] 157. An expression cassette according to any one of embodiments 1 to 124 and 155 to 156, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 20.

[0415] 158. An expression cassette according to any one of embodiments 1 to 124 and 155 to 157, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 20.

[0416] 159. An expression cassette according to any one of embodiments 1 to 124 and 155 to 158, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 20.

[0417] 160. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 21.

[0418] 161. An expression cassette according to any one of embodiments 1 to 124 and 160, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 21.

[0419] 162. An expression cassette according to any one of embodiments 1 to 124 and 160 to 161, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 21.

[0420] 163. An expression cassette according to any one of embodiments 1 to 124 and 160 to 162, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 21.

[0421] 164. An expression cassette according to any one of embodiments 1 to 124 and 160 to 163, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 21.

[0422] 165. An expression cassette according to any one of embodiments 1 to 124, wherein the transcription terminator comprises a nucleotide sequence having at least 80% identity with SEQ ID NO: 47.

[0423] 166. An expression cassette according to any one of embodiments 1 to 124 and 165, wherein the transcription terminator comprises a nucleotide sequence having at least 85% identity with SEQ ID NO: 47.

[0424] 167. An expression cassette according to any one of embodiments 1 to 124 and 165 to 166, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 47.

[0425] 168. An expression cassette according to any one of embodiments 1 to 124 and 165 to 167, wherein the transcription terminator comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 47.

[0426] 169. An expression cassette according to any one of embodiments 1 to 124 and 165 to 168, wherein the transcription terminator comprises a nucleotide sequence having 100% identity with SEQ ID NO: 47.

[0427] 170. An expression cassette according to any one of embodiments 1 to 169, wherein the sequence of interest encodes a protein of interest, and the sequence of interest has a T at the +4 position.

[0428] 171. An expression cassette according to any one of embodiments 1 to 170, wherein the sequence of interest encodes an enzyme.

[0429] 172. An expression cassette according to any one of embodiments 1 to 171, wherein the sequence of interest encodes isobutanol and / or butanol pathway enzymes.

[0430] 173. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes a component of an acetyllactate synthase complex (e.g., EC 2.2.1.6).

[0431] 174. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes a ketool reductase (e.g., EC 1.1.1.86).

[0432] 175. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes a dihydroxy acid dehydratase (e.g., EC 4.2.1.9).

[0433] 176. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes phenylpyruvate decarboxylase (e.g., EC 4.1.1.43).

[0434] 177. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes indolepyruvate decarboxylase (e.g., EC 4.1.1.74).

[0435] 178. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes 4-hydroxyphenylpyruvate decarboxylase (e.g., EC 4.1.1.80).

[0436] 179. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes a branched 2-keto acid decarboxylase (e.g., EC 4.1.1.72).

[0437] 180. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes an alcohol dehydrogenase (e.g., EC 1.1.1.1).

[0438] 181. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes an acetolactate synthase complex having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 60.

[0439] 182. An expression cassette according to any one of embodiments 1 to 172, wherein the sequence of interest encodes an acetolactate synthase complex having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 62.

[0440] 183. Expression cassettes 1 to 165 according to any one of the embodiments, wherein the sequence of interest encodes a mevalonate pathway enzyme.

[0441] 184. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes acetyl-CoA thiolytic enzyme (e.g., EC 2.3.1.9).

[0442] 185. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes hydroxymethylglutaryl-CoA (HMG-CoA) synthase (e.g., EC 2.3.3.10).

[0443] 186. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes hydroxymethylglutaryl-CoA (HMG-CoA) reductase (e.g., EC 1.1.1.34).

[0444] 187. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes mevalonate kinase (e.g., EC 2.7.1.36).

[0445] 188. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes mevalonate kinase (e.g., EC 2.7.4.2).

[0446] 189. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes mevalonate pyrophosphate decarboxylase (e.g., EC 4.1.1.33).

[0447] 190. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes geraniol diphosphate synthase (e.g., EC 2.5.1.1).

[0448] 191. An expression cassette according to any one of embodiments 1 to 165 and 183, wherein the sequence of interest encodes farnesyl diphosphate synthase (e.g., EC 2.5.1.10).

[0449] 192. An expression cassette according to any one of embodiments 1 to 165, wherein the sequence of interest encodes a fatty acid / polyketide pathway enzyme.

[0450] 193. An expression cassette according to any one of embodiments 1 to 165 and 192, wherein the sequence of interest encodes acetyl-CoA carboxylase (e.g., EC 6.4.1.2).

[0451] 194. An expression cassette according to any one of embodiments 1 to 165 and 192, wherein the sequence of interest encodes malonyl-CoA synthase (e.g., EC 6.2.1.14).

[0452] 195. An expression cassette according to any one of embodiments 1 to 165 and 192, wherein the sequence of interest encodes a subunit or regulatory unit of a fatty acid synthase (e.g., EC 2.3.1.85).

[0453] 196. An expression cassette according to any one of embodiments 1 to 165 and 192, wherein the sequence of interest encodes lovastatin nonone ketone synthase (e.g., EC 2.3.1.161).

[0454] 197. An expression cassette according to any one of embodiments 1 to 165 and 192, wherein the sequence of interest encodes an acyl-CoA hydrolase / thioesterase (e.g., EC 3.1.2.20).

[0455] 198. Expression cassettes 1 to 165 according to any one of the embodiments, wherein the sequence of interest encodes a nonribosomal peptide.

[0456] 199. An expression cassette according to any one of embodiments 1 to 165 and 198, wherein the sequence of interest encodes L-cysteine-D-valine synthase (e.g., EC 6.3.2.26).

[0457] 200. An expression cassette according to any one of embodiments 1 to 165 and 198, wherein the sequence of interest encodes isopentine N synthase (e.g., EC 1.21.3.1).

[0458] 201. An expression cassette according to any one of embodiments 1 to 165 and 198, wherein the sequence of interest encodes a nonribosomal peptide synthase (e.g., EC 6.3.2).

[0459] 202. An expression cassette according to any one of embodiments 1 to 165, wherein the sequence of interest encodes an enzyme that phosphorylates the Entner-Doudoroff pathway.

[0460] 203. An expression cassette according to any one of embodiments 1 to 171, wherein the sequence of interest encodes an enzyme in the non-phosphorylated Entner-Doudoroff pathway.

[0461] 204. An expression cassette according to any one of embodiments 1 to 165 and 203, wherein the sequence of interest encodes a gluconate dehydratase (e.g., EC 4.2.1.39).

[0462] 205. An expression cassette according to any one of embodiments 1 to 165, wherein the sequence of interest encodes an enzyme involved in isoprene synthesis.

[0463] 206. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes a dimethylallyltransferase (e.g., EC 2.5.1.1).

[0464] 207. The expression cassette according to embodiment 206, wherein the sequence of interest encodes farnesyl pyrophosphate synthase having dimethylallyltransferase activity.

[0465] 208. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes a dimethylallyl cis-transferase (e.g., EC 2.5.1.28).

[0466] 209. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes geraniol diphosphate phosphatase (e.g., EC 3.1.7.11 and / or EC 3.6.1).

[0467] 210. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes nerolithyl bisphosphatase (e.g., EC 3.1.7.13 and / or EC 3.6.1).

[0468] 211. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes isoprene synthase (e.g., EC 4.2.3.27 and / or EC 1.17.7.4).

[0469] 212. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes limonene synthase (e.g., EC 4.2.3.16 and / or EC 4.2.3.20).

[0470] 213. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes myrcene synthase (e.g., EC 4.2.3.15).

[0471] 214. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes farnesene synthase (e.g., EC 4.2.3.47 and / or EC 4.2.3.46).

[0472] 215. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes linalool synthase (e.g., EC 4.2.3.25 and / or EC 4.2.3.25).

[0473] 216. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes pinene synthase (e.g., EC 4.2.3.14).

[0474] 217. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes ocimene synthase (e.g., EC 4.2.3.B69 and / or EC 4.2.3.B40).

[0475] 218. An expression cassette according to any one of embodiments 1 to 171 and 205, wherein the sequence of interest encodes 1,8-cineole synthase (e.g., EC 4.2.3.108).

[0476] 219. An expression cassette according to any one of embodiments 1 to 165 and 205, wherein the sequence of interest encodes phytoene synthase / lycopene cyclase (e.g., EC 2.5.1.32).

[0477] 220. An expression cassette according to any one of embodiments 1 to 165 and 205, wherein the sequence of interest encodes lycopene cyclase (e.g., EC 5.5.1.19).

[0478] 221. An expression cassette according to any one of embodiments 1 to 165 and 205, wherein the sequence of interest encodes a phytoene desaturase (e.g., EC 1.3.99.31).

[0479] 222. An expression cassette according to any one of embodiments 1 to 165 and 205, wherein the sequence of interest encodes geraniol geraniol diphosphate synthase (e.g., EC 2.5.1.29).

[0480] 223. A nucleic acid, which: (a) Contains an expression box according to any one of embodiments 1 to 222; or (b) is configured to form an expression cassette according to any one of embodiments 1 to 222 after homologous recombination with the fungal genome.

[0481] 224. The nucleic acid according to embodiment 223 is a vector, such as a cloning or expression vector.

[0482] 225. The nucleic acid according to embodiment 223 is a fungal genome.

[0483] 226. The nucleic acid according to embodiment 223 is a yeast artificial chromosome (YAC).

[0484] 227. A fungal cell comprising (e.g., engineered to include) an expression cassette according to any one of embodiments 1 to 222.

[0485] 228. The fungal cell according to embodiment 227, which is a yeast ( Saccharomyces )cell.

[0486] 229. The fungal cell according to embodiment 227 or embodiment 228, which is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae )cell.

[0487] 230. The fungal cell according to any one of embodiments 227 to 229, which is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae BY4741 cells.

[0488] 231. A fungal cell according to any one of embodiments 227 to 230, wherein the expression cassette is located within the chromosome.

[0489] 232. A fungal cell according to any one of embodiments 227 to 231, wherein the expression cassette is extrachromosomal.

[0490] 233. A method for expressing a sequence of interest in a fungus, the method comprising: (a) Culture fungal cells according to any one of embodiments 227 to 232 in an aerobic medium under growth conditions; and (b) The fungus is cultured in a hypoxic medium to induce the expression of the sequence of interest.

[0491] 234. The method according to embodiment 233, wherein the hypoxic culture medium is hypoxic.

[0492] 235. The method according to embodiment 233, wherein the hypoxic culture medium is anaerobic.

[0493] 236. A method for expressing a sequence of interest in a fungus, the method comprising: (a) Culture fungal cells according to any one of embodiments 227 to 232 in an aerobic or anaerobic medium under growth conditions; and (b) The fungus is cultured in an anaerobic medium to induce the expression of the sequence of interest.

[0494] 237. A method for producing a product of interest, the method comprising: (a) In an aerobic medium, fungal cells according to any one of embodiments 227 to 232 are cultured under growth conditions, wherein the sequence of interest encodes a protein of interest, the protein catalyzing a reaction in a pathway between the precursor and the product; (b) The fungal cells were cultured in a hypoxic medium to induce the expression of the sequence of interest; and (c) The reaction is catalyzed by the protein of interest, thereby producing the product of interest.

[0495] 238. The method according to embodiment 237, wherein the hypoxic culture medium is hypoxic.

[0496] 239. The method according to embodiment 237, wherein the hypoxic culture medium is anaerobic.

[0497] 240. A method for producing a product of interest, the method comprising: (a) In an aerobic or anaerobic medium, fungal cells according to any one of embodiments 227 to 232 are cultured under growth conditions, wherein the sequence of interest encodes a protein of interest, the protein catalyzing a reaction in a pathway between the precursor and the product; (b) The fungal cells are cultured in an anaerobic medium to induce the expression of the sequence of interest; and (c) The reaction is catalyzed by the protein of interest, thereby producing the product of interest.

[0498] 241. The method according to any one of embodiments 237 to 240, wherein the protein of interest is an enzyme that catalyzes the reaction in the synthesis of isoprene-like substances.

[0499] 242. The method according to any one of embodiments 237 to 241, wherein steps (b) and (c) are performed simultaneously.

[0500] 243. The method according to any one of embodiments 237 to 242 further includes collecting the product of interest.

[0501] 244. The method according to any one of embodiments 237 to 243 further includes purifying the protein of interest from the fungus.

[0502] 245. The method according to embodiment 244, wherein the catalysis is carried out in vitro.

[0503] 8. Examples

[0504] 8.1. Example 1 – Evaluation of Anaerobic Activation Promoters

[0505] To measure a set of anaerobic activated promoters in Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae To determine the expression level in the ORF encoding green fluorescent protein (GFP), the anaerobic activation promoter (DAN1p) of the DAN1 gene (including the original DAN1p Kozak upstream region) was cloned into the yeast expression plasmid pRS416. A 12 bp sequence containing a restriction site was included between the Kozak upstream region and the +1 nucleotide of the GFP coding region for efficient insertion of the promoter into a GFP-enabled operative link. The gene terminator PGK1t was placed downstream of the GFP ORF. Figure 1 The diagram illustrates the general structure of the expressive constructs of this embodiment and other embodiments.

[0506] The final plasmid construct was named pDAN1p-GFP-PGK1t and introduced into *Saccharomyces cerevisiae* using the standard protocol of the Frozen EZ Yeast Transformation II Kit (Zymo Research Corporation, Irvine, CA). S. cerevisiaeCells were seeded on CM-URA medium for selection. As a positive control, the DAN1p in the construct was replaced with the strongly constitutive promoter TEF1p to construct the plasmid pTEF1p-GFP-PGK1t. The pRS416 plasmid containing the GPD1 promoter and CYC1 terminator but lacking the GFP gene was used as a negative control. To measure GFP expression levels from the promoter and terminator combination, fluorescence of yeast cells carrying pDAN1p-GFP-PGK1t or the control plasmid was measured during aerobic and anaerobic (below 2 ppm, close to 0 ppm) growth in uracil-free CM glucose medium (catalog number C8140, Alpha Teknova, Inc., Hollister, CA).

[0507] In parallel, a set of anaerobic activation promoters (DAN4p, TIR1p, TIR3p, TIR4p, tHEM13p, HEM13p, HES1p, AAC3p, and ANB1p) were operatively ligated to the GFP ORF and PGK1t terminators in plasmid pRS416, respectively. Fluorescence in yeast cells carrying the constructed plasmids was measured under aerobic (dissolved oxygen >5 ppm) and anaerobic (dissolved oxygen <2 ppm) conditions.

[0508] GFP expression (relative to negative control) and the ratio of GFP expression under anaerobic to aerobic conditions (normalized to negative control) show that... Figure 2 The results are summarized in Table 3.

[0509]

[0510] The expression experiments showed that, compared with the negative control, the three promoters (DAN1p, TIR1p and HES1p) showed GFP expression levels that were more than 10 times higher than those of the negative control, and that GFP expression under anaerobic conditions was more than 10 times higher than that under aerobic conditions.

[0511] 8.2. Example 2 – Evaluation of gene terminators PGK1t and SPG5t in combination with Dan1p promoter variants

[0512] The DAN1p in pDAN1p-GFP-PGK1t was replaced with DAN1p variants DAN1pm1, DAN1pm5, and DAN1pm6 (Nevoigt et al., 2006, Biotechnol. Bioeng. https: / / doi.org / 10.1002 / bit.21129). The terminator PGK1t was replaced with SPG5t to determine whether the GFP expression pattern observed in Example 1 was independent of the gene terminator. As described in Example 1, the promoter was operatively linked to the GFP ORF in plasmid pRS416, and fluorescence in yeast cells carrying the constructed plasmid was measured under aerobic and anaerobic conditions. TEF1p was paired with GFP ORF and PGK1t as a positive control. The negative control was the same as in Example 1.

[0513] GFP expression (relative to the negative control) and the ratio of GFP expression under anaerobic to aerobic conditions are shown in Figure 3 The following is a summary in Table 4:

[0514] DAN1p variants DAN1pm1, DAN1pm5, and DAN1pm6 showed increased expression intensity under anaerobic conditions, although with a slight loss of specificity for anaerobic expression compared to aerobic expression. This loss of specificity was independent of the terminator used in the gene expression cassette.

[0515] 8.3. Example 3 – Evaluation of the Kozak upstream region sequence

[0516] In eukaryotes, the sequence of the nine nucleotides immediately upstream of the start codon (corresponding to the last nine nucleotides of the 5' untranslated region (UTR), referred to herein as the "Kozak upstream region") is known to be involved in the regulation of gene translation (protein synthesis) initiation. In this embodiment, the constructs studied in Example 2, containing DAN1 variants DAN1pm1, DAN1pm5, and DAN1pm6, were engineered by replacing the DAN1p-originating Kozak upstream region with the following Kozak upstream region sequences: TCTGAATA (K1) (DAN1pm5 and DAN1pm6), TCTTATAACC (K2) (DAN1pm1 and DAN1pm5), or TCTCAACC (K3) (DAN1pm5). The constructs also contain a terminator SPG5t promoter operatively linked to an ORF encoding GFP, and the terminator SPC5t was introduced into each construct. The constructs were introduced into yeast as described in Example 1. Negative and positive controls were as described in Example 2. The combination of DAN1p and its source Kozak upstream region was also tested.

[0517] GFP expression (relative to negative control) and the ratio of anaerobic to aerobic GFP expression are shown in Figure 4 The figure is annotated with arrows to highlight the changes in relative GFP expression and anaerobic:aerobic expression ratio observed when the Kozak upstream region, which is intrinsically linked to the DAN1p variant, was replaced with K1, K2, or K3.

[0518] All tested DAN1p variants / Kozak upstream region substitutions improved both relative GFP expression and the ratio of anaerobic to aerobic GFP expression. Specifically, exchanging the intrinsic Kozak upstream region with a non-intrinsic region in the DAN1pm5 expression construct resulted in up to a 3-fold increase in GFP expression levels and up to a 2-fold increase in specificity for anaerobic expression compared to aerobic expression. In any combination tested, DAN1pm6 showed the highest relative expression increase (4-fold) when the intrinsic Kozak upstream region of DAN1p was replaced with the Kozak upstream region K1, with a slight increase in anaerobic specificity. On the other hand, DAN1pm1, which showed the highest expression when the intrinsic Kozak upstream region was linked, showed only a slight increase in relative expression levels when the intrinsic Kozak upstream region in the expression construct was replaced with the Kozak upstream region K2. Overall, the modifications resulted in up to a 5-fold increase in expression levels (DAN1pm6(K1)) compared to combinations of DAN1p and its intrinsic Kozak upstream region.

[0519] The greatest improvement in anaerobic specificity was observed in DAN1m5(K1) and DAN1m5(K2), with specificity approximately twice that of DAN1m5 and about 33-50% higher than that of the original DAN1p.

[0520] 8.4. Example 4 – Evaluation of the hypoxia-inducible promoter DAN1p in combination with different gene terminators

[0521] To test the effect of terminators on the expression level of the hypoxia-inducible DAN1p promoter, the terminator PGK1t in pDAN1p-GFP-PGK1t (Example 1) was replaced with a set of different gene terminators (CPS1t, VPS13t, HIS5t, PRM9t, ADH1t, IDP1t, PDC6t, GAT2t, LSC2t, SPG5t, UBX6t, SpO1t, and PRM5t). Yeast was engineered with expression constructs containing DAN1p and each terminator, and fluorescence measurements were performed as described in Example 1.

[0522] GFP expression (relative to negative control) is shown in Figure 5 The following is a summary in Table 5:

[0523] The results showed that replacing the terminator PGK1t with SPG5t, PRM9t, IDP1t, PRM5t, SPO1t, LSC2t, UBX6t, or ADH1t resulted in a relative increase of at least 50% in the expression level of GFP operatively linked to DAN1p in the construct, suggesting that these terminators may enhance the expression intensity of hypoxia-inducible promoters compared to PGK1t.

[0524] 8.5. Example 5 – Evaluation of the hypoxia-inducible promoter DAN1pm1 in combination with different gene terminators

[0525] To assess whether the results found in Example 4 apply to promoters with baseline expression higher than DAN1p, the experiments described in Example 4 were repeated with the DAN1 promoter variant DAN1pm1.

[0526] The tested GFP expression (relative to the negative control) and the ratio of anaerobic to aerobic GFP expression are shown in... Figure 6 middle. Figure 7 The GFP expression of the tested DAN1pm1 / terminator combination (X-axis) and the corresponding DAN1p / terminator combination (Y-axis) is shown (relative to the negative control).

[0527] Figure 6 The results of the DAN1pm1 / terminator shown in the figure indicate that the terminators SPG5t, UBX6t, SPO1t, and PRM5t enhance GFP expression by up to two-fold compared to PGK1t, while the selectivity of the reporter gene for anaerobic expression is almost not reduced compared to aerobic expression. These results are similar to those observed with combinations of terminators and DAN1p (Example 4).

[0528] Figure 7 The comparison between DAN1pm1 and DAN1p shown in the figure indicates that when paired with terminators UBX6t, PRM5t, SPG5t, and SPO1t, both promoters DAN1p and DAN1pm1 exhibit enhanced GFP expression compared to PGK1t.

[0529] 8.6. Example 6 – Evaluation of hypoxia-inducible promoters in combination with PGK1t or SPG5t

[0530] The DAN1p promoter in the PGK1t and SPG5t constructs of Example 4 was replaced with hypoxia-inducible promoters ANB1p, HEM13p, AAC3p, DAN1pm1, DAN1pm5, and DAN1pm6. Furthermore, the DAN1pm1, DAN1pm5, and DAN1pm6 constructs were modified by deleting a 12 bp useful sequence between the Kozak upstream region and the start codon of the GFP coding region. The constructs with the 12 bp deletion were named DAN1pm1-12, DAN1pm5-12, and DAN1pm6-12, respectively. In these constructs with the 12 bp deletion, the promoter (including the Kozak upstream region) was placed directly adjacent to the coding region.

[0531] As described in Example 1, an expression cassette containing a promoter and terminator operatively linked to GFP was introduced into yeast. GFP expression under anaerobic conditions was measured as described in Example 1.

[0532] GFP expression (relative to negative control) of each promoter paired with each terminator under anaerobic conditions is shown in Figure 8 The following is a summary in Table 6:

[0533] Figure 8 The PGK1t and SPG5t results shown indicate that the terminator SPG5t enhances GFP expression compared to PGK1t when paired with all DAN1p variants (DAN1pm1, DAN1pm5, and DAN1pm6) and all other hypoxia-inducible promoters tested except AAC3p. Regardless of the terminator used, the DAN1p variants consistently outperformed unmodified DAN1p, tHEM13p, and HEM13p by approximately 3–4 times. The DAN1p variant construct modified with a 12 bp deletion slightly outperformed the DAN1p variant retaining the 12 bp sequence.

[0534] 8.7. Example 7 – GFP expression under the control of DAN1pm5 or TIR1p promoters combined with non-original Kozak regions

[0535] Expression constructs were prepared according to the methods of Examples 1-6, wherein the GFP coding region was operatively linked to the DAN1pm5 or TIR1p promoter and a non-original Kozak region. GFP expression was measured under aerobic and anaerobic conditions.

[0536] 8.8. Example 8 – GFP expression under the control of a hypoxia promoter in combination with an expression-enhancing terminator

[0537] Expression constructs were prepared according to the methods of Examples 1-6, wherein the GFP coding region was operatively linked to a hypoxia promoter other than DAN1p and an expression-enhancing terminator. GFP expression was measured under aerobic and anaerobic conditions.

[0538] 8.9. Example 9 – Expression of non-GFP gene products under hypoxia promoter control

[0539] Expression constructs were prepared according to the methods of Examples 1-6, wherein the coding region of a polypeptide other than GFP was operatively linked to a hypoxia promoter. Other constructs further comprising a non-original Kozak upstream region and / or a non-original terminator were prepared. Expression of the genes was determined under aerobic and anaerobic conditions.

[0540] 8.10. Example 10 – Evaluation of a new combination of promoters and terminators

[0541] Various combinations of 14 terminators with 9 anaerobic inducible promoters were evaluated. Each pair used a wild-type terminator of the promoter as a control. Combination-specific plasmids were generated by replacing Dan1p or PGK1t in the pDAN1p-GFP-PGK1t plasmid. These new plasmids were then transformed into *Saccharomyces cerevisiae* using a PEG-mediated transformation protocol. S. cerevisiae In strain BY4741. To measure the relative expression levels of the combinations of interest, yeast cells carrying CEN / ARS-based plasmids containing the GFP gene of interest were grown anaerobically (<2 ppm O2) for at least 24 hours, and the fluorescence levels of the yeast cells were measured using a Sony SH-800 cell sorter equipped with a 488 nm laser. To obtain reliable measurements of yeast population fluorescence, fluorescence measurements were performed on at least 50,000 yeast cells. The measured fluorescence of the yeast cell population containing the combinations of interest was divided by the measured intrinsic fluorescence of yeast cells without GFP to obtain the relative expression levels of the genetic constructs of interest.

[0542] To measure the ratio of anaerobic to aerobic expression, yeast cells carrying the DAN1p-GFP plasmid were grown under aerobic conditions for at least a period of time, and their fluorescence was measured in the same manner as described above. The measured fluorescence of the anaerobic yeast cells was then divided by the measured fluorescence of the aerobic yeast cells to obtain the expression ratio between the two growth conditions.

[0543] The promoters and terminators used in the above combination are listed in Table 7:

[0544] The GFP expression levels (relative to the negative control) of the selected promoter-terminator combinations and the ratio of anaerobic to aerobic GFP expression are shown in... Figure 9 middle.

[0545] 8.11. Example 11 – Screening of Kozak sequence libraries

[0546] Kozak sequence libraries were generated as follows: Constructs containing DAN1pm5 or TIR1p were engineered to replace six nucleotides at positions -6 to -1 of the native Kozak upstream region with different Kozak upstream region sequences, resulting in approximately 4000 unique variants for each promoter. The libraries were screened for Kozak sequence enrichment using flow cytometry, with four rounds of selection for constructs containing DAN1pm5 and five rounds for constructs containing TIR1p. In each round, samples were sorted, with 300,000 cells collected under each condition. Gating was set to isolate 10% of the total population under each condition (aerobic-low, aerobic-high, anaerobic-low, and anaerobic-high), and the top 10% of cells with the highest anaerobic expression were passaged for further growth. Table 8 summarizes each TIR1p-Kozak sequence and its count at the end of the 5th round of anaerobic expression cells, while Table 9 summarizes each DAN1pm5-Kozak sequence and its count at the end of the 4th round of anaerobic expression cells.

[0547]

[0548] 8.12. Example 12 – Hypoxia-inducible expression of isobutanol pathway enzymes

[0549] The utility of promoter-terminator combinations in directing non-GFP gene expression was evaluated using enzyme genes in the isobutanol pathway. Details of this pathway are available in [link to relevant documentation]. Figure 10 The following is a brief description. In short, under aerobic conditions, pyruvate can be converted to valine; however, under anaerobic conditions, the pathway stalls after the production of 2,3-dihydroxyisovaleric acid (DIV), leading to DIV accumulation. Expression constructs were prepared in which the coding regions of acetyllactate synthase (alsS) or ketolactone reductase (KARI) were integrated into plasmids, where they were operatively linked to the hypoxia-inducible promoters DAN1p or TIR1p. The following plasmid constructs were prepared: integrator 1 or IC1 (SEQ ID NO: 56), containing an alsS coding sequence operatively linked to the PRM5t terminator and the DAN1p-m6 promoter with the Kozak sequence DK73; and integrator 2 or IC2 (SEQ ID NO: 57), containing a KARI coding sequence operatively linked to the SPG5t terminator and the TIR1p promoter with the Kozak sequence TK5. The plasmid constructs were transformed into Saccharomyces cerevisiae, alone or in combination, using the Frozen EZ Yeast Transformation II Kit (Zymo Research Corporation, Irvine, CA). S. cerevisiae Four strains were produced: BY4741 WT control, BY4741 ΔYPRCdelta15, IC2 only, and IC1+IC2.

[0550] For aerobic fermentation, three bioparameters of each strain were inoculated into 2 mL of YPD medium in 24-well deep-well plates and grown at 30°C and 200 rpm for 24 h. For subsequent anaerobic fermentation, the strains were inoculated from aerobic plates into 2 mL of YPD medium in 24-well deep-well plates at a 1:10 ratio. Anaerobic conditions were created using the BD GasPak™ EZ bag system. Each plate was grown at 30°C and 200 rpm for 24 h. Samples were prepared by adding 1 mL of culture to MP Biomedical lysis matrix Y and then placing them in an MP Biomedical Fastprep 24 5G instrument. Cells were lysed at 6.0 m / sec for 40 sec and then centrifuged at 13,000 rpm for 5 min. The resulting supernatant was then filtered through a 0.22 µm syringe filter. Analysis was performed using an Agilent LCMS-QQQ6400 series instrument with an ACQUITY UPLC CSH C18 column (1.7 µm, 100 mm x 2 µm). The mobile phase was 95% acetonitrile, and the buffer contained 0.1% formic acid.

[0551] The results are as follows Figure 11 As shown in the diagram. In short, no 2,3-dihydroxyisovaleric acid (DIV) accumulation was observed in BY4741 ΔYPRCdelta15, indicating that the deletion in this strain does not affect the pathway described. No accumulation was observed in the IC2 strain alone under both aerobic and anaerobic conditions, suggesting that the presence of alsS is crucial for DIV production. The IC1+IC2 strain, possessing both alsS and KARI genes under hypoxic promoter control, exhibited DIV accumulation under anaerobic conditions.

[0552] 9. Sequence

[0553] Exemplary sequences mentioned in this article are provided in Table 10 below (where “SEQ” refers to SEQ ID NO).

[0554]

Claims

1. An expression cassette comprising the following operatively connected components: (a) Hypoxia-inducible promoters; (b) The sequence of interest; (c) Transcription terminators; and (d) Kozak upstream region; in: (i) The transcription terminator and / or the Kozak upstream region are heterologous to the hypoxia-inducible promoter; and (ii) The expression of the sequence of interest provided by the expression cassette under hypoxia conditions is at least 10 times greater than that under aerobic conditions compared to aerobic conditions.

2. The expression cassette according to claim 1, wherein the transcription terminator is heterologous to the hypoxia-inducible promoter.

3. The expression cassette according to claim 1 or 2, wherein the transcription terminator is heterologous to the sequence of interest.

4. The expression cassette according to any one of claims 1 to 3, wherein the Kozak upstream region is heterologous to the hypoxia-inducible promoter.

5. The expression cassette according to any one of claims 1 to 4, wherein the Kozak upstream region is heterologous to the sequence of interest.

6. The expression cassette according to any one of claims 1 to 5, wherein the expression of the sequence of interest provided by the expression cassette under hypoxic conditions is at least 20 times greater under aerobic conditions than under aerobic conditions.

7. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

1.

8. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

2.

9. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

3.

10. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

4.

11. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

6.

12. The expression cassette according to any one of claims 1 to 6, wherein the hypoxia-inducible promoter comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

11.

13. The expression cassette according to any one of claims 1 to 12, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

15.

14. The expression cassette according to any one of claims 1 to 12, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

17.

15. The expression cassette according to any one of claims 1 to 12, wherein the transcription terminator comprises a nucleotide sequence having at least 90% identity with SEQ ID NO:

14.

16. The expression cassette according to any one of claims 1 to 15, wherein the Kozak upstream region comprises any one of sequences DK1 to DK1365.

17. The expression cassette according to any one of claims 1 to 15, wherein the Kozak upstream region comprises any one of sequences TK1 to TK262.

18. The expression box according to any one of claims 1 to 15, wherein the Kozak upstream region comprises TCTGATATA (K1 -9 to -1).

19. The expression box according to any one of claims 1 to 15, wherein the Kozak upstream region comprises TCTATAACC (K2 -9 to -1).

20. The expression box according to any one of claims 1 to 15, wherein the Kozak upstream region comprises TCTCCAACC (K3 -9 to -1).

21. The expression cassette according to any one of claims 1 to 20, wherein the sequence of interest encodes an isobutanol pathway enzyme.

22. The expression cassette of claim 21, wherein the isobutanol pathway enzyme comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

60.

23. The expression cassette of claim 21, wherein the isobutanol pathway enzyme comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

62.

24. The expression cassette according to any one of claims 1 to 23, wherein the expression cassette provides a higher ratio of hypoxic to aerobic expression compared to an expression cassette having an upstream Kozak region that is intrinsic to the hypoxia-inducible promoter and is otherwise identical.

25. The expression box of claim 24, wherein the higher ratio is a ratio that is 1 to 2 times higher.

26. The expression cassette according to any one of claims 1 to 25, wherein the expression cassette provides a higher ratio of hypoxic expression to aerobic expression compared to an expression cassette having a transcription terminator that is intrinsic to the hypoxia-inducible promoter and is otherwise identical.

27. The expression box of claim 26, wherein the higher ratio is a ratio that is 1 to 2 times higher.

28. A nucleic acid, which: (a) Containing an expression box according to any one of claims 1 to 27; or (b) is configured to form an expression cassette according to any one of claims 1 to 27 after homologous recombination with the fungal genome.

29. The nucleic acid according to claim 28, wherein it is a vector, such as a cloning or expression vector.

30. The nucleic acid according to claim 28, wherein it is a fungal genome.

31. The nucleic acid according to claim 28, wherein it is a yeast artificial chromosome (YAC).

32. A fungal cell comprising an expression cassette according to any one of claims 1 to 27 or a nucleic acid according to any one of claims 26 to 29.

33. The fungal cell of claim 32, which is a Saccharomyces cerevisiae (S. cerevisiae) cell. Saccharomyces cerevisiae ) cell.

34. The fungal cell according to claim 32 or 33, wherein it is a Saccharomyces cerevisiae BY4741 cell.

35. The fungal cell according to any one of claims 32 to 34, wherein the expression cassette is located within the chromosome.

36. The fungal cell according to any one of claims 32 to 34, wherein the expression cassette is located within an extrachromosomal nucleic acid.

37. A fungal culture comprising fungal cells according to any one of claims 32 to 36 in a culture medium.

38. The fungal culture of claim 37, wherein the culture medium is anaerobic or anaerobic.

39. The fungal culture of claim 37, wherein the culture medium is aerobic.

40. A method for expressing a sequence of interest in a fungal culture, the method comprising: (a) Cultivating the fungal culture according to claim 37 under aerobic growth conditions; and (b) The fungal culture is cultured under anaerobic conditions to induce the expression of the sequence of interest.

41. The method of claim 40, wherein step (b) is performed after the cell density in the fungal culture reaches 2 x 10 10 7 cells / ml.

42. A method for producing a product of interest, the method comprising: (a) Cultivating a fungal culture according to claim 37 under aerobic growth conditions, wherein the sequence of interest encodes a protein of interest, the protein of interest catalyzing a reaction in a pathway between the precursor and the product; (b) The fungal culture is cultured under anaerobic conditions to induce the expression of the sequence of interest; and (c) The reaction is catalyzed by the protein of interest, thereby producing the product of interest.

43. The method of claim 42, wherein step (b) is performed after the cell density in the fungal culture reaches 2 x 10<6> cells / ml. 10 7 cells / ml.

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