C2 carbon source responsive promoters

Abstract

The present disclosure relates to promoters engineered to increase responsiveness to C2 carbon sources. Engineered promoters comprise at least one external carbon source responsive element (CSRE) located upstream and proximal to a transcription initiation site of a gene operably associated therewith. The engineered promoters can be used in heterologous nucleic acid molecules, vectors, or expression cassettes to promote expression of genes in microorganisms. The disclosure also relates to methods for producing the engineered promoters and methods for expressing genes in recombinant microbial host cells using the engineered promoters.

Description

[0001] Cross-citation of relevant applications and literature

[0002] This patent application claims priority to U.S. Provisional Patent Application 63 / 508,378, filed June 15, 2023, the entire contents of which are incorporated herein by reference. This patent application also includes a list of sequences of electronic (HTML) versions, which are also fully incorporated herein by reference. Technical Field

[0003] This disclosure relates to promoters capable of inducing gene expression in recombinant microbial hosts in the presence of a C2 carbon source (such as ethanol). Background Technology

[0004] Microorganisms serve as platforms for expressing heterologous genes (which may encode heterologous polypeptides). However, the expression system of heterologous genes should preferably be controlled to limit the metabolic burden on the microbial host. This is why inducible expression systems are generally preferred.

[0005] Saccharomyces cerevisiae ( Komagataella phaffii (formerly known as Pichia pastoris) Pichia pastoris () is a universal expression system for recombinant peptides, allowing expression similar to that of Saccharomyces cerevisiae () Saccharomyces cerevisiae Post-translational modification and secretion occur in a manner consistent with the expression of recombinant peptides. One distinguishing feature of *Phaeocotyle phloxera* as a recombinant expression host is its ability to achieve high density and high protein content, resulting in higher yields of expressed recombinant peptides. Furthermore, low levels of endogenously secreted peptides produce high-purity recombinant peptides in the extracellular fraction. *Phaeocotyle phloxera* is also distinguished by its ability to efficiently utilize non-fermentable carbon sources such as glycerol, particularly methanol, through the action of alcohol oxidases (AOX). In this respect, the AOX1 expression system is well-established in *Phaeocotyle phloxera* and widely used for heterologous peptide expression. The methanol oxidation pathway is tightly regulated, so that the expression of genes required for methanol metabolism is induced only in the presence of methanol (such as AOX1 alcohol oxidase) and is inhibited by various carbon sources, including glucose. In biotechnology, this tight control over methanol-related genes has been used for time-controlled production of heterologous proteins: under the control of the AOX1 promoter, heterologous peptide expression is induced only when methanol is added to the system. This has shown significant advantages for cytotoxic peptides and also allows for the biological process control of protein production. Specifically, in aerobic fermentation, the feed is converted from other carbon sources to methanol, thereby inducing peptide expression. However, methanol poses technical, environmental, and safety risks due to its flammability and toxicity.

[0006] Therefore, there is a need to develop a microbial expression system, particularly a microbial promoter, that is inducible in the presence of an inducer other than methanol.

[0007] In some implementations, the inducer should be compatible with large-scale commercial operations (e.g., having lower flammability and / or toxicity than methanol). Summary of the Invention

[0008] This disclosure relates to promoters that have been engineered to increase expression intensity in the presence of a C2 carbon source such as ethanol. The engineered promoters include an external carbon source-responsive element.

[0009] According to a first aspect, the present invention relates to an engineered promoter, which (i) is derived from a parent promoter having a transcription start site, and (ii) is used to express a gene. The engineered promoter has at least one external carbon source responsive element (CSRE). The at least one external CSRE has a nucleic acid sequence of formula (I): N1N2CCN3N4TN5N6N7CCGN8 (I) Wherein, N1 is any nucleic acid residue; N2 is any nucleic acid residue, preferably C or T; N3 is any nucleic acid residue, preferably A, G, or T; N4 is any nucleic acid residue, preferably C or T; N5 is any nucleic acid residue, preferably A, C, or G; N6 is any nucleic acid residue, preferably A or G; N7 is any nucleic acid residue, preferably G or T; and N8 is any nucleic acid residue, preferably A or G. At least one external CSRE comprises a first external CSRE located upstream of and proximal to the transcription start site. In one embodiment, the gene contains an open reading frame having a start codon. In another embodiment, the first external CSRE is located at up to 390 base pairs upstream of the start codon (-390). In yet another embodiment, the engineered promoter comprises a TATA box. In yet another embodiment, in the presence of a C2 carbon source such as ethanol, the engineered promoter is capable of inducing gene transcription at a higher level than the parental promoter. In yet another embodiment, at least one external CSRE comprises a nucleic acid sequence of any one of SEQ ID NO: 26 to 35. In yet another embodiment, the engineered promoter according to any one of claims 1 to 6 comprises at least two, three, four, five, six, seven, eight, nine, or ten external CSREs. In some embodiments, the parent promoter is an ethanol-responsive promoter, for example... adh2 Gene promoter (adh2p). In some specific embodiments, the engineered promoter has the nucleic acid sequence SEQ ID NO: 6, 7, 8, 9, 10, 11, 19, 20, 21, 22 or 23. In some embodiments, the parental promoter is a constitutive promoter, for example... sti1 Gene promoter (sti1p). In some specific implementations, the engineered promoter has the nucleic acid sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17 or 18.

[0010] According to a second aspect, this disclosure provides a heterologous nucleic acid molecule having an engineered promoter described herein that is operatively associated with a gene. In some embodiments, the gene encodes a polypeptide.

[0011] According to a third aspect, this disclosure provides a vector comprising the engineered promoter or the heterologous nucleic acid molecule described herein.

[0012] According to the fourth aspect, this disclosure provides an expression cassette comprising the engineered promoter or the heterologous nucleic acid molecule described herein.

[0013] According to a fifth aspect, this disclosure provides a recombinant microbial host cell comprising the engineered promoter, the heterologous nucleic acid molecule, the vector, or the expression cassette described herein. In one embodiment, the recombinant microbial host cell has native alcohol dehydrogenase activity. In another embodiment, the recombinant microbial host is yeast. In yet another embodiment, the recombinant microbial host cell is derived from the genus *Komata* (…). Komagataella sp.), and in yet another implementation, from *Favora foetida*.

[0014] According to a sixth aspect, this disclosure relates to a method for increasing the responsiveness of an engineered promoter for expressing a gene to a C2 carbon source. The method includes introducing a first external carbon source responsive element (CSRE) upstream and proximal to the transcription start site in a parental promoter having a transcription start site. The first external CSRE has a nucleic acid sequence of formula (I): N1N2CCN3N4TN5N6N7CCGN8 (I) Wherein, N1 is any nucleic acid residue; N2 is any nucleic acid residue, preferably C or T; N3 is any nucleic acid residue, preferably A, G, or T; N4 is any nucleic acid residue, preferably C or T; N5 is any nucleic acid residue, preferably A, C, or G; N6 is any nucleic acid residue, preferably A or G; N7 is any nucleic acid residue, preferably G or T; and N8 is any nucleic acid residue, preferably A or G. In one embodiment, the gene comprises an open reading frame having a start codon. In another embodiment, the method comprises introducing a first external CSRE at a position of up to 390 base pairs (-390) upstream of the start codon. In another embodiment, the parental promoter comprises a TATA box. In yet another embodiment, the first external CSRE comprises a nucleic acid sequence of any one of SEQ ID NO: 26 to 35. In some embodiments, the method comprises introducing two, three, four, five, six, seven, eight, nine, or ten external CSREs into the parental promoter. In another embodiment, each external CSRE has an independent nucleic acid sequence selected from any one of SEQ ID NO: 26 to 35. In one embodiment, the parent promoter is an ethanol-responsive promoter. In another embodiment, the parent promoter is a constitutive promoter.

[0015] According to a seventh aspect, this disclosure provides a method for expressing a gene in a recombinant microbial host cell described herein. The method includes (i) contacting the recombinant microbial host cell with a C2 carbon source, such as ethanol, to allow gene expression. In one embodiment, the method further includes, prior to step (i), (i') propagating the recombinant microbial host cell with an alternative carbon source different from the C2 carbon source. In one embodiment, the alternative carbon source includes glucose, fructose, and / or glycerol. In another embodiment, the gene encodes a polypeptide. In another embodiment, the polypeptide is an intracellular polypeptide or a secreted polypeptide. In yet another embodiment, the secreted polypeptide is in free form or associated with the surface of the recombinant yeast host cell. In yet another embodiment, the polypeptide associated with the surface of the recombinant yeast host cell is a tethered polypeptide. In another embodiment, the polypeptide is an enzyme. In some embodiments, the method further includes, after step (i), (ii) substantially isolating the polypeptide from the recombinant microbial host cell. Attached Figure Description

[0016] Therefore, the nature of the invention has been generally described, and preferred embodiments of the invention will now be illustrated by way of the accompanying drawings, wherein: Figure 1A schematic diagram of one of the engineered schemes applied to the alcohol dehydrogenase 2 (ADH2) promoter is provided. Triangles symbolically depict the engineered carbon source responsive elements (CSREs) and their relative distances (not depicted) on the nucleotide chain from the functional core promoter region proximal to the adh2 gene. In the engineered promoter variants, denoted as eADH2p-01, eADH2p-02, eADH2p-03, eADH2p-03.1, eADH2p-05, and eADH2p-10, additional CSREs are introduced according to the diagram, progressively closer to the core promoter. The relative positions of the additional CSREs are not drawn to scale.

[0017] Figure 2 Results of reporter gene enzyme activity assays performed on shake flask supernatant fractions of cultures from strains M32338, M32816, M32818, and M32820 are provided. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity (bars) is reported as relative fluorescence units (RFU) compared to the negative control. Productivity (reporter gene enzyme activity versus OD) is also shown. 600 The ratio (diamond shape). Error bars represent the standard deviation of the mean.

[0018] Figure 3 Results of reporter gene enzyme activity assays were presented for supernatant fractions from cultures of strains M32338, isolate T13859, and strain M32820. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity is reported as relative fluorescence units (RFU) compared to the negative control. Error bars indicate the standard deviation of the mean.

[0019] Figure 4 Results of reporter gene enzyme activity assays from supernatant fractions of bioreactor fermentations using strains M31676, M32338, and M32820 are presented. Methanol was used as the carbon source for strain M31676, and ethanol was used for strains M32338 and M32820. Reporter gene enzyme activity units (bars) were calculated by comparison with a standard curve of a commercial lipase sample. Productivity (the ratio of reporter gene enzyme activity to stem cell weight, in diamond shapes) is also shown. Error bars represent the error in proliferation according to established uncertainty proliferation rules.

[0020] Figure 5Results of reporter gene enzyme activity assays performed on supernatant fractions from 96-well culture plates of strains M34673, M32338, M32702, M32816, M32818, M32820, M33401, and M33403 are provided. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity (bars) is reported as relative fluorescence units (RFU) compared to the negative control. Productivity (reporter gene enzyme activity versus OD) is also shown. 600 The ratio (square). Error bars represent the standard deviation of the mean.

[0021] Figure 6 A schematic diagram of another engineering approach for the ADH2 promoter is provided. The triangle symbolically represents the engineered CSRE and its distance from the nucleotide chain. adh2 Relative distances of functional core promoter regions proximal to the gene (not depicted). In engineered promoter variants denoted as eADH2p-03.1 to eADH2p-03.7, a CSRE is introduced, progressively closer to the core promoter, as shown in the illustration. The relative positions of the CSREs are not plotted to scale.

[0022] Figure 7 Reporter gene enzyme activity assays were performed on supernatant fractions from 96-well plates of strains M34673, M32388, M33399 and isolates T15016, T15015, T15014, T15011, T15012, and T15013. For each engineered promoter, the distance between the open reading frame (ORF) and the added CSRE was included on the x-axis label. Ethanol was used as the carbon source for all strains and isolates. Reporter gene enzyme activity (bars) is reported as relative fluorescence units (RFU) compared to the negative control. Productivity (reporter gene enzyme activity versus OD) is also shown. 600 The ratio (square). Error bars represent the standard deviation of the mean.

[0023] Figure 8 Reporter gene enzyme activity assays were performed on shake flask supernatants from cultures of strains M31676, M32338, M32696, and M32716. Methanol was used as the carbon source for strain M31676, and glucose was used for strains M32338, M32696, and M32716. Reporter gene enzyme activities (bars) are reported as relative fluorescence units (RFU) compared to the negative control. Error bars indicate the standard deviation of the mean.

[0024] Figure 9Reporter enzyme activity assays were performed on supernatant fractions from bioreactor fermentations of strains M31676, M32388, M32347, M32696, and M32716. Methanol was used as the carbon source for strain M31676, and ethanol was used for strains M32338, M32347, M32696, and M32716. Reporter enzyme activity units (bars) were calculated by comparison with a standard curve of a commercial lipase sample. Productivity (the ratio of reporter enzyme activity to stem cell weight, in diamond shapes) is also shown. Error bars represent the error in proliferation according to established uncertainty proliferation rules.

[0025] Figure 10 A schematic diagram of one of the engineering schemes for the constitutive stationary phase inducible 1 (SPI1) promoter is provided. A triangle symbolically represents the engineered CSRE and its distance on the nucleotide chain. spi1 Relative distances of the functional core promoter regions proximal to the gene (not depicted). In the engineered promoter variant denoted as eSPI1p-03, additional CSREs are introduced as shown in the diagram, gradually moving closer to the core promoter.

[0026] Figure 11 Reporter gene enzyme activity assays were performed on supernatant fractions of cultures from strains M34673, M32696, M33406, and M35140. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity (bars) is reported as relative fluorescence units (RFU) compared to the negative control. Productivity (reporter gene enzyme activity versus OD) is also shown. 600 The ratio (square). Error bars represent the standard deviation of the mean.

[0027] Figure 12 Reporter gene enzyme activity assays were performed on supernatant fractions of cultures from 96-well plates of strains M17500 (wild-type), M32685, and M33193. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity (bars) is reported as absorbance at 510 nm. Productivity (reporter gene enzyme activity versus OD) is also shown. 600 The ratio (black circles). The error bars represent the standard deviation of the mean.

[0028] Figure 13 Reporter gene enzyme activity assays were performed on supernatant fractions of cultures from 96-well plates of strains M17500 (wild-type), M33232, and M33328. Ethanol was used as the carbon source for all strains. Reporter gene enzyme activity (bars) is reported as absorbance at 400 nm. Productivity (reporter gene enzyme activity versus OD) is also shown. 600The ratio (black circles). The error bars represent the standard deviation of the mean. Detailed Implementation

[0029] This disclosure relates to promoters (e.g., C2 carbon source responsive promoters) for expressing genes (which may be natural or heterologous) in recombinant microbial host cells using a C2 carbon source as an inducer. In the context of this disclosure, an inducer is a chemical or biological entity that, upon contact with a recombinant microbial host cell, enhances the ability of an engineered promoter to promote the expression of downstream genes operatively linked to the engineered promoter. In some embodiments, more than one inducer may affect the ability of an engineered promoter to express downstream genes. In some embodiments, the promoter may be used in methanol-free expression systems, such as expression systems that do not use methanol as an inducer and can be used without adding methanol to the culture medium. This disclosure also provides the use of ethanol-responsive promoters and the linkage of such expression systems to aerobic fermentation processes, wherein ethanol or another C2 carbon source is the carbon source. In some embodiments, the use of ethanol-responsive promoter-based expression systems in aerobic fermentation has the added benefit of the presence of ethanol contributing to microbial contamination control.

[0030] Within the context of this disclosure, the term "C2 carbon source" refers to a carbon source that can be assimilated by a recombinant microbial host and contains two (2) carbon atoms. Embodiments of C2 carbon sources include, but are not limited to, ethanol, acetate, and combinations thereof.

[0031] engineered promoters

[0032] The engineered promoters of this disclosure exhibit increased expression intensity in the presence of a C2 carbon source (e.g., ethanol and / or acetate). This increased expression intensity is observed in the absence of methanol but in the presence of a C2 carbon source such as ethanol. As used herein, "increased expression intensity in the presence of a C2 carbon source such as ethanol" refers to increased expression of genes operatively linked to one or more engineered promoters in recombinant microbial host cells in the presence of a C2 carbon source. This increase in gene expression can be observed when compared to the expression intensity of the parental promoter in the presence of a C2 carbon source.

[0033] In some embodiments, the engineered promoters of this disclosure exhibit derepression in the presence of a non-C2 carbon source. In the context of this disclosure, a non-C2 carbon source refers to a carbon source that can be assimilated by the recombinant microbial host cell and contains more than two (2) carbon atoms. Embodiments of non-C2 carbon sources include, but are not limited to, glucose, fructose, glycerol, and combinations thereof. In such embodiments, the engineered promoter exhibits a higher level of derepression in the presence of a non-C2 carbon source (e.g., glucose, fructose, and / or glycerol) than the corresponding derepression level of the parental promoter.

[0034] In embodiments in which the gene encodes a polypeptide with enzymatic activity, the regulation of expression intensity associated with the engineered promoter can be reflected by an increase of at least 10% in the enzymatic activity of the polypeptide (when compared with the enzymatic activity of the same polypeptide under the control of the parental promoter).

[0035] In some embodiments, the engineered promoters of this disclosure exhibit increased expression intensity in the presence of glucose. This increased expression intensity is observed in the presence of glucose, in the absence of methanol. As used herein, "increased expression intensity in the presence of glucose" refers to an increase in the expression of a gene operatively linked to one or more engineered promoters in the recombinant microbial host cell in the presence of glucose. This increase in gene expression is observed when compared to the expression intensity of the parental promoter in the presence of glucose. As mentioned above, once glucose is consumed, the expression level of the engineered promoter further increases (due to derepression).

[0036] In some embodiments, the engineered promoters of this disclosure exhibit increased expression intensity in the presence of fructose. This increased expression intensity is observed in the presence of fructose in the absence of methanol. As used herein, "increased expression intensity in the presence of fructose" refers to an increase in the expression of a gene operatively linked to one or more engineered promoters in the recombinant microbial host cell in the presence of fructose. This increase in gene expression is observed when compared to the expression intensity of the parental promoter in the presence of fructose. As mentioned above, once fructose is consumed, the expression level of the engineered promoter increases further (due to derepression).

[0037] In some embodiments, the engineered promoters of this disclosure exhibit increased expression intensity in the presence of glycerol. This increased expression intensity is observed in the presence of glycerol in the absence of methanol. As used herein, “increased expression intensity in the presence of glycerol” refers to an increase in the expression of a gene operatively linked to one or more engineered promoters in the recombinant microbial host cell in the presence of glycerol. This increase in gene expression is observed when compared to the expression intensity of the parental promoter in the presence of glycerol. As mentioned above, the expression level of the engineered promoter can be further increased (given derepression) once glycerol is consumed. The engineered promoters of this disclosure contain at least one external carbon source responsive element (CSRE). Carbon source-dependent regulation of promoter activation (when present) is mediated by specific nucleotide motifs in the promoter sequence, in which transcription factors such as Adr1, Cat8 (also known as Cat8-1), Sip4 (also known as Cat8-2), or Mig1 bind. These motifs, referred to as “carbon source responsive elements” (CSREs), mediate the repression, derepression, or activation of the corresponding promoter downstream gene. Cat8 and Sip4 in yeast (including Saccharomyces cerevisiae) have been described as having conserved DNA-binding domains and participating in gene desuppression in the presence of non-fermentable carbon sources.

[0038] In the context of this disclosure, a carbon source responsive element (CSRE) refers to a nucleic acid motif that can be represented by formula (I): N1N2CCN3N4TN5N6N7CCGN8 (I) The common sequence of the external CSRE was obtained by comparing the nucleic acid sequences of the external CSREs used in the comparative examples. Table 1 provides the alignment of the external CSREs used in this example.

[0039] Table 1. The examples use an external CSRE and a common sequence derived therefrom for alignment. In the common sequence, N1 is any nucleic acid residue; N2 is any nucleic acid residue, preferably C or T; N3 is any nucleic acid residue, preferably A, G, or T; N4 is any nucleic acid residue, preferably C or T; N5 is any nucleic acid residue, preferably A, C, or G; N6 is any nucleic acid residue, preferably A or G; N7 is any nucleic acid residue, preferably G or T; and N8 is any nucleic acid residue, preferably A or G.

[0040]

[0041] In formula (I), N1 refers to any naturally occurring nucleic acid residue. In some embodiments, N1 is T. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 28, 31, or 33. In some embodiments, N1 is C. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 27, 30, or 34. In some embodiments, N1 is A. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 29 or 35. In some embodiments, N1 is G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 32.

[0042] In formula (I), N2 refers to any naturally occurring nucleic acid residue. In some embodiments, N2 refers to C or T. In some specific embodiments, N2 refers to C. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 28, 29, 31, 32, or 34. In some specific embodiments, N2 refers to T. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 27, 30, 33, or 35.

[0043] In formula (I), N3 refers to any naturally occurring nucleic acid residue. In some embodiments, N3 refers to A, G, or T. In some specific embodiments, N3 refers to A. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 30, 33, or 35. In some specific embodiments, N3 refers to G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, or 32. In some specific embodiments, N3 refers to T. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 31 or 34.

[0044] In formula (I), N4 refers to any naturally occurring nucleic acid residue. In some embodiments, N4 refers to C or T. In some specific embodiments, N4 refers to C. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 27. In some specific embodiments, N4 refers to T. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35.

[0045] In formula (I), N5 refers to any naturally occurring nucleic acid residue. In some embodiments, N5 refers to A, C, or G. In some specific embodiments, N5 refers to A. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 31, 32, or 33. In some specific embodiments, N5 refers to C. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 27, 29, 30, 34, or 35. In some specific embodiments, N5 refers to G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 28.

[0046] In formula (I), N6 refers to any naturally occurring nucleic acid residue. In some embodiments, N6 refers to A or G. In some specific embodiments, N6 refers to A. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 27, 29, 31, 33, or 35. In some specific embodiments, N6 refers to G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 28, 30, 32, or 34.

[0047] In formula (I), N7 refers to any naturally occurring nucleic acid residue. In some embodiments, N7 refers to G or T. In some specific embodiments, N7 refers to G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 27, 29, 30, 33, or 34. In some specific embodiments, N7 refers to T. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 28, 31, 32, or 35.

[0048] In formula (I), N8 refers to any naturally occurring nucleic acid residue. In some embodiments, N8 refers to A or G. In some specific embodiments, N8 refers to A. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32. In some specific embodiments, N8 refers to G. In such embodiments, the CSRE may have the nucleic acid sequence of SEQ ID NO: 26, 33, 34, or 35.

[0049] The engineered promoters disclosed herein contain an external CSRE. In the context of this disclosure, when used in conjunction with the term "CSRE," the term "external" means the fact that a CSRE has been added to the parent promoter to produce the engineered promoter. A natural CSRE that may be present in the parent promoter is not considered an external CSRE. A natural CSRE that has been modified or replaced is also not considered an external CSRE.

[0050] In one embodiment, the engineered promoter comprises a single external CSRE. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 26. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the single external CSRE may have the nucleic acid sequence of SEQ ID NO: 35. In some implementations, the engineered promoter containing a single external CSRE may have nucleic acid sequences of SEQ ID NO: 6, 11, 12, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24.

[0051] In one embodiment, the engineered promoter comprises at least two external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises two external carbon source responsive elements (CSREs). In this embodiment, the two external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In embodiments where the CSREs have the same nucleic acid sequence, the two external CSREs may have nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In embodiments where the CSREs have different nucleic acid sequences, the two external CSREs may be any two of CSREs having nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 26 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In one implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 27 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 28 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 29 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In one implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 30 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 31 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another implementation, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 32 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35.In some embodiments, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 33 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In other embodiments, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 34 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the two external CSREs may comprise one CSRE having the nucleic acid sequence of SEQ ID NO: 35 and another CSRE having the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In some embodiments, the engineered promoter comprising the two external CSREs may have the nucleic acid sequence of SEQ ID NO: 7.

[0052] In one embodiment, the engineered promoter comprises at least three external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises three external carbon source responsive elements (CSREs). In this embodiment, the three external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the three external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, two of the three external CSREs may have the same nucleic acid sequence, and the third external CSRE may have a different nucleic acid sequence. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the third external CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the third outer CSRE may have the nucleic acid sequence of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the three outer CSREs may have different nucleic acid sequences. In embodiments where the CSREs have different nucleic acid sequences, the three external CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In one specific embodiment, the three external CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, or 28. In yet another embodiment, the three external CSREs may have different nucleic acid sequences, with one CSRE having the nucleic acid sequence of SEQ ID NO: 26, another CSRE having the nucleic acid sequence of SEQ ID NO: 27, and another CSRE having the nucleic acid sequence of SEQ ID NO: 28. In some embodiments, the engineered promoter comprising the three external CSREs may have the nucleic acid sequence of SEQ ID NO: 8 or 13.

[0053] In one embodiment, the engineered promoter comprises at least four external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises four external carbon source responsive elements (CSREs). In this embodiment, the four external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the four external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the four external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the four outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs may be independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the four outer CSREs may have different nucleic acid sequences. In another embodiment, the four outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the four outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, or 29. In yet another embodiment, the four outer CSREs may have different nucleic acid sequences, one CSRE having the nucleic acid sequence of SEQ ID NO: 26, another CSRE having the nucleic acid sequence of SEQ ID NO: 27, another CSRE having the nucleic acid sequence of SEQ ID NO: 28, and yet another CSRE having the nucleic acid sequence of SEQ ID NO: 29. In some implementations, the engineered promoter containing four external CSREs may have the nucleic acid sequence of SEQ ID NO: 14.

[0054] In one embodiment, the engineered promoter comprises at least five external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises five external carbon source responsive elements (CSREs). In this embodiment, the five external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the five external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the five external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the five outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the five outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35.In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the five outer CSREs may have different nucleic acid sequences. In another embodiment, the five outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the five outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, or 30. In yet another embodiment, the five external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the engineered promoter comprising the five external CSREs may have the nucleic acid sequence of SEQ ID NO: 9 or 15.

[0055] In one embodiment, the engineered promoter comprises at least six external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises six external carbon source responsive elements (CSREs). In this embodiment, the six external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the six external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the six external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the six outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the six outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35.In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least five of the six outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27 and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34 or 35.

[0056] In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the six outer CSREs may have different nucleic acid sequences. In another embodiment, the six outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the six outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, or 31.In yet another embodiment, the six external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the engineered promoter comprising the six external CSREs may have the nucleic acid sequence of SEQ ID NO: 16.

[0057] In one embodiment, the engineered promoter comprises at least seven external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises seven external carbon source responsive elements (CSREs). In this embodiment, the seven external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the seven external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the seven external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the seven outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the seven outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35.In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least five of the seven outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35.In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least six of the seven outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35.In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs may be independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In another embodiment, the seven outer CSREs may have different nucleic acid sequences. In yet another embodiment, the seven outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the seven outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, or 32.In yet another embodiment, the seven external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 31, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 32.

[0058] In one embodiment, the engineered promoter comprises at least eight external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises eight external carbon source responsive elements (CSREs). In this embodiment, the eight external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In embodiments where the CSREs have the same nucleic acid sequence, the eight external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the eight external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30 and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34 or 35.

[0059] In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the eight outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35.In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the eight outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35.In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least five of the eight outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35.In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least six of the eight outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35.In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least seven of the eight outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences.For example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35.In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs may be independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the eight outer CSREs may have different nucleic acid sequences. In an embodiment, the eight outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the eight outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, or 33. In yet another embodiment, the eight external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 31, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 32, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 33.

[0060] In one embodiment, the engineered promoter comprises at least nine external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises nine external carbon source responsive elements (CSREs). In this embodiment, the nine external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In an embodiment where the CSREs have the same nucleic acid sequence, the nine external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another embodiment, at least two of the nine external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35.In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least five of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35.In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least six of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35.In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least seven of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35.In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least eight of the nine outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35.In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the nine outer CSREs may have different nucleic acid sequences.In one implementation, the nine external CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another specific implementation, the nine external CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In yet another embodiment, the nine external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 31, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 32, another CSRE has the nucleic acid sequence of SEQ ID NO: 33, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 34.

[0061] In one embodiment, the engineered promoter comprises at least ten external carbon source responsive elements (CSREs). In another embodiment, the engineered promoter comprises ten external carbon source responsive elements (CSREs). In yet another embodiment, the engineered promoter comprises more than ten external carbon source responsive elements (CSREs). In each embodiment, the ten external CSREs may have the same nucleic acid sequence or different nucleic acid sequences. In embodiments where the CSREs have the same nucleic acid sequence, the ten external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In each embodiment, at least two of the ten external CSREs may have the same nucleic acid sequence, and the remaining external CSREs may have different nucleic acid sequences. For example, two of the external CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining external CSREs may be independently selected from any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35.In one example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, two of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least three of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35.In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, three of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least four of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35.In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, four of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least five of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35.In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, five of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least six of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35.In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In one example, six of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least seven of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35.In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from any one of the nucleic acid sequences of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In one example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In another example, seven of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least eight of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences. For example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35.In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In one example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35. In yet another example, eight of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, at least nine of the ten outer CSREs may have the same nucleic acid sequence, and the remaining outer CSREs may have different nucleic acid sequences.For example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 26, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 27, 28, 29, 30, 31, 32, 33, 34, or 35. In another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 27, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 28, and the remaining outer CSREs may be independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 29, 30, 31, 32, 33, 34, or 35. In yet another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 29, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 30, 31, 32, 33, 34, or 35. In one example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 30, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 31, 32, 33, 34, or 35. In another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 31, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 32, 33, 34, or 35. In another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 32, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 33, 34, or 35. In another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 33, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 34, or 35. In yet another example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 34, and the remaining outer CSREs are independently selected from the nucleic acid sequences of any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 35.In one example, nine of the outer CSREs may have the nucleic acid sequence of SEQ ID NO: 35, and the remaining outer CSREs may be independently selected from any one of SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, or 34. In an embodiment, the ten outer CSREs may have different nucleic acid sequences. In an embodiment, the ten outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In a specific embodiment, the ten outer CSREs may have nucleic acid sequences independently selected from SEQ ID NO: 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In yet another embodiment, the ten external CSREs may have different nucleic acid sequences: one CSRE has the nucleic acid sequence of SEQ ID NO: 26, another CSRE has the nucleic acid sequence of SEQ ID NO: 27, another CSRE has the nucleic acid sequence of SEQ ID NO: 28, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 29, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 30, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 31, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 32, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 33, yet another CSRE has the nucleic acid sequence of SEQ ID NO: 34, and yet another CSRE has the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the engineered promoter comprising the ten external CSREs may have the nucleic acid sequence of SEQ ID NO: 10.

[0062] The engineered promoters disclosed herein are derived from parental promoters. In embodiments, the parental promoter is not inhibited under aerobic conditions and / or in the presence of a C2 carbon source such as ethanol. The parental promoter can be a promoter naturally present in any living organism or can be a synthetic promoter. The parental promoter may already contain one or more carbon source responsive elements (CSREs) as described herein. The parental promoter may lack any CSRE. In some specific embodiments, the parental promoter is an inducible promoter, for example, when a recombinant yeast host cell is contacted with an inducer or a combination of inducers, the expression of the gene operably linked thereto increases. In some specific embodiments, the parental promoter is a C2 carbon source inducible promoter (such as an ethanol-inducible promoter), for example, when a recombinant yeast host cell is contacted with an inducer (a C2 carbon source, such as ethanol), the expression of the gene operably linked thereto increases. In some embodiments, the parental promoter is not an ethanol-inducible promoter, for example, when a recombinant yeast host cell is contacted with an inducer, the expression of the gene operably linked thereto does not increase (e.g., remains substantially the same or decreases). In some embodiments, the parental promoter is a glucose-inducible promoter, for example, when the recombinant yeast host cell is contacted with glucose (e.g., an inducer), the expression of the gene operatively linked to it increases. In some embodiments, the parental promoter is a fructose-inducible promoter, for example, when the recombinant yeast host cell is contacted with fructose (e.g., an inducer), the expression of the gene operatively linked to it increases. In some embodiments, the parental promoter is a glycerol-inducible promoter, for example, when the recombinant yeast host cell is contacted with glycerol (e.g., an inducer), the expression of the gene operatively linked to it increases. In some embodiments, the parental promoter is a constitutive promoter, for example, a promoter whose expression intensity remains substantially the same regardless of the presence of an inducer (e.g., a C2 carbon source, such as ethanol) or a non-C2 carbon source (e.g., glucose, fructose, or glycerol, for example).

[0063] In embodiments where the recombinant microbial host cell is derived from *Saccharomyces cerevisiae*, the parental promoter can be an inducible promoter or a constitutive promoter. In such embodiments, the parental promoter can be obtained from or derived from natural promoters present in *Saccharomyces cerevisiae*. Inducible promoters include, but are not limited to, glucose-regulated promoters (e.g., hxt7 The promoter of the gene (called hxt7p); ctt1 The promoter of a gene (called ctt1p); glo1 The promoter of a gene (called glo1p); ygp1 The promoter of the gene (called ygp1p); gsy2 The promoter of a gene (called gsy2p); gpm1The promoter of the gene (called gpm1p), the promoter of the pgk1 gene (called pgk1p)), molasses-regulated promoters (e.g., mol1 The promoter of a gene (called mol1p Heat shock regulated promoters (e.g., glo1 The promoter of a gene (called glo1p); sti1 The promoter of a gene (called sti1p); ygp1 The promoter of the gene (called ygp1p); gsy2 Gene promoters (called gsy2p), oxidative stress-responsive promoters (e.g., cup1 The promoter of the gene (called cup1p); the promoter of the ctt1 gene (called ctt1p); trx2 The promoter of a gene (called trx2p); gpd1 The promoter of a gene (called gpd1p); hsp12 The promoter of the gene (called hsp12p); the promoter of the hsp150 gene (called hsp150p). ssc1 Gene promoters (called ssc1p), osmotic stress-responsive promoters (e.g., ctt1 The promoter of a gene (called ctt1p); glo1 The promoter of a gene (called glo1p); gpd1 The promoter of a gene (called gpd1p); ygp1 The promoter of the gene (called ygp1p); hor7 The promoter of a gene (called hor7p); stl1 Gene promoters (called stl1p), nitrogen-regulated promoters (e.g., ygp1 The promoter of the gene (called ygp1p); adh1 Gene promoters (called adh1p); anaerobic regulatory promoters (e.g., tir1 The promoter of a gene (called tir1p) pau5 The promoter of a gene (called pau5p) dan1 The promoter of a gene (called dan1p) tdh1 The promoter of a gene (called tdh1p) spi1 The promoter of a gene (called spi1p) hxk1 The promoter of the gene (called hxk1p) anb1 The promoter of a gene (called anb1p) hxt6 The promoter of the gene (called hxt6p) trx1 The promoter of a gene (called trx1p) aac3 The promoter of a gene (called aac3p) hor7The promoter of a gene (called hor7p) adh1 The promoter of a gene (called adh1p) tdh2 The promoter of a gene (called tdh2p) tdh3 The promoter of a gene (called tdh3p) gdp1 The promoter of a gene (called gpd1p) cdc19 The promoter of the gene (called cdc19p) eno2 The promoter of a gene (called eno2p) pdc1 The promoter of a gene (called pdc1p) hxt3 The promoter of the gene (called hxt3p) or tpi1 The promoter of a gene (called tpi1p Ethanol-regulated promoters (including ethanol-responsive promoters); redox-regulated promoters (including but not limited to) gpd2 Gene promoters (called gpd2p); sulfite-regulated promoters (including but not limited to) fzf1 The promoter of the gene (called fzf1p) ssu1 The promoter of the gene (called ssu1p) and ssu1-r Gene promoters (called ssu1-rp); and stress-responsive promoters (including but not limited to) yap1 The promoter of a gene (called yap1p) ssa3 The promoter of the gene (called ssa3p) and hsp104 Gene promoters (called hsp104p). Constitutive promoters include, but are not limited to, those that... tef2 The promoter of a gene (called tef2p) cwp2 The promoter of a gene (called cwp2p) ssa1 The promoter of a gene (called ssa1p) eno1 The promoter of a gene (called eno1p) hxk1 The promoter of the gene (called hxk1p) pgk1 The promoter of a gene (called pgk1p) adh1 The promoter of a gene (called adh1p) rev1 The promoter of a gene (called rev1p ), cyc1 The promoter of the gene (called cyc1p) and ste5 The promoter of a gene (called ste5p).

[0064] The recombinant microbial host cell in this context is a methyltrophic yeast (such as *Phaeodactylum fafesumi* or *Hansenula polymorpha*). Ogataea polymorphaIn the embodiments of the present invention, the parental promoter may be an inducible promoter or a constitutive promoter. In such embodiments, the parental promoter may be obtained or derived from natural promoters present in *Phaemophyton flavus*. Inducible promoters include, but are not limited to, glucose-regulated promoters, fructose-regulated promoters, glycerol-regulated promoters, heat shock-regulated promoters, oxidative stress-responsive promoters, osmotic stress-responsive promoters, nitrogen-regulated promoters, and ethanol-regulated promoters. In one embodiment, ethanol-regulated promoters include, but are not limited to, promoters derived from... adh2 The promoter of a gene, also known as adh3 Gene (called adh2p). Constitutive promoters include, but are not limited to, those from... spi1 The gene promoter (called spi1p). In one implementation, the parental promoter is derived from... gap1 The gene promoter (called gap1p). In one implementation, the parental promoter is derived from... hgt1 The gene promoter (called hgt1p). In one implementation, the parental promoter is derived from... glc3 The gene promoter (called glc3p). In one implementation, the parental promoter is derived from... acb2 The gene promoter (called acb2p). In one implementation, the parental promoter is derived from... pex8 The gene promoter (called pex8p). In one implementation, the parental promoter is derived from... urc1 The gene promoter (called urc1p). In one implementation, the parental promoter is derived from... tpo3 The gene promoter (called top3p). In one implementation, the parental promoter is derived from... bio2 The gene promoter (called bio2p). In one implementation, the parental promoter is derived from... gut1 The gene promoter (called gut1p). In one implementation, the parental promoter is derived from... cat1 The gene promoter (called cat1p). In one implementation, the parental promoter is derived from... icl1 The gene promoter (called icl1p). In one implementation, the parental promoter is derived from... gcw14 The gene promoter (called gcw14p). In one implementation, the parental promoter is derived from... sor1 The promoter of the gene (called sor1p) comes from the methanol oxidase of Hansenula polymorpha. mox1 The gene promoter (called mox1p) comes from gap1 The polymorphic Hansenula polymorpha promoter (called OpGAP1p) is derived from... gapdh The polymorphic Hansenula polymorpha promoter (called OpGAPDHp) is derived from... gcw14 The polymorphic Hansenula yeast promoter (called OpGCW14p) is derived from adh1 The polymorphic Hansenula polymorpha promoter (called OpADH1p) is derived from... icl1 The polymorphic Hansenula polymorpha promoter (called OpICL1p) or derived from tef1 The polymorphic Hansenula yeast promoter (called OpTEF1p).

[0065] The engineered promoters disclosed herein are designed to be operatively linked (or associated) with a gene to increase / drive its expression in the presence of an inducer (C2 carbon source, such as ethanol) and / or de-inhibited by a non-C2 carbon source such as glucose, fructose, or glycerol. In the heterologous nucleic acid molecules described herein, the promoter and the nucleic acid molecule containing the gene are operatively linked to each other. In the context of this disclosure, the expression “operatively linked” or “operatively associated” means the fact that the engineered promoter is physically associated with the gene (e.g., in cis orientation) in a manner that allows or increases gene expression in the presence of a C2 carbon source such as ethanol. The engineered promoter is typically located upstream of the gene (5'). Therefore, the gene operatively linked to the promoter is typically located downstream of the engineered promoter (3'). In the context of this disclosure, one or more engineered promoters may be used to express the operatively linked gene. When more than one promoter is included, each promoter is operatively linked to the gene. In the context of this disclosure, one or more genes (e.g., operons) may be operatively linked to the engineered promoter.

[0066] Genes operatively linked to engineered promoters may encode polypeptides (in some embodiments, the polypeptide may be an enzyme) or RNA molecules (e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), guide RNA (gRNA), small nuclear RNA (snRNA), or ribozymes).

[0067] The engineered promoter disclosed herein has an upstream boundary and a downstream boundary. The upstream boundary is located in the 5' direction of the engineered promoter. The engineered promoter extends upstream to include elements necessary to initiate / drive transcription in the presence of an inducer (a C2 carbon source, such as ethanol) or a non-C2 carbon source (e.g., glucose, fructose, or glycerol). The downstream boundary is located in the 3' direction of the promoter and is designed to be operatively linked to the upstream boundary (located in the 5' direction) of the gene intended for expression. The promoter typically includes a core promoter, which is defined as the minimal region required to guide transcription initiation. Within the core promoter, a polypeptide-binding domain (consensus sequence) responsible for RNA polymerase binding, a transcription start site (TSS), and a 5' untranslated region (5' UTR, which may also be referred to as a leader sequence) will be found. In some embodiments of eukaryotic and archaea promoters, the core promoter includes a TATA box (which may have been previously verified or presumed) that defines the binding site for TATA-binding proteins and ultimately the binding site for RNA polymerase. As is known in the art, the TATA box can be located by determining the presence of the TATA consensus sequence in the core promoter. In yeast, the TATA concordant sequence (derived from the Saccharomyces cerevisiae concordant sequence) has a nucleic acid sequence of formula (II): TATAN a AN b N c (II) Where N a Is it A or T? N b Is it A or T? N c Is it A or G? When a gene is expressed under the control of an engineered promoter and encodes a polypeptide, the gene includes an open reading frame (ORF) and a start site (e.g., a start codon). In *Phaeodactylum fabri*... adh2 In the promoter of the gene, the 5' nucleotide of the TATA box is located 82 base pairs upstream of the ORF start site (-82). In *Phaeodactylum phagnum*... spi1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 93 base pairs upstream of the ORF start site (-93), and the TSS is located 45 base pairs upstream of the ORF start site (-45). In *Phaeodactylum phagnum*... gap1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 69 base pairs upstream of the ORF start site (-69). In *Phaeodactylum phragmus*... hgt1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 64 base pairs upstream of the ORF start site (-64). In *Phaeodactylum phagnum*... glc3In the promoter of the gene, the 5' nucleotide of the TATA box is located 57 base pairs upstream of the ORF start site (-57). In *Phaeodactylum phragmus*... acb2 In the promoter of the gene, the 5' nucleotide of the TATA box is located 60 base pairs upstream of the ORF start site (-60). In *Phaeodactylum phagnum*... pex8 In the promoter of the gene, the 5' nucleotide of the TATA box is located 73 base pairs upstream of the ORF start site (-73). In *Phaeodactylum phragmus*... urc1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 90 base pairs upstream of the ORF start site (-90). In *Phaeodactylum phragmus*... tpo3 In the promoter of the gene, the 5' nucleotide of the TATA box is located 70 base pairs upstream of the ORF start site (-70). In *Phaeodactylum phragmites*... bio2 In the promoter of the gene, the 5' nucleotide of the TATA box is located 68 base pairs upstream of the ORF start site (-68). In *Phaeodactylum phagnum*... gut1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 60 base pairs upstream of the ORF start site (-60). In *Phaeodactylum phagnum*... cat1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 78 base pairs upstream of the ORF start site (-78). In *Phaeodactylum phragmus*... icl1 In the promoter of the gene, the 5' nucleotide of the TATA box is located 96 base pairs upstream of the ORF start site (-96). In *Phaeodactylum phragmus*... gcw14 In gene promoters, the 5' nucleotide of the TATA box is located 93 base pairs upstream of the ORF start site (-93). In bacterial promoter implementations, the core promoter includes the Pribnow box, which defines the region where RNA polymerase initially binds.

[0068] As indicated above, promoters (engineered promoters and parental promoters) have a transcription start site (TSS) near their 3' boundary. As is known in the art, the promoter's TSS can be determined by mapping with the nuclease S1. The promoter also includes a 5' UTR located downstream of the TSS. While the 5' UTR is transcribed into the coding strand of mRNA, it is generally not transcribed into a polypeptide. In such embodiments, the 5' UTR is located between the TSS and the gene to be expressed.

[0069] In the context of this disclosure, the engineered promoter contains a first external CSRE, and this first external CSRE is located upstream (5' direction) of the transcription start site. Therefore, the first external CSRE is not located within the 5' UTR region of the parental promoter. In some embodiments, none of the external CSREs that may be present in the engineered promoter are located within the core region of the engineered promoter. In some embodiments, only one of the external CSREs is located within the core region of the engineered promoter. In other embodiments, all external CSREs are located outside and upstream (5' direction) of the core promoter of the engineered promoter.

[0070] Within the context of this disclosure, the first external CSRE is located proximal to the transcription start site of the parental promoter. As illustrated herein, the position of the first external CSRE relative to the transcription start site influences the performance of the resulting engineered promoter in promoting the transcription of the gene operably linked to it. Within the context of this disclosure, the term "proximal" means that the position of the first external CSRE is sufficiently close to the transcription start site (and by extending into the TATA box or start codon) to increase the transcription of the operably linked gene and / or the stability of the transcribed RNA molecule in the presence of an inducing agent (C2 carbon source, such as ethanol) or a non-C2 carbon source (e.g., glucose, glycerol, or fructose). In some embodiments, this increase in transcription can be determined by measuring the transcriptional level of the gene operably linked to the engineered promoter. Furthermore, this increase in transcription can be determined by measuring the amount / activity of the polypeptide encoded by the gene operably linked to the engineered promoter. In the case where the gene encodes an enzyme, this increase in transcription can be determined by measuring the enzymatic activity of the polypeptide encoded by the gene operably linked to the engineered promoter.

[0071] In the context of this disclosure, the insertion position of at least one external CSRE will be defined by providing its upstream position relative to the transcription start site, TATA box, or start codon, or by referring to a specific region upstream of the transcription start site, TATA box, or start codon. For example, the statement "at least one external CSRE is located at at most XX base pairs upstream of the transcription start site / TATA box / start codon" indicates that the insertion position of at least one CSRE is located between the position immediately upstream of the transcription start site / TATA box / start codon and the XXth base pair upstream of the transcription start site / TATA box / start codon. In another example, the statement "at least one external CSRE is located between approximately position AA upstream of the transcription start site / TATA box / start codon and approximately position BB upstream of the transcription start site / TATA box / start codon" indicates that the insertion position of at least one CSRE is located between the AAth base pair upstream of the transcription start site / TATA box / start codon and the BBth position relative to the transcription start site / TATA box / start codon.

[0072] In some embodiments, the engineered promoter of this disclosure includes a first external CSRE located at up to 397 base pairs (e.g., -397) upstream of the transcription start site. In some cases, the engineered promoter may include one or more external CSREs that may be located more than 397 base pairs (e.g., -397) upstream of the transcription start site (provided that it includes at least one external CSRE located at up to 397 base pairs upstream of the transcription start site). In some further embodiments, the engineered promoter includes a first external CSRE located at up to 350 base pairs (e.g., -350) upstream of the transcription start site. In some cases, the engineered promoter may include one or more external CSREs that may be located more than 350 base pairs (e.g., -350) upstream of the transcription start site (provided that it includes at least one external CSRE located at up to 350 base pairs upstream of the transcription start site).

[0073] In some embodiments, the first external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the first external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some further embodiments, the first external CSRE is located between 46 base pairs (e.g., -46) upstream of the transcription start site and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 37 base pairs (e.g., -37) upstream of the transcription start site and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 55 base pairs (e.g., -55) and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 94 base pairs (e.g., -94) and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 77 base pairs (e.g., -77) and approximately 156 base pairs (e.g., -156) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site.In some specific embodiments, the first external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 143 base pairs (e.g., -143) and 144 base pairs (e.g., -144) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific implementations, the first external CSRE is located between approximately 111 base pairs (e.g., -111) upstream of the transcription start site and approximately 190 base pairs (e.g., -190) upstream of the transcription start site.In some other embodiments, the first external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 189 base pairs (e.g., -189) and approximately 268 base pairs (e.g., -268) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 237 base pairs (e.g., -237) upstream of the transcription start site and 238 base pairs (e.g., -238) upstream of the transcription start site.In some specific embodiments, the first external CSRE is located between approximately 239 base pairs (e.g., -239) upstream of the transcription start site and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 278 base pairs (e.g., -278) upstream of the transcription start site and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the first external CSRE is located between approximately 318 base pairs (e.g., -318) upstream of the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the first external CSRE is located between 357 base pairs (e.g., -357) upstream of the transcription start site and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0074] In some embodiments, the engineered promoter of this disclosure comprises at least two external CSREs. This document provides embodiments of the location and nucleic acid sequence of the first external CSRE, which can be used in engineered promoters comprising two or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are contiguous, or they may be provided at different positions and are discontinuous. In some embodiments, the second external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the second external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 55 base pairs (e.g., -55) and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 94 base pairs (e.g., -94) and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 77 base pairs (e.g., -77) and approximately 156 base pairs (e.g., -156) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site.In some specific embodiments, the second external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 93 base pairs (e.g., -93) upstream of the transcription start site and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 132 base pairs (e.g., -132) upstream of the transcription start site and approximately 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 94 base pairs (e.g., -94) upstream of the transcription start site and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 133 base pairs (e.g., -133) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 143 base pairs (e.g., -143) and 144 base pairs (e.g., -144) upstream of the transcription start site. In some specific implementations, the second external CSRE is located between approximately 109 base pairs (e.g., -109) upstream of the transcription start site and approximately 188 base pairs (e.g., -188) upstream of the transcription start site.In some other embodiments, the second external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 189 base pairs (e.g., -189) and approximately 268 base pairs (e.g., -268) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 228 base pairs (e.g., -228) upstream of the transcription start site and 229 base pairs (e.g., -229) upstream of the transcription start site.In some specific embodiments, the second external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the second external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the second external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site. In yet another specific embodiment, an engineered promoter having at least two external CSREs comprises a first external CSRE located between 129 base pairs (e.g., -129) and 208 base pairs (e.g., -208) upstream of the transcription start site; and a second external CSRE located between 198 base pairs (e.g., -198) and 277 base pairs (e.g., -277) upstream of the transcription start site. For example, an engineered promoter with at least two external CSREs includes a first external CSRE located between 168 base pairs (e.g., -168) upstream of the transcription start site and 169 base pairs (e.g., -169) upstream of the transcription start site; and a second external CSRE located between 237 base pairs (e.g., -237) upstream of the transcription start site and 238 base pairs (e.g., -238) upstream of the transcription start site.

[0075] In some embodiments, the engineered promoter of this disclosure comprises at least three external CSREs. Embodiments of the positions and nucleic acid sequences of the first and second external CSREs are provided herein, and these can be used in engineered promoters comprising three or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or they may be provided at different positions and are discontinuous. In some embodiments, the third external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the third external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 116 base pairs (e.g., -116) upstream of the transcription start site and 117 base pairs (e.g., -117) upstream of the transcription start site.In some specific embodiments, the third external CSRE is located between approximately 81 base pairs (e.g., -81) upstream of the transcription start site and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 120 base pairs (e.g., -120) upstream of the transcription start site and approximately 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 87 base pairs (e.g., -87) upstream of the transcription start site and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 126 base pairs (e.g., -126) upstream of the transcription start site and approximately 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 93 base pairs (e.g., -93) upstream of the transcription start site and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 132 base pairs (e.g., -132) upstream of the transcription start site and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 94 base pairs (e.g., -94) upstream of the transcription start site and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 133 base pairs (e.g., -133) upstream of the transcription start site and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 143 base pairs (e.g., -143) and 144 base pairs (e.g., -144) upstream of the transcription start site. In some specific implementations, the third external CSRE is located between approximately 109 base pairs (e.g., -109) upstream of the transcription start site and approximately 188 base pairs (e.g., -188) upstream of the transcription start site.In some other embodiments, the third external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 189 base pairs (e.g., -189) and approximately 268 base pairs (e.g., -268) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 228 base pairs (e.g., -228) upstream of the transcription start site and 229 base pairs (e.g., -229) upstream of the transcription start site.In some specific embodiments, the third external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the third external CSRE is located between approximately 318 base pairs (e.g., -318) upstream of the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the third external CSRE is located between 357 base pairs (e.g., -357) upstream of the transcription start site and 358 base pairs (e.g., -358) upstream of the transcription start site. In yet another specific embodiment, an engineered promoter having at least three external CSREs comprises a first external CSRE located between 129 base pairs (e.g., -129) and 208 base pairs (e.g., -208) upstream of the transcription start site; a second external CSRE located between 198 base pairs (e.g., -198) and 277 base pairs (e.g., -277) upstream of the transcription start site; and a third external CSRE located between 318 base pairs (e.g., -318) and 397 base pairs (e.g., -397) upstream of the transcription start site. For example, an engineered promoter with at least three external CSREs includes a first external CSRE located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site; a second external CSRE located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site; and a third external CSRE located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.In yet another specific embodiment, an engineered promoter having at least three external CSREs comprises a first external CSRE located between 55 base pairs (e.g., -55) upstream of the transcription start site and 134 base pairs (e.g., -134) upstream of the transcription start site; a second external CSRE located between 81 base pairs (e.g., -81) upstream of the transcription start site and 160 base pairs (e.g., -160) upstream of the transcription start site; and a third external CSRE located between 87 base pairs (e.g., -87) upstream of the transcription start site and 166 base pairs (e.g., -166) upstream of the transcription start site. For example, an engineered promoter with at least three external CSREs includes a first external CSRE located between 94 base pairs (e.g., -94) and 95 base pairs (e.g., -95) upstream of the transcription start site; a second external CSRE located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site; and a third external CSRE located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site.

[0076] In some embodiments, the engineered promoter of this disclosure comprises at least four external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, and third external CSREs are provided herein, and these can be used in engineered promoters comprising four or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are contiguous, or they may be provided at different positions and are discontinuous. In some embodiments, the fourth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the fourth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the fourth external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the fourth external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the fourth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the fourth external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the fourth external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the fourth external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0077] In some embodiments, the engineered promoter of this disclosure comprises at least five external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, and fourth external CSREs are provided herein, and these can be used in engineered promoters comprising five or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or they may be provided at different positions and are discontinuous. In some embodiments, the fifth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the fifth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the fifth external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the fifth external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the fifth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the fifth external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the fifth external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the fifth external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site. In yet another specific embodiment, the engineered promoter having at least five external CSREs comprises a first external CSRE located between 93 base pairs (e.g., -93) and 172 base pairs (e.g., -172) upstream of the transcription start site; a second external CSRE located between 111 base pairs (e.g., -111) and 190 base pairs (e.g., -190) upstream of the transcription start site; and a third external CSRE located 129 base pairs upstream of the transcription start site. The third external CSRE is located between the transcription start site (e.g., -129) and 208 base pairs (e.g., -208) upstream of the transcription start site; the fourth external CSRE is located between 198 base pairs (e.g., -198) upstream of the transcription start site and 277 base pairs (e.g., -277) upstream of the transcription start site; and the fifth external CSRE is located between 318 base pairs (e.g., -318) upstream of the transcription start site and 397 base pairs (e.g., -397) upstream of the transcription start site.For example, an engineered promoter with at least five external CSREs includes a first external CSRE located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site; a second external CSRE located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site; a third external CSRE located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site; a fourth external CSRE located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site; and a fifth external CSRE located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0078] In some embodiments, the engineered promoter of this disclosure comprises at least six external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, fourth, and fifth external CSREs are provided herein, and these can be used in engineered promoters comprising six or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or they may be provided at different positions and are discontinuous. In some embodiments, the sixth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the sixth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the sixth external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the sixth external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the sixth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the sixth external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the sixth external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the sixth external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0079] In some embodiments, the engineered promoter of this disclosure comprises at least seven external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, fourth, fifth, and sixth external CSREs are provided herein, and these can be used in engineered promoters comprising seven or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or they may be provided at different positions and are discontinuous. In some embodiments, the seventh external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the seventh external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the seventh external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the seventh external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the seventh external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the seventh external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the seventh external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the seventh external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0080] In some embodiments, the engineered promoter of this disclosure comprises at least eight external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, fourth, fifth, sixth, and seventh external CSREs are provided herein and can be used in engineered promoters comprising eight or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or they may be provided at different positions and are discontinuous. In some embodiments, the eighth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the eighth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 55 base pairs (e.g., -55) upstream of the transcription start site and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 94 base pairs (e.g., -94) upstream of the transcription start site and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the transcription start site and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the eighth outer CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the eighth outer CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the eighth outer CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the eighth outer CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the eighth external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the eighth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the eighth external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the eighth external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the eighth external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0081] In some embodiments, the engineered promoter of this disclosure comprises at least nine external CSREs. This document provides embodiments of the positions and nucleic acid sequences of the first, second, third, fourth, fifth, sixth, seventh, and eighth external CSREs, which can be used in engineered promoters comprising nine or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and be continuous, or they may be provided at different positions and be discontinuous. In some embodiments, the ninth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the ninth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 7 base pairs (e.g., -7) and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 55 base pairs (e.g., -55) and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 94 base pairs (e.g., -94) and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 77 base pairs (e.g., -77) and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the ninth external CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the ninth external CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the ninth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the ninth external CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the ninth external CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the ninth external CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0082] In some embodiments, the engineered promoter of this disclosure comprises at least ten external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, fourth, fifth, sixth, seventh, eighth, and ninth external CSREs are provided herein, and can be used in engineered promoters comprising ten or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are continuous, or may be provided at different positions and are discontinuous. In some embodiments, the tenth external CSRE is located between the transcription start site and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some alternative embodiments, the tenth external CSRE is located between approximately 7 base pairs (e.g., -7) upstream of the transcription start site and approximately 350 base pairs (e.g., -397) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 7 base pairs (e.g., -7) and approximately 86 base pairs (e.g., -86) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 46 base pairs (e.g., -46) and 47 base pairs (e.g., -47) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 37 base pairs (e.g., -37) and approximately 116 base pairs (e.g., -116) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 76 base pairs (e.g., -76) and 77 base pairs (e.g., -77) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 47 base pairs (e.g., -47) and approximately 126 base pairs (e.g., -126) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 86 base pairs (e.g., -86) and 87 base pairs (e.g., -87) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 55 base pairs (e.g., -55) and approximately 134 base pairs (e.g., -134) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 94 base pairs (e.g., -94) and 95 base pairs (e.g., -95) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 77 base pairs (e.g., -77) and approximately 156 base pairs (e.g., -156) upstream of the transcription start site.In some other embodiments, the tenth outer CSRE is located between 116 base pairs (e.g., -116) and 117 base pairs (e.g., -117) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 81 base pairs (e.g., -81) and approximately 160 base pairs (e.g., -160) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 120 base pairs (e.g., -120) and 121 base pairs (e.g., -121) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 87 base pairs (e.g., -87) and approximately 166 base pairs (e.g., -166) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 126 base pairs (e.g., -126) and 127 base pairs (e.g., -127) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 93 base pairs (e.g., -93) and approximately 172 base pairs (e.g., -172) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 132 base pairs (e.g., -132) and 133 base pairs (e.g., -133) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 94 base pairs (e.g., -94) and approximately 173 base pairs (e.g., -173) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 99 base pairs (e.g., -99) and approximately 178 base pairs (e.g., -178) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 104 base pairs (e.g., -104) and approximately 183 base pairs (e.g., -183) upstream of the transcription start site. In some other embodiments, the tenth external CSRE is located between 143 base pairs (e.g., -143) upstream of the transcription start site and 144 base pairs (e.g., -144) upstream of the transcription start site.In some specific embodiments, the tenth outer CSRE is located between approximately 109 base pairs (e.g., -109) and approximately 188 base pairs (e.g., -188) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 111 base pairs (e.g., -111) and approximately 190 base pairs (e.g., -190) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 150 base pairs (e.g., -150) and 151 base pairs (e.g., -151) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 114 base pairs (e.g., -114) and approximately 193 base pairs (e.g., -193) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 119 base pairs (e.g., -119) and approximately 198 base pairs (e.g., -198) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 124 base pairs (e.g., -124) and approximately 203 base pairs (e.g., -203) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 129 base pairs (e.g., -129) and approximately 208 base pairs (e.g., -208) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site. In some specific implementations, the tenth external CSRE is located between approximately 189 base pairs (e.g., -189) upstream of the transcription start site and approximately 268 base pairs (e.g., -268) upstream of the transcription start site.In some other embodiments, the tenth outer CSRE is located between 228 base pairs (e.g., -228) and 229 base pairs (e.g., -229) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 198 base pairs (e.g., -198) and approximately 277 base pairs (e.g., -277) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 239 base pairs (e.g., -239) and approximately 318 base pairs (e.g., -318) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 278 base pairs (e.g., -278) and 279 base pairs (e.g., -279) upstream of the transcription start site. In some specific embodiments, the tenth outer CSRE is located between approximately 318 base pairs (e.g., -318) and approximately 397 base pairs (e.g., -397) upstream of the transcription start site. In some other embodiments, the tenth outer CSRE is located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.In yet another specific embodiment, the engineered promoter having at least ten external CSREs includes a first external CSRE located between 94 base pairs (e.g., -94) upstream of the transcription start site and 173 base pairs (e.g., -173) upstream of the transcription start site; a second external CSRE located between 99 base pairs (e.g., -99) upstream of the transcription start site and 178 base pairs (e.g., -178) upstream of the transcription start site; a third external CSRE located between 104 base pairs (e.g., -104) upstream of the transcription start site and 183 base pairs (e.g., -183) upstream of the transcription start site; a fourth external CSRE located between 109 base pairs (e.g., -109) upstream of the transcription start site and 188 base pairs (e.g., -188) upstream of the transcription start site; and a fourth external CSRE located between 114 base pairs (e.g., -114) upstream of the transcription start site and 193 base pairs (e.g., -173) upstream of the transcription start site. The fifth external CSRE is located between 93) and 119 base pairs (e.g., -119) upstream of the transcription start site and 198 base pairs (e.g., -198) upstream of the transcription start site; the seventh external CSRE is located between 124 base pairs (e.g., -124) upstream of the transcription start site and 203 base pairs (e.g., -203) upstream of the transcription start site; the eighth external CSRE is located between 129 base pairs (e.g., -129) upstream of the transcription start site and 208 base pairs (e.g., -208) upstream of the transcription start site; the ninth external CSRE is located between 198 base pairs (e.g., -198) upstream of the transcription start site and 277 base pairs (e.g., -277) upstream of the transcription start site; and the tenth external CSRE is located between 318 base pairs (e.g., -318) upstream of the transcription start site and 397 base pairs (e.g., -397) upstream of the transcription start site.For example, an engineered promoter with at least ten external CSREs includes a first external CSRE located between 133 base pairs (e.g., -133) and 134 base pairs (e.g., -134) upstream of the transcription start site; a second external CSRE located between 138 base pairs (e.g., -138) and 139 base pairs (e.g., -139) upstream of the transcription start site; a third external CSRE located between 143 base pairs (e.g., -143) and 144 base pairs (e.g., -144) upstream of the transcription start site; a fourth external CSRE located between 148 base pairs (e.g., -148) and 149 base pairs (e.g., -149) upstream of the transcription start site; and a fourth external CSRE located between 153 base pairs (e.g., -153) and 154 base pairs (e.g., -154) upstream of the transcription start site. The fifth external CSRE; the sixth external CSRE located between 158 base pairs (e.g., -158) and 159 base pairs (e.g., -159) upstream of the transcription start site; the seventh external CSRE located between 163 base pairs (e.g., -163) and 164 base pairs (e.g., -164) upstream of the transcription start site; the eighth external CSRE located between 168 base pairs (e.g., -168) and 169 base pairs (e.g., -169) upstream of the transcription start site; the ninth external CSRE located between 237 base pairs (e.g., -237) and 238 base pairs (e.g., -238) upstream of the transcription start site; and the tenth external CSRE located between 357 base pairs (e.g., -357) and 358 base pairs (e.g., -358) upstream of the transcription start site.

[0083] Some parental / engineered promoters include a TATA box, and in some embodiments, the CSRE is positioned relative to the TATA box. In some embodiments, the engineered promoter of this disclosure includes a first external CSRE located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and up to 363 base pairs (e.g., -363) upstream of the TATA box. In some cases, the engineered promoter may include one or more external CSREs that may be located more than 363 base pairs upstream of the TATA box (e.g., -363) (provided that it includes at least one external CSRE located at at least 363 base pairs upstream of the TATA box). In some embodiments, the engineered promoter of this disclosure includes a first external CSRE located at up to 275 base pairs upstream of the TATA box (e.g., -275). In some cases, the engineered promoter may include one or more external CSREs that may be located more than 275 base pairs upstream of the TATA box (e.g., -275) (provided that it includes at least one external CSRE located at at least 275 base pairs upstream of the TATA box).

[0084] In some alternative embodiments, the first external CSRE is located between approximately 38 base pairs downstream of the TATA box (e.g., +38) and approximately 363 base pairs upstream of the TATA box (e.g., -363). In some specific embodiments, the first external CSRE is located between approximately 38 base pairs downstream of the TATA box (e.g., +38) and approximately 41 base pairs upstream of the TATA box (e.g., -41). In some further embodiments, the first external CSRE is located between 1 base pair upstream of the TATA box (e.g., -1) and 2 base pairs upstream of the TATA box (e.g., -2). In some specific embodiments, the first external CSRE is located between approximately 8 base pairs downstream of the TATA box (e.g., +8) and approximately 71 base pairs upstream of the TATA box (e.g., -71). In some other embodiments, the first external CSRE is located between 31 base pairs upstream of the TATA box (e.g., -31) and 32 base pairs upstream of the TATA box (e.g., -32). In some embodiments, the first external CSRE is located between approximately 2 base pairs (e.g., -2) upstream of the TATA box and approximately 81 base pairs (e.g., -81) upstream of the TATA box. In other embodiments, the first external CSRE is located between 41 base pairs (e.g., -41) upstream of the TATA box and approximately 42 base pairs (e.g., -42) upstream of the TATA box. In some embodiments, the first external CSRE is located between approximately 10 base pairs (e.g., -10) upstream of the TATA box and approximately 89 base pairs (e.g., -89) upstream of the TATA box. In other embodiments, the first external CSRE is located between 49 base pairs (e.g., -49) upstream of the TATA box and approximately 50 base pairs (e.g., -50) upstream of the TATA box. In some embodiments, the first external CSRE is located between approximately 32 base pairs (e.g., -32) upstream of the TATA box and approximately 111 base pairs (e.g., -111) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 71 base pairs (e.g., -71) upstream of the TATA box and 72 base pairs (e.g., -72) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 36 base pairs (e.g., -36) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 75 base pairs (e.g., -75) upstream of the TATA box and 76 base pairs (e.g., -76) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 42 base pairs (e.g., -42) upstream of the TATA box and approximately 121 base pairs (e.g., -121) upstream of the TATA box.In some other embodiments, the first external CSRE is located between 81 base pairs (e.g., -81) upstream of the TATA box and 82 base pairs (e.g., -82) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 59 base pairs (e.g., -59) upstream of the TATA box and approximately 138 base pairs (e.g., -138) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 98 base pairs (e.g., -98) upstream of the TATA box and 99 base pairs (e.g., -99) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 60 base pairs (e.g., -60) upstream of the TATA box and approximately 139 base pairs (e.g., -139) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 99 base pairs (e.g., -99) upstream of the TATA box and 100 base pairs (e.g., -100) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 65 base pairs (e.g., -65) upstream of the TATA box and approximately 144 base pairs (e.g., -144) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 104 base pairs (e.g., -104) upstream of the TATA box and 105 base pairs (e.g., -105) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 70 base pairs (e.g., -70) upstream of the TATA box and approximately 149 base pairs (e.g., -149) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 109 base pairs (e.g., -109) upstream of the TATA box and 110 base pairs (e.g., -110) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 75 base pairs (e.g., -75) upstream of the TATA box and approximately 154 base pairs (e.g., -154) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 114 base pairs (e.g., -114) upstream of the TATA box and 115 base pairs (e.g., -115) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the TATA box and approximately 156 base pairs (e.g., -156) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 116 base pairs (e.g., -116) upstream of the TATA box and 117 base pairs (e.g., -117) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 80 base pairs (e.g., -80) upstream of the TATA box and approximately 159 base pairs (e.g., -159) upstream of the TATA box.In some other embodiments, the first external CSRE is located between 119 base pairs (e.g., -119) upstream of the TATA box and 120 base pairs (e.g., -120) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 85 base pairs (e.g., -85) upstream of the TATA box and approximately 164 base pairs (e.g., -164) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 124 base pairs (e.g., -124) upstream of the TATA box and 125 base pairs (e.g., -125) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 90 base pairs (e.g., -90) upstream of the TATA box and approximately 169 base pairs (e.g., -169) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 129 base pairs (e.g., -129) upstream of the TATA box and 130 base pairs (e.g., -130) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 134 base pairs (e.g., -134) upstream of the TATA box and 135 base pairs (e.g., -135) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 155 base pairs (e.g., -155) upstream of the TATA box and approximately 234 base pairs (e.g., -234) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 194 base pairs (e.g., -194) upstream of the TATA box and 195 base pairs (e.g., -195) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 203 base pairs (e.g., -203) upstream of the TATA box and 204 base pairs (e.g., -204) upstream of the TATA box. In some specific embodiments, the first external CSRE is located between approximately 205 base pairs (e.g., -205) upstream of the TATA box and approximately 284 base pairs (e.g., -284) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 244 base pairs (e.g., -244) upstream of the TATA box and 245 base pairs (e.g., -245) upstream of the TATA box.In some specific embodiments, the first external CSRE is located between approximately 284 base pairs (e.g., -284) upstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some other embodiments, the first external CSRE is located between 323 base pairs (e.g., -323) upstream of the TATA box and 324 base pairs (e.g., -324) upstream of the TATA box.

[0085] In some embodiments, the engineered promoter of this disclosure comprises at least two external CSREs. This document provides embodiments of the location and nucleic acid sequence of the first external CSRE, which can be used in engineered promoters comprising two or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are sequential, or they may be provided at different positions and are discontinuous. In some alternative embodiments, the second external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 41 base pairs (e.g., -41) upstream of the TATA box. In some further embodiments, the second external CSRE is located between 1 base pair (e.g., -1) upstream of the TATA box and 2 base pairs (e.g., -2) upstream of the TATA box. In some embodiments, the second external CSRE is located between approximately 8 base pairs downstream of the TATA box (e.g., +8) and approximately 71 base pairs upstream of the TATA box (e.g., -71). In other embodiments, the second external CSRE is located between 31 base pairs upstream of the TATA box (e.g., -31) and 32 base pairs upstream of the TATA box (e.g., -32). In some embodiments, the second external CSRE is located between approximately 2 base pairs upstream of the TATA box (e.g., -2) and approximately 81 base pairs upstream of the TATA box (e.g., -81). In other embodiments, the second external CSRE is located between 41 base pairs upstream of the TATA box (e.g., -41) and 42 base pairs upstream of the TATA box (e.g., -42). In some embodiments, the second external CSRE is located between approximately 10 base pairs upstream of the TATA box (e.g., -10) and approximately 89 base pairs upstream of the TATA box (e.g., -89). In some other embodiments, the second external CSRE is located between 49 base pairs (e.g., -49) upstream of the TATA box and 50 base pairs (e.g., -50) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 32 base pairs (e.g., -32) upstream of the TATA box and approximately 111 base pairs (e.g., -111) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 71 base pairs (e.g., -71) upstream of the TATA box and 72 base pairs (e.g., -72) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 36 base pairs (e.g., -36) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box.In some other embodiments, the second external CSRE is located between 75 base pairs (e.g., -75) upstream of the TATA box and 76 base pairs (e.g., -76) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 42 base pairs (e.g., -42) upstream of the TATA box and approximately 121 base pairs (e.g., -121) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 81 base pairs (e.g., -81) upstream of the TATA box and 82 base pairs (e.g., -82) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 59 base pairs (e.g., -59) upstream of the TATA box and approximately 138 base pairs (e.g., -138) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 98 base pairs (e.g., -98) upstream of the TATA box and 99 base pairs (e.g., -99) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 60 base pairs (e.g., -60) upstream of the TATA box and approximately 139 base pairs (e.g., -139) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 99 base pairs (e.g., -99) upstream of the TATA box and approximately 100 base pairs (e.g., -100) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 65 base pairs (e.g., -65) upstream of the TATA box and approximately 144 base pairs (e.g., -144) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 104 base pairs (e.g., -104) upstream of the TATA box and approximately 105 base pairs (e.g., -105) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 70 base pairs (e.g., -70) upstream of the TATA box and approximately 149 base pairs (e.g., -149) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 109 base pairs (e.g., -109) upstream of the TATA box and approximately 110 base pairs (e.g., -110) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 75 base pairs (e.g., -75) upstream of the TATA box and approximately 154 base pairs (e.g., -154) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 114 base pairs (e.g., -114) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box. In some specific implementations, the second external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the TATA box and approximately 156 base pairs (e.g., -156) upstream of the TATA box.In some other embodiments, the second external CSRE is located between 116 base pairs (e.g., -116) upstream of the TATA box and 117 base pairs (e.g., -117) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 80 base pairs (e.g., -80) upstream of the TATA box and approximately 159 base pairs (e.g., -159) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 119 base pairs (e.g., -119) upstream of the TATA box and 120 base pairs (e.g., -120) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 85 base pairs (e.g., -85) upstream of the TATA box and approximately 164 base pairs (e.g., -164) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 124 base pairs (e.g., -124) upstream of the TATA box and 125 base pairs (e.g., -125) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 90 base pairs (e.g., -90) upstream of the TATA box and approximately 169 base pairs (e.g., -169) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 129 base pairs (e.g., -129) upstream of the TATA box and 130 base pairs (e.g., -130) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 134 base pairs (e.g., -134) upstream of the TATA box and 135 base pairs (e.g., -135) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 155 base pairs (e.g., -155) upstream of the TATA box and approximately 234 base pairs (e.g., -234) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 194 base pairs (e.g., -194) upstream of the TATA box and 195 base pairs (e.g., -195) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 203 base pairs (e.g., -203) upstream of the TATA box and 204 base pairs (e.g., -204) upstream of the TATA box.In some specific embodiments, the second external CSRE is located between approximately 205 base pairs (e.g., -205) upstream of the TATA box and approximately 284 base pairs (e.g., -284) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 244 base pairs (e.g., -244) upstream of the TATA box and approximately 245 base pairs (e.g., -245) upstream of the TATA box. In some specific embodiments, the second external CSRE is located between approximately 284 base pairs (e.g., -284) upstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some other embodiments, the second external CSRE is located between 323 base pairs (e.g., -323) upstream of the TATA box and approximately 324 base pairs (e.g., -324) upstream of the TATA box. In yet another embodiment, an engineered promoter having two or more external CSREs comprises a first external CSRE located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box; and a second external CSRE located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box. In yet another embodiment, an engineered promoter having two or more external CSREs comprises a first external CSRE located between 134 base pairs (e.g., -134) upstream of the TATA box and 135 base pairs (e.g., -135) upstream of the TATA box; and a second external CSRE located between 203 base pairs (e.g., -203) upstream of the TATA box and 204 base pairs (e.g., -204) upstream of the TATA box.

[0086] In some embodiments, the engineered promoter of this disclosure comprises at least three external CSREs. This document provides embodiments of the positions and nucleic acid sequences of the first and second external CSREs, which can be used in engineered promoters comprising three or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are sequential, or they may be provided at different positions and are discontinuous. In some alternative embodiments, the third external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 41 base pairs (e.g., -41) upstream of the TATA box. In some further embodiments, the third external CSRE is located between 1 base pair (e.g., -1) upstream of the TATA box and 2 base pairs (e.g., -2) upstream of the TATA box. In some embodiments, the third external CSRE is located between approximately 8 base pairs downstream of the TATA box (e.g., +8) and approximately 71 base pairs upstream of the TATA box (e.g., -71). In some other embodiments, the third external CSRE is located between 31 base pairs upstream of the TATA box (e.g., -31) and 32 base pairs upstream of the TATA box (e.g., -32). In some embodiments, the third external CSRE is located between approximately 2 base pairs upstream of the TATA box (e.g., -2) and approximately 81 base pairs upstream of the TATA box (e.g., -81). In some other embodiments, the third external CSRE is located between 41 base pairs upstream of the TATA box (e.g., -41) and 42 base pairs upstream of the TATA box (e.g., -42). In some embodiments, the third external CSRE is located between approximately 10 base pairs upstream of the TATA box (e.g., -10) and approximately 89 base pairs upstream of the TATA box (e.g., -89). In some other embodiments, the third external CSRE is located between 49 base pairs (e.g., -49) upstream of the TATA box and 50 base pairs (e.g., -50) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 32 base pairs (e.g., -32) upstream of the TATA box and approximately 111 base pairs (e.g., -111) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 71 base pairs (e.g., -71) upstream of the TATA box and 72 base pairs (e.g., -72) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 36 base pairs (e.g., -36) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box.In some other embodiments, the third external CSRE is located between 75 base pairs (e.g., -75) upstream of the TATA box and 76 base pairs (e.g., -76) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 42 base pairs (e.g., -42) upstream of the TATA box and approximately 121 base pairs (e.g., -121) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 81 base pairs (e.g., -81) upstream of the TATA box and 82 base pairs (e.g., -82) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 59 base pairs (e.g., -59) upstream of the TATA box and approximately 138 base pairs (e.g., -138) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 98 base pairs (e.g., -98) upstream of the TATA box and 99 base pairs (e.g., -99) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 60 base pairs (e.g., -60) upstream of the TATA box and approximately 139 base pairs (e.g., -139) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 99 base pairs (e.g., -99) upstream of the TATA box and approximately 100 base pairs (e.g., -100) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 65 base pairs (e.g., -65) upstream of the TATA box and approximately 144 base pairs (e.g., -144) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 104 base pairs (e.g., -104) upstream of the TATA box and approximately 105 base pairs (e.g., -105) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 70 base pairs (e.g., -70) upstream of the TATA box and approximately 149 base pairs (e.g., -149) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 109 base pairs (e.g., -109) upstream of the TATA box and approximately 110 base pairs (e.g., -110) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 75 base pairs (e.g., -75) upstream of the TATA box and approximately 154 base pairs (e.g., -154) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 114 base pairs (e.g., -114) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box. In some specific implementations, the third external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the TATA box and approximately 156 base pairs (e.g., -156) upstream of the TATA box.In some other embodiments, the third external CSRE is located between 116 base pairs (e.g., -116) upstream of the TATA box and 117 base pairs (e.g., -117) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 80 base pairs (e.g., -80) upstream of the TATA box and approximately 159 base pairs (e.g., -159) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 119 base pairs (e.g., -119) upstream of the TATA box and 120 base pairs (e.g., -120) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 85 base pairs (e.g., -85) upstream of the TATA box and approximately 164 base pairs (e.g., -164) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 124 base pairs (e.g., -124) upstream of the TATA box and 125 base pairs (e.g., -125) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 90 base pairs (e.g., -90) upstream of the TATA box and approximately 169 base pairs (e.g., -169) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 129 base pairs (e.g., -129) upstream of the TATA box and 130 base pairs (e.g., -130) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 134 base pairs (e.g., -134) upstream of the TATA box and 135 base pairs (e.g., -135) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 155 base pairs (e.g., -155) upstream of the TATA box and approximately 234 base pairs (e.g., -234) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 194 base pairs (e.g., -194) upstream of the TATA box and 195 base pairs (e.g., -195) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 203 base pairs (e.g., -203) upstream of the TATA box and 204 base pairs (e.g., -204) upstream of the TATA box.In some specific embodiments, the third external CSRE is located between approximately 205 base pairs (e.g., -205) upstream of the TATA box and approximately 284 base pairs (e.g., -284) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 244 base pairs (e.g., -244) upstream of the TATA box and 245 base pairs (e.g., -245) upstream of the TATA box. In some specific embodiments, the third external CSRE is located between approximately 284 base pairs (e.g., -284) upstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some other embodiments, the third external CSRE is located between 323 base pairs (e.g., -323) upstream of the TATA box and 324 base pairs (e.g., -324) upstream of the TATA box. In yet another embodiment, an engineered promoter having three or more external CSREs comprises a first external CSRE located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box; a second external CSRE located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box; and a third external CSRE located between approximately 284 base pairs (e.g., -284) upstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In yet another embodiment, an engineered promoter having three or more external CSREs comprises a first external CSRE located between 134 base pairs (e.g., -134) and 135 base pairs (e.g., -135) upstream of the TATA box; a second external CSRE located between 203 base pairs (e.g., -203) and 204 base pairs (e.g., -204) upstream of the TATA box; and a third external CSRE located between 323 base pairs (e.g., -323) and 324 base pairs (e.g., -324) upstream of the TATA box.

[0087] In some embodiments, the engineered promoter of this disclosure comprises at least four external CSREs. This document provides embodiments of the positions and nucleic acid sequences of the first, second, and third external CSREs, which can be used in engineered promoters comprising four or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are sequential, or they may be provided at different positions and are discontinuous. In some alternative embodiments, the fourth external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 41 base pairs (e.g., -41) upstream of the TATA box. In some further embodiments, the fourth external CSRE is located between 1 base pair (e.g., -1) upstream of the TATA box and 2 base pairs (e.g., -2) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 8 base pairs downstream of the TATA box (e.g., +8) and approximately 71 base pairs upstream of the TATA box (e.g., -71). In some other embodiments, the fourth external CSRE is located between 31 base pairs upstream of the TATA box (e.g., -31) and 32 base pairs upstream of the TATA box (e.g., -32). In some specific embodiments, the fourth external CSRE is located between approximately 2 base pairs upstream of the TATA box (e.g., -2) and approximately 81 base pairs upstream of the TATA box (e.g., -81). In some other embodiments, the fourth external CSRE is located between 41 base pairs upstream of the TATA box (e.g., -41) and 42 base pairs upstream of the TATA box (e.g., -42). In some specific embodiments, the fourth external CSRE is located between approximately 10 base pairs upstream of the TATA box (e.g., -10) and approximately 89 base pairs upstream of the TATA box (e.g., -89). In some other embodiments, the fourth external CSRE is located between 49 base pairs (e.g., -49) upstream of the TATA box and 50 base pairs (e.g., -50) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 32 base pairs (e.g., -32) upstream of the TATA box and approximately 111 base pairs (e.g., -111) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 71 base pairs (e.g., -71) upstream of the TATA box and 72 base pairs (e.g., -72) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 36 base pairs (e.g., -36) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box.In some other embodiments, the fourth external CSRE is located between 75 base pairs (e.g., -75) upstream of the TATA box and 76 base pairs (e.g., -76) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 42 base pairs (e.g., -42) upstream of the TATA box and approximately 121 base pairs (e.g., -121) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 81 base pairs (e.g., -81) upstream of the TATA box and 82 base pairs (e.g., -82) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 59 base pairs (e.g., -59) upstream of the TATA box and approximately 138 base pairs (e.g., -138) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 98 base pairs (e.g., -98) upstream of the TATA box and 99 base pairs (e.g., -99) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 60 base pairs (e.g., -60) upstream of the TATA box and approximately 139 base pairs (e.g., -139) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 99 base pairs (e.g., -99) upstream of the TATA box and approximately 100 base pairs (e.g., -100) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 65 base pairs (e.g., -65) upstream of the TATA box and approximately 144 base pairs (e.g., -144) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 104 base pairs (e.g., -104) upstream of the TATA box and approximately 105 base pairs (e.g., -105) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 70 base pairs (e.g., -70) upstream of the TATA box and approximately 149 base pairs (e.g., -149) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 109 base pairs (e.g., -109) upstream of the TATA box and approximately 110 base pairs (e.g., -110) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 75 base pairs (e.g., -75) upstream of the TATA box and approximately 154 base pairs (e.g., -154) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 114 base pairs (e.g., -114) upstream of the TATA box and approximately 115 base pairs (e.g., -115) upstream of the TATA box. In some specific implementations, the fourth external CSRE is located between approximately 77 base pairs (e.g., -77) upstream of the TATA box and approximately 156 base pairs (e.g., -156) upstream of the TATA box.In some other embodiments, the fourth external CSRE is located between 116 base pairs (e.g., -116) upstream of the TATA box and 117 base pairs (e.g., -117) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 80 base pairs (e.g., -80) upstream of the TATA box and approximately 159 base pairs (e.g., -159) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 119 base pairs (e.g., -119) upstream of the TATA box and 120 base pairs (e.g., -120) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 85 base pairs (e.g., -85) upstream of the TATA box and approximately 164 base pairs (e.g., -164) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 124 base pairs (e.g., -124) upstream of the TATA box and 125 base pairs (e.g., -125) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 90 base pairs (e.g., -90) upstream of the TATA box and approximately 169 base pairs (e.g., -169) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 129 base pairs (e.g., -129) upstream of the TATA box and 130 base pairs (e.g., -130) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 95 base pairs (e.g., -95) upstream of the TATA box and approximately 174 base pairs (e.g., -174) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 134 base pairs (e.g., -134) upstream of the TATA box and 135 base pairs (e.g., -135) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 155 base pairs (e.g., -155) upstream of the TATA box and approximately 234 base pairs (e.g., -234) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 194 base pairs (e.g., -194) upstream of the TATA box and 195 base pairs (e.g., -195) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 164 base pairs (e.g., -164) upstream of the TATA box and approximately 243 base pairs (e.g., -243) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 203 base pairs (e.g., -203) upstream of the TATA box and 204 base pairs (e.g., -204) upstream of the TATA box.In some specific embodiments, the fourth external CSRE is located between approximately 205 base pairs (e.g., -205) upstream of the TATA box and approximately 284 base pairs (e.g., -284) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 244 base pairs (e.g., -244) upstream of the TATA box and 245 base pairs (e.g., -245) upstream of the TATA box. In some specific embodiments, the fourth external CSRE is located between approximately 284 base pairs (e.g., -284) upstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some other embodiments, the fourth external CSRE is located between 323 base pairs (e.g., -323) upstream of the TATA box and 324 base pairs (e.g., -324) upstream of the TATA box.

[0088] In some embodiments, the engineered promoter of this disclosure comprises at least five external CSREs. Embodiments of the positions and nucleic acid sequences of the first, second, third, and fourth external CSREs are provided herein and can be used in engineered promoters comprising five or more external CSREs. When multiple CSREs are included in an engineered promoter, the CSREs may be independently located at the same position and are sequential, or they may be provided at different positions and are discontinuous. In some alternative embodiments, the fifth external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 363 base pairs (e.g., -363) upstream of the TATA box. In some specific embodiments, the fifth external CSRE is located between approximately 38 base pairs (e.g., +38) downstream of the TATA box and approximately 41 base pairs (e.g., -41) upstream of the TATA box. In some further embodiments, the fifth external CSRE is located between 1 base pair (e.g., -1) upstream of the TATA box and 2 base pairs (e.g., -2) upstream of the TATA box. In some specific embodiments, the fifth external CSRE is located between approximately 8 base pairs downstream of the TATA box (e.g., +8) and approximately 71 base pairs upstream of the TATA box (e.g., -71). In some other embodiments, the fifth external CSRE is located between 31 base pairs upstream of the TATA box (e.g., -31) and 32 base pairs upstream of the TATA box (e.g., -32). In some specific embodiments, the fifth external CSRE is located between approximately 2 base pairs upstream of the T...

Claims

1. An engineered promoter (i) derived from a parent promoter having a transcription start site, and (ii) for expressing a gene, wherein the engineered promoter has at least one external carbon source responsiveness element (CSRE), wherein the at least one external CSRE has a nucleic acid sequence of Formula (I): N1N2CCN3N4TN5N6N7CCGN8 (I) N1 is any nucleic acid residue; N2 is any nucleic acid residue, preferably C or T; N3 is any nucleic acid residue, preferably A, G, or T; N4 is any nucleic acid residue, preferably C or T; N5 is any nucleic acid residue, preferably A, C, or G; N6 is any nucleic acid residue, preferably A or G; N7 is any nucleic acid residue, preferably G or T; and N8 is any nucleic acid residue, preferably A or G; and wherein the at least one external CSRE comprises a first external CSRE located upstream of and proximal to the transcription start site.

2. The engineered promoter of claim 1, wherein the gene comprises an open reading frame having a start codon.

3. The engineered promoter of claim 2, wherein the first external CSRE is located up to 390 base pairs upstream of the start codon (-390).

4. The engineered promoter of any one of claims 1 to 3, comprising a TATA box.

5. The engineered promoter of any one of claims 1 to 4, wherein the engineered promoter is capable of inducing transcription of the gene at a higher level than the parent promoter in the presence of a C2 carbon source.

6. The engineered promoter of any one of claims 1 to 5, wherein the at least one external CSRE comprises a nucleic acid sequence of any one of SEQ ID NOs: 26 to 35.

7. The engineered promoter of any one of claims 1 to 6, comprising at least two external CSREs, three external CSREs, four external CSREs, five external CSREs, six external CSREs, seven external CSREs, eight external CSREs, nine external CSREs, or ten external CSREs.

8. The engineered promoter of any one of claims 1 to 7, wherein the parent promoter is an ethanol-responsive promoter.

9. The engineered promoter of claim 8, wherein the parent promoter is adh2 the promoter of the gene (adh2p).

10. The engineered promoter of claim 9, having a nucleic acid sequence of SEQ ID NO: 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, or 23.

11. The engineered promoter of any one of claims 1 to 7, wherein the parent promoter is a constitutive promoter.

12. The engineered promoter of claim 11, wherein the parent promoter is sti1 the promoter of the gene sti1p.

13. The engineered promoter of claim 12, having a nucleic acid sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, or 18.

14. A heterologous nucleic acid molecule having an engineered promoter according to any one of claims 1 to 13 in operable association with a gene.

15. The heterologous nucleic acid molecule of claim 14, wherein the gene encodes a polypeptide.

16. A vector comprising an engineered promoter according to any one of claims 1 to 13 or a heterologous nucleic acid molecule according to claim 14 or 15.

17. An expression cassette comprising an engineered promoter according to any one of claims 1 to 13 or a heterologous nucleic acid molecule according to claim 14 or 15.

18. A recombinant microbial host cell comprising an engineered promoter according to any one of claims 1 to 13, a heterologous nucleic acid molecule according to claim 14 or 15, a vector according to claim 16 or an expression cassette according to claim 17.

19. The recombinant microbial host cell of any one of claims 1 to 18 having native alcohol dehydrogenase activity.

20. The recombinant microbial host cell of claim 18 or 19, which is a yeast.

21. The recombinant microbial host cell of claim 20, which is from the genus Komagataella.

22. The recombinant microbial host cell of claim 21, which is from K. phaffii.

23. A method for increasing the responsiveness of an engineered promoter for expressing a gene to a C2 carbon source, the method comprising introducing a first external carbon source responsiveness element (CSRE) upstream and proximal to a transcription start site in a parent promoter having the transcription start site, wherein the first external CSRE has a nucleic acid sequence of formula (I): N1N2CCN3N4TN5N6N7CCGN8 (I) N1 is any nucleic acid residue; N2 is any nucleic acid residue, preferably C or T; N3 is any nucleic acid residue, preferably A, G or T; N4 is any nucleic acid residue, preferably C or T; N5 is any nucleic acid residue, preferably A, C or G; N6 is any nucleic acid residue, preferably A or G; N7 is any nucleic acid residue, preferably G or T; and N8 is any nucleic acid residue, preferably A or G.

24. The method of claim 23, wherein the gene comprises an open reading frame having a start codon.

25. The method of claim 23 or 24, comprising introducing the first external CSRE up to 390 base pairs upstream of the start codon (-390).

26. The method of any one of claims 23 to 25, wherein the parent promoter comprises a TATA box.

27. The method of any one of claims 23 to 26, wherein the first external CSRE comprises the nucleic acid sequence of any one of SEQ ID NOs: 26 to 35.

28. The method of any one of claims 23-27, comprising introducing two external CSREs, three external CSREs, four external CSREs, five external CSREs, six external CSREs, seven external CSREs, eight external CSREs, nine external CSREs, or ten external CSREs in the parental promoter.

29. The method of claim 28, wherein the external CSREs each have a nucleic acid sequence independently selected from any one of SEQ ID NOs: 26-35.

30. The method of any one of claims 23-29, wherein the parental promoter is an ethanol-responsive promoter.

31. The engineered promoter of any one of claims 23-29, wherein the parental promoter is a constitutive promoter.

32. A method for expressing a gene in the recombinant microbial host cell of any one of claims 18-22, the method comprising (i) contacting the recombinant microbial host cell with a C2 carbon source to allow expression of the gene.

33. The method of claim 32, further comprising, prior to step (i), (i’) propagating the recombinant microbial host cell with an alternative carbon source that is different from the C2 carbon source.

34. The method of claim 33, wherein the alternative carbon source comprises glucose, fructose, and / or glycerol.

35. The method of any one of claims 32-34, wherein the gene encodes a polypeptide.

36. The method of claim 35, wherein the polypeptide is an intracellular polypeptide or a secreted polypeptide.

37. The method of claim 36, wherein the secreted polypeptide is in free form or associated with the surface of the recombinant yeast host cell.

38. The method of claim 37, wherein the polypeptide associated with the surface of the recombinant yeast host cell is a tethered polypeptide.

39. The method of any one of claims 35-38, wherein the polypeptide is an enzyme.

40. The method of any one of claims 32-39, further comprising, after step (i), (ii) substantially isolating the polypeptide from the recombinant microbial host cell.

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