Regulated gene expression systems and constructs thereof

1. A nitrogen-sensitive expression system comprising:

a nucleic acid sequence comprising an upstream truncated or downstream truncated variant of SEQ ID NO: 234 or a sequence having at least 95% sequence identity to said truncated sequence;

wherein the upstream truncated or downstream truncated variant of SEQ ID NO: 234 or the sequence having at least 95% sequence identity thereto comprises (i) a transcription factor region comprising an NtcA binding site, (ii) a core promoter region, and (iii) nitrogen-sensitive promoter activity;

wherein,

the upstream truncated variant of SEQ ID NO: 234 does not contain

(a) nucleotide positions 1 to 78 of SEQ ID NO: 234,

(b) nucleotide positions 1 to 128 of SEQ ID NO: 234,

(c) nucleotide positions 1 to 138 of SEQ ID NO: 234,

(d) nucleotide positions 1 to 148 of SEQ ID NO: 234,

(e) nucleotide positions 1 to 158 of SEQ ID NO: 234,

(f) nucleotide positions 1 to 166 of SEQ ID NO: 234,

(g) nucleotide positions 1 to 168 of SEQ ID NO: 234,

(h) nucleotide positions 1 to 178 of SEQ ID NO: 234,

(i) nucleotide positions 1 to 188 of SEQ ID NO: 234,

(j) nucleotide positions 1 to 198 of SEQ ID NO: 234,

(k) nucleotide positions 1 to 208 of SEQ ID NO: 234,

(l) nucleotide positions 1 to 218 of SEQ ID NO: 234,

(m) nucleotide positions 1 to 228 of SEQ ID NO: 234, or

(n) nucleotide positions 1 to 238 of SEQ ID NO: 234; or

the downstream truncated variant of SEQ ID NO: 234 does not contain

(a) nucleotide positions 347 to 836 of SEQ ID NO: 234,

(b) nucleotide positions 353 to 836 of SEQ ID NO: 234,

(c) nucleotide positions 357 to 836 of SEQ ID NO: 234,

(d) nucleotide positions 367 to 836 of SEQ ID NO: 234,

(e) nucleotide positions 377 to 836 of SEQ ID NO: 234,

(f) nucleotide positions 387 to 836 of SEQ ID NO: 234,

(g) nucleotide positions 397 to 836 of SEQ ID NO: 234,

(h) nucleotide positions 407 to 836 of SEQ ID NO: 234,

(i) nucleotide positions 417 to 836 of SEQ ID NO: 234,

(j) nucleotide positions 427 to 836 of SEQ ID NO: 234,

(k) nucleotide positions 477 to 836 of SEQ ID NO: 234,

(l) nucleotide positions 527 to 836 of SEQ ID NO: 234,

(m) nucleotide positions 577 to 836 of SEQ ID NO: 234,

(n) nucleotide positions 627 to 836 of SEQ ID NO: 234,

(o) nucleotide positions 677 to 836 of SEQ ID NO: 234,

(p) nucleotide positions 727 to 836 of SEQ ID NO: 234,

(q) nucleotide positions 777 to 836 of SEQ ID NO: 234,

(r) nucleotide positions 807 to 836 of SEQ ID NO: 234, or

(s) nucleotide positions 817 to 836 of SEQ ID NO: 234,

wherein,

the core promoter region comprises or is operably linked to the transcription factor region; and

the nitrogen-sensitive promoter activity comprises promoter activity when urea or ammonia is the predominant nitrogen source and promoter repression when nitrate is a predominant nitrogen source;

the nitrogen-sensitive expression system further comprises a transcribable nucleic acid molecule and a 3′ transcription termination nucleic acid molecule;

the core promoter region is operably linked to the transcribable nucleic acid molecule;

the transcribable nucleic acid molecule is operably linked to the 3′ transcription termination nucleic acid molecule;

expression of the transcribable nucleic acid molecule is repressed when nitrate is a predominant nitrogen source and the transcribable nucleic acid molecule is expressed when urea or ammonia is the predominant nitrogen source; and

the transcribable nucleic acid molecule encodes a polypeptide having sucrose phosphate synthase activity; sucrose phosphate phosphatase activity, sucrose phosphate synthase and sucrose phosphate phosphatase activity; trehalose phosphate synthase activity; trehalose phosphate phosphatase activity, gluocosylglycerol phosphate synthase activity; gluocosylglycerol phosphate phosphatase activity;

mannosylfructose phosphate synthase activity; or mannosylfructose phosphate phosphatase activity.

2. The expression system of claim 1 , wherein the transcription factor region comprises at least one of: a GTAN 11 C consensus sequence; a GTAN 8 TAC consensus sequence; a GTAN 8 TGC consensus sequence; a GTN 10 AC consensus sequence; a TGTN 9 ACA consensus sequence; or a TGTN 10 ACA consensus sequence.

3. The expression system of claim 1 , wherein the transcription factor region comprises at least one of a TAN 3 T/A box.

4. The expression system of claim 1 , wherein the nitrogen-sensitive expression system comprises SEQ ID NO: 189 or SEQ ID NO: 190.

5. The expression system of claim 1 , wherein the transcribable nucleic acid molecule comprises:

SEQ ID NO: 1, or at least 95% sequence identity thereto and encoding a polypeptide having sucrose phosphate synthase and sucrose phosphate phosphatase activity; or

a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 2, or a polypeptide at least 95% similar thereto having sucrose phosphate synthase and sucrose phosphate phosphatase activity.

6. An expression cassette comprising:

(a) the expression system of claim 1 ;

(b) a transcribable nucleic acid molecule;

(c) a 3′ transcription termination nucleic acid molecule; and

(d) a nitrate inducible promoter operably linked to a polynucleotide sequence encoding an NtcA polypeptide or an NtcB polypeptide;

wherein,

the core promoter region is operably linked to the transcribable nucleic acid molecule;

the transcribable nucleic acid molecule is operably linked to the 3′ transcription termination nucleic acid molecule;

elements (a)-(c) are positioned in relation to each other such that expression of the expression cassette in a host organism results in production of a polypeptide sequence encoded by the transcribable nucleic acid molecule; and

element (d) is positioned such that expression of the expression cassette in the host organism results in production of the NtcA polypeptide or the NtcB polypeptide.

7. A transgenic host cell:

comprising the expression system of claim 1 ; or

the expression cassette of claim 6 ;

wherein

expression of the polypeptide sequence encoded by the transcribable nucleic acid molecule is repressed in the presence of nitrate; and

expression of the polypeptide sequence encoded by the transcribable nucleic acid molecule is induced in the presence of urea or ammonia.

8. A kit comprising the expression system of claim 1 or the expression cassette of claim 6 and at least one reagent for introducing the expression cassette into a host cell and, optionally, instructions for introducing the expression cassette into a host cell.

9. The nitrogen-sensitive expression system of claim 1 , wherein the upstream truncated variant of SEQ ID NO: 234 does not contain nucleotide positions 1 to 166 of SEQ ID NO: 234.

10. The nitrogen-sensitive expression system of claim 1 , wherein the downstream truncated variant of SEQ ID NO: 234 does not contain nucleotide positions 353 to 836 of SEQ ID NO: 234.

11. The expression cassette of claim 6 , wherein:

the nitrate inducible promoter comprises a nirA promoter having SEQ ID NO: 32; and

polynucleotide sequence encoding an NtcA polypeptide has a sequence of SEQ ID NO: 279.