Regulated gene expression systems and constructs thereof

An expression system and transcription factor technology, which can be used in the introduction of foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc. using vectors, which can solve problems such as limited costs

Inactive Publication Date: 2014-09-10
PROTERRO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Large-scale production of materials by fermentation is often limited by costs associated with regulating gene expression

Method used

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  • Regulated gene expression systems and constructs thereof
  • Regulated gene expression systems and constructs thereof
  • Regulated gene expression systems and constructs thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0148] Example 1: Removal of invertase activity from cells

[0149] To accumulate sucrose, Synechocystis sp. PCC6803 was engineered to remove invertase (β-fructofuranosidase) activity from the cell, which hydrolyzes sucrose into energy that would reenter the cell for normal metabolism. Glucose and fructose. LYB471 was obtained by deleting the gene lim17 (SEQ ID NO: 140) encoding invertase from ATCC27184 (LYB426). It also deletes LYB472 from the wild type line (LYB467). Over time, these lines diverged, resulting in a loss of motility in LYB426 (Kamei et al. 2001).

[0150]LYB443 was first generated by deleting the gene lim17 encoding invertase from LYB426 using a neomycin resistance marker. Using sequential PCR, a deletion was made in lim17 (SEQ ID NO: 140), leaving a large stretch of flanking DNA. A first PCR reaction was performed on the LYB426 whole cell genomic DNA template using Sspinvdel-F (SEQ ID NO: 142) and Sspinvdel-R (SEQ ID NO: 143) as primers. The product of t...

Embodiment 2

[0153] Example 2: Reduced expression of epipolysaccharide

[0154] Furthermore, it is known that Synechocystis sp. PCC6803 produces large amounts of exopolysaccharide (EPS) in response to normal aging of cultures or under environmental stress conditions on the cells (Panoff et al. 1988). The large amount of EPS represents a substantial carbon allocation and overall metabolic flux that can be diverted to sucrose production. Although EPS mutants have been selected, the genetics of the epipolysaccharide produced in Synechocystis sp. PCC6803 is relatively unknown (Panoff and Joset 1989). A large cluster of genes (almost 18 kbp in size) on the endogenous plasmid pSYSM has been postulated to be primarily responsible for EPS production based on their homology to known genes (Kaneko et al. 2003). To this end, putative genes considered responsible for key biosynthetic pathways related to EPS biosynthesis were removed from LYB472 to generate LYB476.

[0155] A similar strategy to dele...

Embodiment 3

[0157] Example 3: Expression of ASF by the nitrite reductase promoter

[0158] The following examples show ASF expression by the Synechocystis sp. PCC6803 or Synechococcus elongatus PCC7942 nitrite reductase promoters.

[0159] A strain of LYB476 (Synechocystis sp.), with a plasmid containing the nitrite reductase promoter from Synechococcus elongatus PCC7942 in front of the asf gene (pLybAL18), but with reduced EPS expression) was passed through 50 ml of 5 mM HEPES buffer The culture medium was adjusted to pH 8.0 in BG11 medium supplemented with 25 μg / ml chloramphenicol and 4 mM ammonium chloride instead of potassium nitrate as nitrogen source. (Plasmid pLybAL18 and its generation are described in U.S. Patent Application Publication No. 2009 / 0181434, which is incorporated herein by reference in its entirety. Plasmid pLybAL18 was introduced by triparental conjugation also as described in U.S. Patent Application Publication No. 2009 / 0181434 LYB476). Incubation was performed a...

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Abstract

The present disclosure is directed to compositions and methods for nitrogen sensitive regulation of expression of a transcribable nucleic acid molecule. One aspect provides a nitrogen-sensitive expression system that includes a transcription factor region comprising an NtcA binding site and a core promoter region comprising a RuBisCo promoter or a variant or a functional fragment thereof. Another aspect provides a method of transforming a host cell with an expression system. Also provided are expression cassettes, transformed host cells, and kits.

Description

[0001] Cross References to Related Applications [0002] This patent application claims the benefit of priority to US Provisional Application Serial No. 61 / 467,873, filed March 24, 2011, which is hereby incorporated by reference in its entirety. [0003] Material incorporated by reference [0004] The Sequence Listing which is part of this disclosure includes a computer readable form containing the nucleotide or amino acid sequences of the invention. The subject matter of the Sequence Listing is hereby incorporated by reference in its entirety. Background of the invention [0005] Large-scale production of materials by fermentation is generally limited by the costs associated with regulating gene expression. Often, the strategies employed require expensive sugar derivatives or other small molecules to trigger protein production to yield the desired product. [0006] Promoters are nucleic acid molecules that contain 5' regulatory elements that play an integral role in the ov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
CPCC12N15/635C12N15/52C12N15/63C12N15/67C12N15/74
Inventor R·J·特纳V·塞尔尚J·艾肯斯D·霍尔兹
Owner PROTERRO INC
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