Difference between revisions of "Part:BBa K4605008"

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==Description==
 
==Description==
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BpsA stands for the Blue-pigment indigoidine synthetase gene.Itself is derived from Streptomyces lavendulae and is used in the synthesis of indigo. It can only be activated from inative apo-form to the active holo-bpsA by the addition of CoA to its PCP, catalyzed by PPTase, which synthesizes two molecules of glutamine into one molecule of indigo. Corynebacterium glutamicum is usually used to express bpsA for high indigo production.
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BpsA stands for the blue pigment indigoidine synthetase gene, encoding a single module type non-ribosomal peptide synthetase called BpsA. Indigoidine synthetase can synthesize two molecules of glutamine into one molecule of indigoidine. Itself is derived from Streptomyces lavendulae.
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Corynebacterium glutamicum is the ideal host for the expression of bpsA to achieve high indigoidine production, because it carries strong fluxes of L-glutamate, a precursor of L-glutamine and L-glutamine is the substrate of the indioigdine synthetase. Meanwhile, C. glutamicum also has the native pcpS gene, which expresses PPTase(4'-phosphopantetheinyl transferase). The PPTase is of great significance because it converts the apo-form of the BpsA into its active holo-form by attaching coenzyme A to the peptide carrier domain (PCP).
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In this project first we will obtain indigoidine, the chemical structure of which is 5,5-diamino-4,4-dihydroxy-3,3-diazadiphenoquinone-(2,2), by introducing pEKEX2 plasmid backbone ligated with bpsA, into C. glutamicum. In the next step, we would genetically modify Komagataeibacter xylinus and introduce PSB1A2 plasmid backbone ligated with bpsA and pcpS for one-step synthesis of colored fibers, and also codon optimize the bpsA and pcpS coding sequences to meet our needs.
  
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In this experiment we will modify Komagataeibacter xylinus to express bpsA for one-step synthesis of colored fibers, and also codon optimize the bpsA coding sequence.
 
  
 
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Revision as of 11:47, 9 October 2023


Strong promoters to improve the bpsA expression

Description

BpsA stands for the blue pigment indigoidine synthetase gene, encoding a single module type non-ribosomal peptide synthetase called BpsA. Indigoidine synthetase can synthesize two molecules of glutamine into one molecule of indigoidine. Itself is derived from Streptomyces lavendulae.

Corynebacterium glutamicum is the ideal host for the expression of bpsA to achieve high indigoidine production, because it carries strong fluxes of L-glutamate, a precursor of L-glutamine and L-glutamine is the substrate of the indioigdine synthetase. Meanwhile, C. glutamicum also has the native pcpS gene, which expresses PPTase(4'-phosphopantetheinyl transferase). The PPTase is of great significance because it converts the apo-form of the BpsA into its active holo-form by attaching coenzyme A to the peptide carrier domain (PCP).

In this project first we will obtain indigoidine, the chemical structure of which is 5,5-diamino-4,4-dihydroxy-3,3-diazadiphenoquinone-(2,2), by introducing pEKEX2 plasmid backbone ligated with bpsA, into C. glutamicum. In the next step, we would genetically modify Komagataeibacter xylinus and introduce PSB1A2 plasmid backbone ligated with bpsA and pcpS for one-step synthesis of colored fibers, and also codon optimize the bpsA and pcpS coding sequences to meet our needs.


Experiment

Expression of bpsA in K. xylinus

With previous basic explorations, we will use a wood vinegar compatible PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104, J23102, etc.), and bpsA sequences to try to express bpsA in K. xylinus while binding to bacterial cellulose membranes.

References

[1] Mohammad Rifqi Ghiffary, Cindy Pricilia Surya Prabowo, Komal Sharma, Yuchun Yan, Sang Yup Lee, and Hyun Uk Kim.High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 [2]Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH. An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synth Biol. 2019 Apr 19;8(4):708-723.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]