Part:BBa_K4605015
Used to express indigoidine successfully.
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 indigoidine in Corynebacterium glutamicum
We have successfully expressed bpsA in Corynebacterium glutamicum. As shown below, the right conical flask shows the fermentation results after introducing empty PEKEX2 into the C.glutamicum, whereas the left conical flask shows the fermentation results of indigoidine production after introducing bpsA plasmid into C.glutamicum. Obviously, the left one expresses bpsA successfully with fully blue in the fermentation broth.
We used DMSO to suspend C.glutamicum, and then sonicated the bacteria to break them apart. After centrifugation, we collected the supernatant to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.
Direct Dyeing
We stained the bacterial cellulose membranes directly with C. glutamicum cultures. We obtained blue BC membrane, indicating that our idea of reconstructing the indigoidine synthesis pathway in K.xylinus is feasible
Characterizations
Expression of bpsA and pcpS in K. xylinus to produce color fibers
Because K. xylinus does not have the native PPTase that is necessary for activating apo-form of indigoidine synthase into its active holo-form by adding coenzyme A to the peptide carrier domain (PCP), we need to transfect the target gene both bpsA and pcpS (encoding PPTase)into K. xylinus using pSB1A2 as a plasmid vector, and synthesize indigoidine fibers using K. xylinus which is capable of producing cellulose in high yield.
In earlier literature research, we noted three strong promoters(J23104,J23100,J23119) compatible with K.xylinus. Thus, to verify if they can also work in our system,we ligate EGFP or YFP to the three promoters separately.
With previous basic explorations, we will use PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104,J23100,J23119 etc.), and CDS sequences to express bpsA and pcpS in K. xylinus while binding to bacterial cellulose membranes.
However,it is a question to use fusion proteins or the separate expression of two gene fragments. At first,considering that the present research universally choose to express bpsA and pcpS separately, we want to explore the possibility of BpsA-PcpS fusion protein .So in dry lab,we use Amino Acid Folding tools to calculate the structure of amino acid sequence. To be specific, we will use ColabFold v1.5.2-patch, a online folding tool to calculate the 3D structure of proteins.
The 3D structure of only bpsA protein is:
The 3D structure of bpsA-pcpS fusion protein is:
Comparing the 3D structure of BspA and BspA-PcpS fusion protein, we find that the fusion protein appears to have some domain structural deformation, this is concentrated in the green segment, although this may be due to the low confidence in the structural predictions of this segment. Consequently,in order to avoid interfering with the growth of bacteria too early, and in combination with the results of structure prediction, we think that bpsA and pcpS should be eventually expressed separately by bacteria, instead of using a fusion protein expression system. On the one hand, this may lead to higher indigoidine production efficiency, on the other hand, it may make the system easier to control.
With previous experiences, we decided to express two genes separately and reconstructed the plasmid using PSB1A2 as the plasmid backbone with the addition of the bpsA as well as the pcpS genes and a strong promoter compatible with K.xylinus.Below is a schematic of our expression system as well as a plasmid profile.
Below is the agarose gel electrophoresis result we got in our experiment, the DNA fragments are between 3000-5000, which proves that we successfully got the target fragments.
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
[2] Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).
[3] Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T. Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synth Biol. 2021 Dec 17;10(12):3422-3434.
[4]Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3431-40.
[5]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.
[6]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 DyesACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622
[7]Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4055
Illegal NgoMIV site found at 4069
Illegal NgoMIV site found at 4174
Illegal NgoMIV site found at 4207
Illegal AgeI site found at 4198 - 1000COMPATIBLE WITH RFC[1000]
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