Part:BBa_K4376003
the composite part is used to express the bpsA gene.
Construction of the bpsA gene and tac inducible promoter to control the production of the blue-pigment indigoidine synthetase. The composite part is inserted into C.glutamicum in plasmid, resulting in the successful biosynthesis of indigodine pigment.
We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.
Nanjing-China 2023's Characterization
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.
Expression of bpsA 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.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
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