Part:BBa_K4605002

Blue-pigment indigoidine synthetase gene from Streptomyces lavendulae

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.

At the same time, we used DMSO to directly suspend the bacteria for ultrasound, and then centrifuge to obtain the upper clearing to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.

Below is a diagram of SDS-PAGE of Corynebacterium glutamicum. From left to right, the first lane is the whole cell lysate of C. glutamicum, the second lane is the whole cell lysate after introduction of the plasmid, the third lane is the supernatant of wild-type C. glutamicum, and the fourth lane is the supernatant after introduction of the plasmid. It indicates that bpsA successfully expressed indigo after introduction of the plasmid.

Direct Dyeing

We stained the bacterial cellulose membranes directly with C. glutamicum cultures.

Co-culturing

In order to lay the groundwork for the subsequent one-step production of colored fibers by expressing bpsA directly in K.xylinus, we first started with a co-culture of K. xylinus and C. glutamicum as a way to further explore the way indigoidine binds to bacterial cellulose as well as the physical and chemical properties. The reason we choose K.xylinus is because it is reported as one of the high cellulose-producing strains by journal articles. Unfortunately, we were not able to obtain colored BC membranes first, but rather colored granular bacterial cellulose.

This is an electron microscope image after direct staining. Microfibers intertwine with each other to form a mesh-like structure, in which the indigoidine-secreting C. glutamicum are encapsulated.

In subsequent experiments, we choose the static culture conditions and utilize BC membranes as a framework to grow C. glutamicum. This novel idea offers us a paradigm to obtain the colored BC membranes with different patterns determined by how we inoculate C. glutamicum.

Expression of bpsA in K. xylinus

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.

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]