iGEM 2022 — Team nfls-nanjing

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{% block title %}Design{% endblock %}
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### Overview

Our experiment adopts the method of biosynthesis to produce indigodine, a kind of dye which is widely used in the clothing industry. We choose to repeat experiment in [1] to successfully synthesize indigodine. We transplanted the *bpsA* gene
encoding the catalyzing enzyme by plasmid into the *C. glutamicum*. Meanwhile, we adapted the procedure of TCA cycle to keep the supply of L-glutamate and L-glutamine. As a result, we could realize the goal of the synthesis and production of
biological dye inside the *C. glutamicum*.

### The construction of the strain of BIRU1& the plasmid of pEK-BA1

The indigodine synthesis gene *bpsA* from *S. lavendulae* was initially expressed in *C. glutamicum* to construct a base strain. The *C. glutamicum* has the *pptaCg* gene which encodes the PPTase that is necessary
for activating BpsA. A codon-optimized *bpsA* gene from *S. lavendulae* was cloned into a plasmid pEKEx1 under the inducible *Ptac* promoter, resulting in the pEK-BA1 plasmid and the transformed strain BIRU1. Then under the
induction of 1mM IPTG, BIRU1 in the glutamic acid medium successfully yielded the indigodine.

### The construction of BIRU2~BIRU4&the plasmid of pCES-GOG

Based on BIRU1, the overexpression of glutamine synthetase I(GSI) which is encoded by *glnA* can catalyze the transformation of L-glutamate to L-glutamine, leading the flux towards the L-glutamine,which provides a high ammonium condition in which gene *glnA*Y405F encodes a variant of GSI that is resistant to the downregulation by adenylation of this gene. Therefore, we can clone *glnA*Y405F on the basis of *PH36* to construct the strain BIRU2, which increased the yield of indigodine compared with BIRU1.

Then we need to conduct the flux of α-ketoglutarate to the synthesis of L-glutamine, so we should inhibit the procedure to enter the TCA cycle. We overexpressed OdhI encoded by *odhI*35 to reduce the α-ketoglutarate dehydrogenase complex (AKGD) activity. The phosphorylation-resistant variant *odhI*T15A which strongly inhibits the activity of AKGD was overexpressed in the BIRU2 strain to construct the BIRU3 strain, which exhibited a higher production of indigodine.

Eventually, we overexpressed the glutamate dehydrogenase(GDH) encoded by *gdh* to increase the conversion of α-ketoglutarate and ammonia to L-glutamate. And it forms the BIRU4 strain, which shows a further progression in the yield. The plasmid pCES-GOG, which is the derivative of pCES-H36, works as vectors of genes like *glnA*Y405F,*odhI*T15A, *gdh*.

We also thought overexpressing a fatty acid transcriptional repressor(FasR) would trigger the overproduction of L-glutamate by redirecting the flux of fatty acid biosynthesis to the TCA cycle. So we attempted to clone *fasR* gene as an operon
with the
*bpsA* gene under the control of *Ptac* in BIRU4 strain to get the BIRU5 strain. However, the result went against our anticipation and it demonstrates a decrease in the yield of indigodine compared with BIRU4.

Therefore, among the five transformed strain above, the yield of BIRU4 topped(as *fig*2 shown), so we would use it for further genome engineering.

### The construction of BIRU11&the knockout of *pyk*, *yggB*

Then we overexpressed the myo-Inositol Permeases (*iolT1* and *iolT2*) and Glucosekinase (*ppgk*) by replacing the original promoters with strong promoters *PO6*, *Psod*, and *Ptuf* in
BIRU4 to construct the strain BIRU6. The replacement of promoters was realized by mutating certain nucleotides. For example, The *PO6* promoter was constructed by mutating two nucleotides at position −113 (A → G) and −112 (C →
G) upstream of the *iolT1* gene sequence according to the previous report. As a result, the yield of production improved.

As we knew that the PEP−pyruvate−oxaloacetate node represents a critical control node in the biosynthesis and the cell growth, we could overexpress PEP carboxylase(encoded by *ppc*) or pyruvate carboxylase(encoded by *pyc*) to boost the
yield. Therefore, we respectively overexpressed *ppc*, *pyc* and both *ppc* and *pyc* in BIRU7, BIRU8 and BIRU9. The result shown in Figure3 indicates that BIRU7 contributes to an improved yield, while BIRU8 and shows the decline.
Therefore, we carry on our experiment on BIRU7.

The eventual efforts were towards the removal of the byproducts, mainly citrate and isocitrate for improper flux direction instead of towards L-glutamate synthesis. So we wanted to delete the *pyk* gene encoding pyruvate kinase to construct BIRU10.
It could rebalance the intermediates of the TCA cycle, increasing the L-glutamate synthesis fluxes. Though no citrate could be detected, the yield of indigodine declined.

So we decided to overexpress isocitrate dehydrogenase encoded by *icd* to transform isocitrate into α-ketoglutarate and NADPH. To solve the bottleneck of isocitrate dehydrogenase when overexpressing *ppc*, we chromosomally exchanged the
native promoter and the start codon(GTG) with *Ptuf* and ATG respectively to construct BIRU11. It raised the yield with no byproducts in the medium.

Finally we constructed BIRU12 on the basis of BIRU11, knocking out the *yggB*, which encodes a mechanosensitvie channel. The knockout of the gene may lead to the conversion of L-glutamate to other components in *C. glutamicum*. However,
the final yield of indigodine in BIRU12 prominently decreased.

As *fig* indicates, the yield of indigodine and DCW(dry cell weight) of BIRU11 outweighs those of other strains, so we adopted the BIRU11 to industrial production.

### References

[1] Ghiffary M R , Prabowo C , Sharma K , et al. High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes[J]. ACS Sustainable Chemistry And Engineering, 2021.
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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Overview

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Our experiment mainly consists of four parts.

1)Codon optimization

2)Vector construction

3)Competent Corynebacterium glutamate production

4)Inducible expression

### Codon optimization

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The initial length of *bpsA* gene was 3849bp, and was synthesized into two parts, which were 1900bp and 1970bp separately. A Flag-tag was added before the stop codon.

### Vector construction

![](https://static.igem.wiki/teams/4376/wiki/engineering-1.png)

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1.Amplification of two *bpsA* fragments. DNA Marker: 2000bp

![](https://static.igem.wiki/teams/4376/wiki/engineering-2.png)

2.Recombinant vector was transformed into DH5α. DNA Marker: 5000bp

![](https://static.igem.wiki/teams/4376/wiki/engineering-3.png)

![](https://static.igem.wiki/teams/4376/wiki/engineering-4.png)

3.Recombinant vector was transformed into Corynebacterium glutamicum. DNA Marker: 5000bp

![](https://static.igem.wiki/teams/4376/wiki/engineering-5.png)

### Competent *Corynebacterium glutamate* production

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Using Bio-rad power station instrument to perform the following electrical transfer steps:

1)Add 5 uL plasmid into competent *Corynebacterium glutamicum* cells at 1.8 kV/mm for 5ms;

2)Immediately add 915 uL of preheated LBHIS Buffer, at 46 ℃, and incubate for 6 min;

3)resuscitation at 200 RPM for 2 hours at 30 ℃;

4)Spread on Kana resistant plates and produce transformants in 1-2 days.

### Inducible expression

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The *C.glu-bpsA* strain was directly streaked on LBG(K+) solid plate with IPTG at final concentration of 1mM, and cultured in incubator at 37℃ for 1-2 days.

![](https://static.igem.wiki/teams/4376/wiki/engineering-6.png)

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# Experiment

#### ●Plasmid Extraction

#### ●Steps:

1)Take 1~5ml of bacterial liquid cultured overnight (12-16h), add it to a centrifuge tube, and centrifuge at 10000rpm for 1min. Discard the culture medium and invert it on absorbent paper to absorb the residual liquid.

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{% block title %}Results{% endblock %}
{% block lead %}A summary of our project.{% endblock %}
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# Overview

In our project we aim to mass produce natural indigodine dye from glutamine in C.glutamicum. We have successfully transformed gene bpsA into plasmid pEKEx1, as well as E.coli and C.glu strains. We have also extracted crude indigodine dye out of the C.glu-bpsA strain, and obtained its absorption spectrum. In addition, we tested the effect of different growth medium on bacterial growth, and the durability of the dye we produced.

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