Design
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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 glnAY405F encodes a variant of GSI that is resistant to the downregulation by adenylation of this gene. Therefore, we can clone glnAY405F 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 odhI35 to reduce the α-ketoglutarate dehydrogenase complex (AKGD) activity. The phosphorylation-resistant variant odhIT15A 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 glnAY405F,odhIT15A, 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 fig2 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.