Team:ECUST China/Design
As a rare natural blue pigment, Phycocyanin has a broad development and application prospect and with 670 million USD market value now, it is promising.
While phycocyanin is limited by it’s high cost and instability. As the only approved rare nature blue pigment by FDA, phycocyanin faces many obstacles. Firstly, the high production cost. At present, phycocyanin production mainly relies on lengthy physicochemical method to extract from algae. Secondary, phycocyanin is sensitive to heat and light which will also cause waste and economic losses.
At the same time, we obtained phycocyanin with high thermal stability which can be more widely used in food, medicine, fluorescent reagent for biological detection,cosmetics and other field to avoid the problem of denaturation caused by temperature or light. ECUST iGEMers invented”magic blue” which is a dual-plasmid-system that is imported into Saccharomyces cerevisiae. And our experiment includes three parts:
● The construction of the expression pathway of phycocyanin
● Regulator
● Improvement of the
thermal stability
We choose Saccharomyces cerevisiae S288C as the host.
The reason is that it’s classified as risk 1 which means it poses little risk and the strain we used is harmless to human beings and surrounding environment. It’s a world-widely used engineered strain which has been proved to be safe.
What’s more yeast is an important unicellular model organism which grows fast and is easy to modify. It can also be easily used in fermentation to achieve the high production.
Phycocyanin is composed of α-subunit of phycocyanin (apoprotein) and 3Z-phycocyanobilin. Phycocyanin α-subunit covalently binds to phycocyanobilin (PCB) at position α-84, and the ligand are tightly linked to the cysteine residues of the apoprotein through thioether bond. However, 3Z-phycocyanobilin binds with apoprotein nonspecifically and the apoprotein also lacks the ability to specifically distinguish the ligand. So the binding reaction between the 3Z-phycocyanobilin and apoprotein is catalyzed by lyases in vivo.
The expression pathway of phycocyanin is as follows: Heme oxygenase 1 (Hoxl encoding) and pcyA catalyze the production of biliverdin IX and phycocyanobilin respectively in mitochondria. Then the product phycocyanobilin is transported out of the mitochondria to the cytoplasmic matrix.The cleavage synthase (encode by cpcE and cpcF) and phycocyanin α subunits(encode by cpcA) are generated in the endoplasmic reticulum and then enter the cytoplasm by default. The 2-3 molecules of phycocyanobilin will bind to the phycocyanin α subuni catalyzed by the cleavage synthase and eventually produce phycocyanin. In yeast, the original substances are heme, heme oxygenase 1, and we introduce phycocyanin synthase (cpcE and cpcF encoded), phycocyanin α subunit (cpcA encoded), and ferritin oxidoreductase (pcyA encoded) to complete the whole metabolic pathway.
Fig. The expression pathway of phycocyanin
Regulator consists of two parts: the regulate of pcyA and the regulate of cpcA、cpcE、cpcF，which work synergistically to achieve the purpose of increasing the expression of the phycocyanin automatically.
We preserve the constitutive promoter GAP of pcyA for the final product of pcyA is a kind of small molecule--phycocyanobilin which will not effect the groth of yeast. And the phycocyanobilin can prepare for the final construction of phycocyan more efficiently for phycocyanin αsubunits call for 2-3 phycocyanobilin to combine. The pre-synthesis of phycocyanobilin can improve the synthesis efficiency when the other molecules (encode by cpcE,cpcA and cpcF) was induced without affecting the growth of S. cerevisiae as much as possible.
Fig. PcyA expression
We learned that GAL1 is a commonly used inducible promoter in S. cerevisiae. The transcription of GAL genes involved in galactose metabolism in S. cerevisiae is tightly regulated.They will not express in the absence of galactose while In the presence of galactose, the expression level of the product will increase by about 1 ,000 fold. A large number of biochemical genetic analyses have preliminarily established the basic regulatory model of GAL gene expression. In addition, the expression of the GAL gene is also regulated by metabolite repression, thus forming a complex regulatory network system.
VP16 is a transcription-activating enzyme that is responsible for the transcription of downstream genes and can enhance the expression of downstream genes.
LexA is a transcriptional regulator in yeast, and the protein expressed by it can bind to downstream LexO, thus initiating transcription of downstream genes.
We selected Lexa-LEXO as a pair of inductor, and started transcription with GAL1 as the inducer promoter also added a VP16 sequence after LexA to increase its expression.
Fig. CpcE expression
Fig. CpcF expression
Fig. CpcA expression
In Saccharomyces cerevisiae, through constructing the biosynthesis pathway of phycocyanin from Synechococcus lividus, we have achieved the constituent biosynthesis of phycocyanin and phycocyanin α-subunit. In order to discuss the theoretical possibility of further improve the thermal stability of our product, site-directed mutagenesis techniques was applied to modify the key amino acid sites. Comparing amino acid sequence of phycocyanin α-subunit between Synechococcus sp. PCC 6715 and Synechocystis sp.PCC 6803,we found 40 different sites might related to thermal stability.
[ Protein sequence ]
| 5 | 10 | 15 | 20 | 25 | 30 | 35 | 40 | |
| MKTPI | TEAIA | AADTQ | GRFLS | NTELQ | AADGR | FKRAV | ASMEA | |
| ARALT | NNAQS | LIDGA | AQAVY | QKFPY | TTTMQ | GSQYA | STPEG | |
| KAKCA | RDIGY | YLRMV | TYCLV | AGGTG | PMDEY | LIAGL | AEINS | |
| TFDLS | PSWYV | EALKY | IKANH | GLSGQ | AAVEA | NAYID | YAINA | LS |
Taking Synechococcus amino acid sequence of phycocyanin α-subunit as template. The 40 different sites were directed mutated respectively by I-Mutant website to predict protein stability changes upon mutations. The change value of the mutant’s free energy is calculated according the formula of △△G = △G (mutant) −△G (wild-type strain). For instance, the L (in position 115) was mutated to T because T was predicted the most stable whose △△G equals -4.84 kcal/mol.
In order to further improve the stability of the yeast recombinant colony and the safety of our products, we knocked out the resistance genes on the two plasmids p426 and pTDH3 and integrated the plasmids without resistance genes into the yeast genome.
HXK2 and PTC1 genes are located on Chromosome VII and Chromosome IV in Saccharomyces cerevisiae S288C respectively. By referring to NCBI, we found that the replacement of these two genes had little influence on yeast growth and phycocyanin synthesis and its properties. We selected a sequence of these two genes (see table) and cloned them into expression box upstream in P426 and pTDH3, respectively. The cloned plasmids were digested with single enzyme and transferred into yeast. The homologous arms at both ends of the plasmid were combined with the corresponding gene sequences of yeast, and the genes on the plasmid were integrated into the yeast genome.
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