Team:ECUST China/Engineering
At the first beginning, in order to solve the two main problems that limit the development of phycocyanin—— the poor stability and high production cost. ECUST_China come out the idea to synthesize phycocyanin through rebuild the metabolism pathway on the host of yeast using synthetic biology. It can not only reduce the economic cost and save time but also using biosynthesis process to improve phycocyanin’s thermostability. What we want to build is a kind of optimal foreign protein expression system and using synthetic biology to rebuild the metabolic pathway in Saccharomyces cerevisiae S288c.
Refered to the survey results and professional suggestions, we finally choose the traditional model organism——Saccharomyces cerevisiae among all the yeast species to meet the market’s interest. In the past three decades, saccharomyces cerevisiae has been rebuilt to express a variety of recombinant proteins and it has already been approved by the U.S. Food and Drug Administration (FDA) and the European Drug Administration (EMEA) of it’s safety. Saccharomyces cerevisiae is widely used in the food industry and has great advantage for it’s nontoxicity property, along with it’s low demand for oxygen, clear genetic background, simple to operate and so on. The yeast we use is Saccharomyces cerevisiae S288C, which is provided by Professor Cai Menghao's laboratory.
The initial pathway we built is based on the combination of phycocyanobilin and apoprotein (phycocyanin α subunit) which is catalyzed by the cleavage synthase. The catalytic pathway is follows. Heme is catalyzed by the heme oxygenase to produce Biliverdin(BV IXα) with ring opening pyrrole structure and then Biliverdin is further catalyzed by phycobilin reductase to form phycocynobilin. The 2-3 molecules of phycocyanobilin will bind to the phycocyanin α subunit catalyzed by the cleavage synthase and eventually produce phycocyanin. Note that cleavage synthase has a lot family members including E/F lyase (cpcE/F), S/U lyase (cpcS/U) and t-lyase (cpcT). And what we choose is cpcE/F to catalyze the binding of phycocyan and its reverse reaction.
In addition, our first experimental design was to use surface display system that anchors phycocyanin protein to the cell wall and makes the yeast turn blue.But after in-depth investigation, we found that the final location of phycocyanin α subunit and the color base—— phycocyanobilin are different in cells. Phycocyanin α subunit will finally go to the cell wall after transmit by endoplasmic reticulum and golgi apparatus through fuse expression with the GPI anchoring domain. While the color base—— phycocyanobilin is a kind of small molecule pigment. For phycocyanobilin is not a kind of protein, so it can not be directed transported through adding a length of signaling peptide.What’s more, phycocyanobilin’s molecular weight is of 586.7g/mol which is far from the small molecule (molecular weight of 10 times urea) ,meaning it is nearly impossible to directly cross the membrane. Thus, the chance of the combination of phycocyanin mainly on the cytosol and phycocyanin α subunit mainly on the cell wall is greatly reduced.
So we reconstructed the experimental pathway to express different genes in different parts of the cell, but finally let the phycocyanin α subunit and phycocyanobilin recombine in the cytoplasm. The expression system of phycocyanin is 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. In yeast, the original substances are heme, heme oxygenase 1 and phycocyanin, so we need to extra introduce phycocyanin lyase (cpcE and cpcF coding), phycocyanin a subunit (cpcA coding) and ferredoxin oxidoreductase (pcyA coding).
[1] 衣俊杰, 臧晓南, 张学成, 等. 具荧光活性的节旋藻藻蓝蛋白-亚基在大肠杆菌中的重组表达[J]. 中国海洋大学学报, 2011, 41(5): 59-62.
[2] Reuter W, Nicker-Reuter. Molecular assembly of the phycobilisomes from the cyanobacteria Magstigocladus laminosus, J Photochem Photobiol B:Biol, 1993, 18:51～66
[3] Zhao K H, Deng M G, Zheng M, etal. Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin. FEBS Lett, 2000,469:9～13
The phycocyanin gene we initially selected was from Galdieria phlegrea (strain 009) whose Tm equals 85 ± 1 ° C( https://doi.org/10.1016/j.ijbiomac.2020.02.045).
While there are no gene nor protein database depend on the Galdieria phlegrea (strain 009). So we use the the protein sequencing on the paper to find similar sequence on other species through BLAST on NCBI. Unexpectedly, we found that the phycocyanin sequence is exactly the same as that from another red algae Cyanidium caldarium and the genetic relationship between the two red algae is very close. So we change our mind to to use the sequence from Cyanidium caldarium to build phycocyanin and only build the α Subunits not the β Subunit.
In addition, we also need E/F lyase to catalyze the binding of phycocyanin to phycocyanin α subuni. But the cpcE/F sequence we can find on NCBI is very limited and often incomplete. Therefore, the genes related to cleavage synthase could not be found in the genome of Cyanidium caldarium and there was no homologou schizoisomerase gene. However, we found cpcE/F in Paulinella chromatophora. Although it is an amoeba, it has photosynthetic endosymbiosis from cyanobacteria.
After in-depth investigation, we found a kind of phycocyanin from the heat-resistant cyanobacteria Synechococcus lividus PCC 6715. It is reported to withstand the high temperature of 95 ° C and we could find the homologous cleavage isomerase gene in its genome along with all the needed genes. Although it is a prokaryote and we don't know whether it can be successfully expressed in yeast, with it’s excellence in heat-resistant and clear genetic information we finally choose Synechococcus lividus PCC 6715 as our gene resources to further explore our project.
We initially wanted to import Saccharomyces cerevisiae YPM plasmid, but considering that this plasmid is not available in our laboratory we turned to choose pTDH3-dCas9 plasmid that was already available. After construction, it was found that the plasmid was overwhelmed to carry all our interest genes so the transformation rate was very low.
Therefore, we finally selected the two plasmid systems using pTDH3-dCas9 and P426-SNR52p-gRNA
plasmids. Initially, considering our pcyA gene was introduced into the endoplasmic reticulum and the
other three genes were introduced into mitochondria. We chose to introduce pcyA into pTDH3-dCas9 and
cpcA, cpcE and cpcF into P426-SNR52p-gRNA plasmid. We found GenScript company to help us synthesize
the genes(pcyA,cpcE,cpcFand cpcE). And then we add the primers and terminators to the genes to
complete the expression vector. (For yeast is a kind of eukaryotic cell, each gene calls for a
dependent primer and terminator). When we added expression boxes to each of the genes synthesized by
the company, we found that the different position of genes on the plasmids had little effect on the
final expression. Therefore, while re-splicing the cpcA expression boxes to pTDH3-dCas9 plasmid.
Because dCas9 would affect the normal expression of our plasmid, we further chose to knock out the
dcas9 gene.
The construction of the plasmids are as follows:
The phycocyanin αsubunits encoded by cpcA and the lyase encoded by cpcE and cpcF are proteins. In the yeast's growth period, if there exist large molecules, such as the subunit of phycocyanin. It will definatly affect the growth of yeast then we can not get the required output of phycocyanin to support the yeast to get blue. So we use the inducible expression system on the gene of cpcA,cpcE and cpcF to solve the problem., thus the growth of S. cerevisiae is not affected as much as possible.
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. VP16 is a transcription-activating enzyme that is responsible for the transcription of downstream genes and can enhance the expression of downstream genes. And LexA is a transcriptional regulator in yeast, and the protein expressed by it can bind to downstream LexO, thus initiating transcription of downstream genes .
Finally 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.
Therefore, there are four expression vectors in the regulation of this module. The first expression box is PGAL1-Lexa-VP16-CYC1, the second expression box is Lexo-PTDH3-CpcA-CYC1, the third expression box is Lexo-PTDH3-CpcE-ADH1, and the fourth expression box is Lexo-PTDH3-CpcF-ADH1. At the same time, we selected the plasmid with 2u which will greatly increase the copy number of plasmids and finally increase the production of phycocyanin.
While 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.
The transformed double plasmids were as follows:
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.
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.
GET IN TOUCH
+86 15821261548
IGEM_ECUST@outlook.com
ID:IGEM_ECUST
ECUST_China
East China University of Science and Technology
Shanghai,China
