Part:BBa_K4273020
pTDH3-Np5598-tTDH1-pPGK1-NlmysD-tPGK1
Np5598 is a gene found in cyanobacteria Nostoc punctiforme that encodes for AGL whereas NlmysD encodes for AlaL in Nostoc linckia. AGL-AlaL allows 4-DG to be converted into the MAAs shinorine or porphyra-334. In our experiment, we found that AlaL encoded by NlmysD has a strong preference toward the amino acid threonine. Therefore, this part could efficiently produce the MAA porphyra-334 primarily, making it the first successful case of producing pure samples of porphyra-334. This part is within our part collection that allows efficiently production of MAAs in S. cerevisiae. Our part collection contains necessary genes to produce gadusol and the MAAs shinorine, porphyra-334, and palythine at a high rate. Xyl1, Xyl2, and Xyl3 are genes that allow S. cerevisiae to utilize xylose to produce S7P. DDGS and OMT converts S7P to the precursor of MAA, 4-deoxygadusol (4-DG). AGL converts 4-DG into M-glycine (MG). AlaL, by adding either serine or threonine, produces shinorine and porphyra-334, respectively. MysH could be added to the circuit of shinorine to produce palythine. S7P could also be converted into gadusol under the catalyzation of EEVS and M-Tox. In this part collection, we included multiple pathways and methods to increase the production of the upstream S7P and downstream MAAs. This part collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.
Usage and Biology
We selected promoters pTDH3, pPGK1, and pTEF2 due to their stability expression in S. cerevisiae (Apel et. al., 2016). These promoters are shown to have stable and strong expression in YPD culture mediums. Among the three, pTDH3 has highest stability and strength, followed by pPGK1. For expression of AGL and AlaL enzymes, we used pTDH3 for AGL and pPGK1 for AlaL. In order to optimize our production, we inserted this part into the yeast’s genome at position 106, chromosome I (Apel et. al., 2016).
Experiment
Design and Absorbance of Nine AGL-AlaL Combinations We expressed AGL-AlaL genes first using plasmid vector and then through genome insertion. Due to the existence of multiple different types of AGL and AlaL with different efficiency and amino acid preference in nature, we selected ligases from three different marine organisms: Nostoc punctiform (Np5598 and Np5597), Nostoc linckia (NlmysC and NImysD) and Actinosynnema mirum (Am4257 and Am4256), and expected to create nine combinations of AGL-AlaL. We used Lee's yeast toolkit to produce Level1 plasmids containing only AGL or AlaL, and used Golden Gate assembly on the basis of Level1 to construct Level2 plasmid containing the 9 combinations of AGL and AlaL. Finally, we transformed these Level2 plasmids into SC.L3 strains, which are S. cerevisiae which could produce 4-DG efficiently from the addition of DDGS and OMT, to form strain SC.L5.
Shinorine and porphyra-334 has similar absorption curves with an absorption peak at 334 nm. We cultivated the SC.L5 yeast using a SC-Ura culture medium with 1% glucose and 1% xylose and tested the absorption spectrum of the supernatant of the fermentation broth. Other than the Am4257-Am4256 combination yeast which failed to grow, there is an obvious absorption peak of 334 nm for the other 8 combinations. We found that the absorption peak of AGL Np5598 series was significantly higher than that of NlmysC and Am4257. In ALAL series, NlmysD has the highest absorption peak, followed by Am4256 and Np5597. We selected the 6 samples with higher peak and lysed them to preform OD scanning on the lysate. The scanning results after lysis further performed that Np5598 is the most efficient AGL and NlmysD is the most efficient AlaL. We think we have found an optimal combination of these two enzymes and have successfully produced MAAs.
Determining the Amino Acid Preference of Np5597 and NlmysD Moreover, because AlaL has a preference for different amino acids to produce shinorine and porphyra-334, we need to further confirm the type of MAA produced by different strains. To do so, we used High-performing Liquid Chromatology (HPLC) and Mass Spectrometer (MS) technology. Due to the lack of standard samples of these MAAs in the market, we extracted MAAs from nori samples using methods we learnt through our human practice activities and used the extractives as standard. We found that the metabolite of Np5598-NlmysD mainly has the absorption spectrum of porphyra-334 according to HPLC, and the result of MS also proved that mainly porphyra-334 exist in the metabolite (m/q = 347). This shows that NlmysD has a strong selective preference toward the amino acid Threonine, and thus will mainly produce porphyra-334 if both Threonine and Serine are present in the environment. This is the first successful case of producing mainly 334 only. Np5597, on the other hand, shows shinorine's absorption peak, meaning it has preference toward serine. Therefore, we decided to use Np5598-NlmysD for porphyra-334 production and Np5598-NlmysD for shinorine production.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3423
Illegal XhoI site found at 2075 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2321
Illegal BsaI.rc site found at 3433
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