Part:BBa_K4273014

NlMysH

Gene from Nostoc linkia NIES-25 involved in biosynthesis of palythine.

encoding 2-oxoglutarate-dependent oxygenase (MysH）that converts shinorine or porphyra-334 to palythine

Improved by Fudan iGEM 2023

Creating synthetic genetic circuits often involves introducing several heterologous genes. Conventional approaches require the regulation of proteins under different promoters and the introduction of multiple plasmids, leading to lengthy cargo DNA.

However, we offer an alternative solution. Instead of sequentially assembling coding sequences(CDSs), we have developed a ribozyme-assisted polycistronic co-expression system (pRAP) by inserting stem-loop and Twister P1 sequences between CDSs. The Twiseter P1 ribozyme cleaves the polycistronic mRNA transcript into individual mono-cistrons. The stem-loop structure prevents mono-cistron mRNA from degradation. This design minimizes self-interaction within the polycistron, ensuring that each cistron initiates translation with comparable efficiency. Consequently, our genetic circuit attains optimal functionality while maintaining minimal size and complexity.

NlMysH encodes a 2-oxoglutarate-dependent oxygenase, which we refer to as the gene MysH in our part registry.

Improved parts

Our improved part is BBa_K4765014 (MysH), which underwent codon optimization from the original part BBa_K4273014 (NlMysH), with a specific focus on its expression for the Escherichia coli K12 strain, resulting in the creation of this improved part. We also intergrated this part into our ribozyme-assisted polycistronic co-expression system (pRAP) to assemble four enzymes, which are in the biosynthetic pathway of MAA, under one promoter within a single plasmid in our composite part BBa_K4765118 (ribozyme connected: MysABCDH)

Usage in Biology

It was observed that among Nostoc punctiforme, Nostoc linckia, and Actinosynnema mirum, N. linckia was capable of producing palythine due to the existence of gene NlmysH, which removes a carboxyl group from shinorine to form palythine. Thus, we inserted pTEF2-NlmysH-tSSA1 to the genetic circuit of producing shinorine, allowing us to form this part. 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.

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, then pTEF2. We used pTDH3 to express AGL, pPGK1 to express AlaL, and pTEF2 to express MysH.

Experiments

After obtaining shinorine, we could produce palythine by removing a carboxyl group from shinorine. Thus, we added pTEF2-NlmysH-tSSA1 to our Np5598-Np5597 combination to obtain the genetic circuit for producing palythine. We transformed the new palythine-coding plasmid into SC.L3 and SC.L6 to test the success of our optimization in SC.L6 (Nqm1::OMT-DDGS). We obtained L3:Np5598-Np5597-NlmysH and L6: Np5598-Np5597-NlmysH, fermented the two strains for 72 hours in SC-Ura culture medium with 1% glucose and 1% xylose. The absorption spectrum of the supernatant broth was tested, and results show that there is an obvious absorption peak at 320 nm. We also compared the production in L3:Np5598-Np5597-NlmysH and L6:Np5598-Np5597-NlmysH using OD 320 value, which shows that L6 strains are much more productive than L3 strains. The OD 320 value of L6 is 2.62 times of L3 strains, whereas L3 strains barely had any increase in UV absorption at 320 nm. Therefore, we concluded that we had successfully produced palythine and had greatly increased its production through optimization.

Figure 1: Palythine production in L9 and L5 strains. We transferred palythine plasmid Np5598-Np5597-NlmysH into the L3 and L6 strains (A). After 72 hours of fermentation, OD scanning results show that an absorption peak at was clear, but only in L9 strains (C). OD 320 value shows that L6 strains were much more effective in absorbing UV at 320 nm compared to L5 strains, and the value of OD320 of L9 is 2.62 times the control, justifying the production and optimization of palythine.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 401
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 626
  Illegal SapI.rc site found at 747
  

Reference

Park SH, Lee K, Jang JW, Hahn JS. Metabolic Engineering of Saccharomyces cerevisiae for Production of Shinorine, a Sunscreen Material, from Xylose. ACS Synth Biol. 2019;8(2):346-357.

Jin C, Kim S, Moon S, Jin H, Hahn JS. Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae. FEMS Yeast Res. 2021;21(7):foab053.

Chen M, Rubin GM, Jiang G, Raad Z, Ding Y. Biosynthesis and Heterologous Production of Mycosporine-Like Amino Acid Palythines. J Org Chem. 2021 Aug 20;86(16):11160-11168.

Osborn AR, Almabruk KH, Holzwarth G, Asamizu S, LaDu J, Kean KM, Karplus PA, Tanguay RL, Bakalinsky AT, Mahmud T. De novo synthesis of a sunscreen compound in vertebrates. Elife. 2015 May 12;4:e05919.

Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Jan 9;45(1):496-508.

Zhang H, Jiang Y, Zhou C, Chen Y, Yu G, Zheng L, Guan H, Li R. Occurrence of Mycosporine-like Amino Acids (MAAs) from the Bloom-Forming Cyanobacteria Aphanizomenon Strains. Molecules. 2022 Mar 7;27(5):1734.

Cress BF, Toparlak ÖD, Guleria S, et al. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015;4(9):987-1000.