Part:BBa_K4273005

NpR5599

The second gene of gene clusters involved in biosynthesis of shinorine in cyanobacteria N. punctiforme.
encoding O-methyltransferase (O-MT) that converts 2-demethyl-4-deoxygadusol (DDG) to 4-deoxygadusol (4-DG)

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 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.

NpR5599 encodes an O-methyltrans-ferase(O-MT), which we refer to as the gene MysB in our part registry.

Improved parts

Our improved part is BBa_K4765011 (MysB), which underwent codon optimization from the original part BBa_K4273005 (NpR5599), 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 and Biology

DDGS is encoded by NpR5600, a gene of gene clusters found in cyanobacteria Nostoc punctiforme. The gene NpR5600 was used in our experiment for encoding 2-demethyl 4-deoxygadusol synthase (DDGS) that converts sedoheptulose 7-phosphate (S7P) to 2-demethyl-4-deoxygadusol (DDG) during the pathway of shinorine production. OMT is encoded by NpR5599, which with the other two N. punctiforme genes, NpR5600 and NpR5598, led to the production of mycosporine-glycine that prevent oxidative stress. In our experiment, the gene NpR5599 was used to encode O-methyltransferase (O-MT) that converts 2-demethyl-4-deoxygadusol (DDG) to 4-deoxygadusol (4-DG).

We selected promoters pTDH3 and pPGK1 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. pTDH3 has highest stability and strength, followed by pPGK1. Therefore, we used pTDH3 for DDGS and pPGK1 for OMT. By introducing another copy of DDGS-OMT at position Nqm1 and inserting the genes into S. cerevisiae's genome, we were able to increase the production of shinorine and porphyra-334 to a great extent.

Experiment

Characterization
For our experiment, we used the strong promoters such as pTDH3 for DDGS and pPGK1 for OMT when we tried to convert S7P to 4DG efficiently, and selected DDGS(NpR5600) and OMT(NpR5599) for homologous recombination into SC.L2 genome. Moreover, we chosed position 308 from chromosome III (Apel et. al., 2016) for genome recombinant. By using this method, we transformed pCRCT-308 plasmid, the DNA fragments of homologous arms, DDGS, and OMT into SC.L2. The recombinant strains were identified by PCR and sequencing (Fig.4C and D), demonstrating that SC.L3 was obtained in the experiment.

Figure 1: Insertion of DDGS and OMT at 308 position. By transforming CRISPR-308 plasmid pCRCT-308, LA, DDGS, OMT and RA, the two genes should be inserted at 308 position (A). We expanded the homogenous arms, DDGS and OMT genes through PCR and transformed them into L2 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-DDGS and OMT (C) and verified this result through the sequencing testing (D), obtaining the L3 strain.

Optimization
To increase the quantity of MAAs production, we further optimized the SC.L3 strains. We notice that Nqm1 has similar functions as TAL1 in shunting S7P into glycolytic pathway. So, to increase the S7P pool, we decided to remove this gene and insert an extra copy of DDGS-OMT simultaneously. The result shows that gene expression can be enhanced by using multiple promoters for increasing MAAs production (Yang et. al., 2018). For modification, pTDH3 was used to express OMT and pPGK1 was used to express DDGS, making the total transcription rate of the two copie roughly equal. Then, we inserted DNA fragments of LA, OMT, DDGS, RA and the pCRCT-Nqm1 plasmid into SC.L3. After PCR, DNA sequencing, and selection of recombinant colonies, we removed the pCRCT-Nqm1 plasmid, obtaining SC.L6 in the end.

Figure 2: Optimizing production by inserting OMT-DDGS and deleting Nqm1 gene. Nqm1 function similarly as TAL1, which reduces S7P concentrations. By transforming CRISPR-Nqm1 plasmid pCRCT-Nqm1, LA, OMT, DDGS, and RA, the two genes should be inserted at Nqm1 position; we switched the promoter of DDGS to pPGK1 and OMT to pTDH3 in order to have equal net transcription rate of DDGS and OMT genes. We expanded the homogenous arms, OMT, DDGS genes through PCR and transformed them into L5 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-OMT, and DDGS (C) and verified this result through the sequencing testing (D), obtaining the L6 strain.

For the production of shinorine and porphyra-334

The production of shinorine and porphyra-334 as our main experimental goal after S7P is converted to 4DG by DDGS and OMT. Shinorine and porphyra-334 are produced by ATP-grasp ligase (AGL) and D-Ala-D-Ala ligase (ALAL) with two enzymatic steps. First, 4DG is converted to mycosporine-glycine(MG) by conjugating glycine to 4DG under the action of AGL. Then, another amino acid is attached to MG by AGL to produce shinorine or porphyra-334, and L-serine for shinorine and L-threonine for porphyra-334. However, due to the fact that there are variety types of AGL and ALAL with different efficiency and amino acid preference in the enviromnment, we selected ligases from three different marine organisms: Nostoc punctiform(Np5598 and Np5597), Nostoc linckia(NlmysC and NImysD) and Actinosynnema mirum(Am4257 and Am4256, expecting to create nine combinations of AGL-AlaL. For results, we found that NlmysD has a strong selective preference toward the amino acid Threonine. Thus, it will mainly produce porphyra-334 if both Threonine and Serine are present in the environment, marking our first successful case of producing only 334. Np5597, on the other hand, shows shinorine's absorption peak, meaning it has preference toward serine.

Figure 3: Shinorine and porphyra-334 production after optimization. We transformed porphyra-334 producing plasmid Np5598-NlmysD and shinorine producing plasmid Np5598-Np5597 into the L3 and L6 strains (A) and compared the absorption spectrum after 72 hours of fermentation. The absorption peak at 334nm of L7 strains displayed significant improvement compared to L5 strains (B) . From OD334, we concluded that in comparison with the L5 assembly, porphyra-334 production in L6 yeast increased by 91.8%, and shinorine production increased by 70.9% (C).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.