Part:BBa_K4273001

NOTOC__ pTDH3-DDGS-tTDH1-pPGK1-OMT-tPGK1

Xyl1 is a xylose assimilation gene encoding xylose reductase (XR) that can converts xylose to xylitol. Xyl1 can provides an alternative carbon source for the pentose phosphate pathway that can increase S7P production. We selected Xyl1 genes from Scheffersomyces stipitis that proved to be successfully heterologous expressed for efficiently xylose-fermenting in S. Cerevisiae. We used yeast toolkit (Lee, 2015) to assemble the Level 1 plasmid including the promoter, coding sequence, and terminator. Xyl1 is under the control of promoter pTDH3 which provides a high and stable transcription efficiency (Apel, et. al, 2016). Then, we used PCR amplification to obtain the DNA fragment Xyl1 from Level1 plasmid, and two homology arms (LA and RA) from yeast genome.

Usage and Biology

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3423
  Illegal XhoI site found at 2075
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2321
  Illegal BsaI.rc site found at 3433