Part:BBa_K814014

caffeine biosynthetic pathway for yeast

The induction of this novel pathway in S. cerevisiae requires two additional enzymes from Coffea canephora: XMT1 and DXMT1. S. cerevisiae will produce the required metabolic precursors to Xanthosine, where after the XMT1 enzyme from C. canephora will use the substrate to synthesize 7-methylxanthosine. DXMT1 will continue the synthesis by converting to 7-Methylxanthine, where in XMT1 will again convert it to Theobromine. Finally DXMT1 will convert the metabolite to caffeine. Both the XMT1 and DXMT1 genes produce bifunctional enzymes and this particular pathway was chosen so that one less enzyme would be required in the synthesis (compared to three in C. arabica).

This device contains XMT1 and DXMT1 generators designed for yeast. This part can be used for caffeine biosynthesis in yeast.

Figure 1. SDS Page

Figure 2. HPLC trace of caffeine standard and attempted production of caffeine in yeast.

McCarthy, A, & McCarthy, J G. (2007). The structure of two n-methyltransferases from the caffeine biosynthetic pathway. Plant physiology, 144(2), 879-889.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1400
  Illegal NotI site found at 3540
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 251
  Illegal BglII site found at 2355
  Illegal BamHI site found at 257
  Illegal BamHI site found at 2361
  Illegal XhoI site found at 1388
  Illegal XhoI site found at 3528
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization by 2021iGEM_SHSID

Improvement of an existing part

Figure 14. The blast results about the DNA sequence of our new part BBa_K3993013 and the old parts BBa_K814014..

Compared to the old part BBa_K814014, set up a caffeine biosynthetic pathway for yeast, we design a new part BBa_K3993013, which contains the pTDH3 promoters and a new protein speB. The speB protein catalyzes the formation of putrescine from agmatine and play an important role in metabolism. The group iGEM12_Minnesota aimed to produce caffeine by using both the XMT1 and DXMT1 genes in S. cerevisiae. However, they didn’t successfully detected caffeine after 72h.

Based on the these groups’ contribution, our team design the new composite part BBa_K3993013 to express speB. After the composite part was inserted in a particular plasmid vector and transformed into BY4741. The different regulatory properties in the fermentation are measured, so as to achieve the purpose of our project.

First of all, we constructed a composite part BBa_K3993013 which contains several regulatory elements and transformed it into BY4741. Furthermore, in order to have a general idea of fermentation, we collected metabolites (supernatant) and analyze metabolites by LC-MS. The putrescine was detected.

In addition, our project set up a cell factory system. Cell factory has advantages in the production of tropane alkaloids due to its low environmental pollution, high yield, small space occupation and convenient operation.

Usage and Biology

Kl tropane alkaloids (TAs) refers to a kind of alkaloids containing the tropane alkyl skeleton formed by the combination of pyrrole ring and piperidine ring in structure. It is a natural product of plant and has a long history and important medicinal value. tropane alkaloids have great market demand and often appear in global shortages. A method that can produce Tas in scale is expected. Using synthetic biology to create a microbial cell factory to produce TAs is a highly potential strategy.

The Tropane alkaloid (TAs) is obtained by a series of chemical reactions through the formation of Putrescine (1, 4-butylenediamine, Putrescine) from Arginine. Putrescine is an essential polyamine for ribosomal biogenesis and mRNA translation, but is regulated by polyamines and remains at low concentrations during normal cell growth. In this study, by overexpressing the natural genes involved in arginine metabolism and polyamine biosynthesis, the regulatory mechanism of polyamine biosynthesis is adjusted, so as to engineer the production of excessive putrescine strains.

Figure1. Principle diagram of TAs..

Construct design

The Tropine part of Tropane alkaloids (TAs) is obtained from arginine to putrescine (1,4-butanediamine, putrescine), and then through a series of chemical reactions. In this project, natural genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The engineer strains that produced excess putrescine. (Figure 2).

Figure 2. DNA sequence map of plasmid pYES2-AsADC-SPE1-SpeB..

BBa_K3993000

Usage and Biology

his protein is involved in step 1 of the subpathway that synthesizes putrescine from L-ornithine. Catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine, which is the precursor for the polyamines, spermidine and spermine. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis. Homodimer and only the dimer is catalytically active, as the active sites are constructed of residues from both monomers

BBa_K3993001

Usage and Biology

This protein is involved in step 1 of the subpathway that synthesizes putrescine from agmatine. This subpathway is part of the pathway putrescine biosynthesis via agmatine pathway, which is itself part of Amine and polyamine biosynthesis. The expression of AUH activity is antagonistically regulated by cyclic AMP and agmatine. In the presence of the cAMP receptor protein, cAMP represses the expression of AUH, while agmatine induces it.

BBa_K3993002

Usage and Biology

This protein is involved in step 1 of the subpathway that synthesizes agmatine from L-arginine. This subpathway is part of the pathway agmatine biosynthesis, which is itself part of Amine and polyamine biosynthesis.

BBa_K3993003

Usage and Biology

Yeast CEN/ARS vector (Leu2) that contains multiple cloning site ( MCS ) and TDH3 promoter.

BBa_K3993004

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems, and can be used for modulating gene expression in yeast.

BBa_K3993005

Usage and Biology

Selection for in-frame fusion expression constructs.

BBa_K3993006

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems, and can be used for modulating gene expression in yeast.

BBa_K3993007

Usage and Biology

TEF1 gene have a strong promoter activity, and it has been shown to be constitutively expressed even in the presence of glucose. The TEF1 promoter could be used for production of homologous or heterologous proteins under conditions where the expression of a large number of genes involved in the use of less favoured carbon sources are repressed.

BBa_K3993008

Usage and Biology

TEF1 terminator is a common terminator in yeast.

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 3. Gel electrophoresis of amplified fragments..

Lane 1 is promoter PPGK1, Lane 2 is terminator TADH1, and Lane 3 is promoter PTEF1, Lane 4 is target gene SPE1, Lane 5 is terminator TTEF1. Lane 6 is promoter PTDH3, Lane 7 is the target gene AsADC, Lane 8 is the target gene speB.

2. overlap PCR to assembly the promoter-gene-terminator fragments

Figure 4. Gel electrophoresis of amplified fragments..

Lane 1 : promoter PPGK1 + the target gene AsADC , named seg1; Lane 2 : terminator TADH1 + promoter PTEF1, named seg2; Lane 3 : target gene SPE1 + terminator TTEF1. named seg3; Lane 4 : promoter PTDH3 + target gene speB , named seg4;

To do the 3rd round of overlap-PCR using the fragments recycled in figure4 as templates.

Figure 5. Gel electrophoresis of amplified fragments..

Lane 1 : seg1 + seg2 , named frag1; Lane 2 : seg3 + seg4 , named frag2;

To increase the yield of frag2, we performed the 4th round overlap-PCR.

Figure 6. Gel electrophoresis of amplified fragments..

Lane 1 to 3 is frag2; Lane 4 to 6 is terminator TCYC1; Lane 7 to 9 is the backbone plasmid pYES2.

To make up the long fragments, we performed the 5th round overlap-PCR.

Figure 7. Gel electrophoresis of amplified fragments..

Lane 1, 3, 5 : frag2 + terminator TCYC1; Lane 2, 4, 6 : seg4 + terminator TCYC1,named half1. This figure shows that the overlap PCR of seg4 and terminator TCYC1 was successful, while frag2 + terminator TCYC1 was not successful. So the 6th round of overlap-PCR was performed to overlap frag2+half1.

Figure 8. Gel electrophoresis of amplified fragments..

Lane 1 : frag2 + half1; In order to obtain our target plasmid, there are multi-fragment assembly plan A and B. In plan A, PCR amplification products of frag1, frag2, half1 and the backbone plasmid pYES2 were recovered from gel and the corresponding recombinant plasmids were transformed into competent cells for resistance screening of kana. In plan B, the sequence fragments were frag1, frag2, terminator TCYC1 and the backbone plasmid pYES2.

Figure 9.Verification of the plasmids via colony PCR..

Five single colonies in plan A and one single colony in plan B were picked up for further cultivate and plasmid extraction. Identification by electrophoresis showed that plasmid 1,4,5 of plan A and 6 of plan B were the candidate with correct size, in which plasmid 5 of plan A was confirmed by sequencing (Figure 9).

3. sequence information of the final plasmids

Figure 10: Blast DNA sequences with theoretical sequences and actual sanger sequencing documents of pYES2-ASADC-SPE1-SpeB..

The blast result shows that the plasmid is constructed successfully.

Proof of function

1. Metabolite production tests

Figure 11. The peak of putrescine detected by LC-MS..

Plasmids pYES2-AsADC-SPE1-SpeB were transferred to BY4741 chemically competent yeast cells and screened on YPD/Hyg plates. Transformants were picked into YPD/Hyg medium for activation. Metabolite production tests were carried out in YNB-SC fermentation medium (containing 10 times arginine raw material). 48h metabolites (supernatant) were collected and the putrescine analyze metabolites by LC-MS. The results showed that at 32 min, the peak pattern of putrescine appeared (as shown in the figure 3), indicating that the engineered strain we constructed successfully produced putrescine.

2. Modeling for predicting the performance of our engineered bacteria to produce tropine

Firstly, we get the polynomial linear regression-2 shown in figure 12. (Data from the published articles, according to references3/4 )

Figure 12..

The R-squared reaches 0.9855, which can be used to predict the performance of our engineered bacteria to produce tropine. Substituting the time and the OD600 value we tested in the laboratory into the model to get figures 13.

Figure 13..

The results show that when the OD600 of our engineered bacteria reaches a certain value, the output of tropine will increase sharply, indicating that our engineered bacteria have great industrial application prospects.