Team:SHSID/Engineering
Engineering
Background
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.
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 our project, natural
genes involved in arginine metabolism and polyamine biosynthesis was designed to overexpress in yeast. The
engineer strains that produced excess putrescine. The following genes were used to improve putrescine
production:
(1) SPE1 gene: Overexpression of the SPE1 gene can increase the expression of Spe1p
which converts ornithine to putrescine.
(2) AsADC and speB gene: Arginine is decarboxylated by arginine decarboxylase (ADC)
to agmatine (Agmatine), agmatine hydrolase ( AUH) can remove the guanidine group from agmatine and directly
produce putrescine. In this study, ADC from oats (AsADC) and AUH from E. coli (speB) were selected, both of
which have been shown to be active in Saccharomyces cerevisiae.
Build
The three genes SPE1, speB, and AsADC were inserted into yeast expression vector
pYES2. different promoters and terminators were applied for the transcription of the three genes,
respectively (Figure 1).
Figure 2. DNA sequence map of plasmid pYES2-AsADC-SPE1-SpeB.
Test
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.
Figure 3. The peak of putrescine detected 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.
Learn
The three genes that can improve putrescine production were successfully introduced
into the yeast strain. The yeast strain generates putrescine during fermentation experiment. What is
unsatisfactory is the peak area in LC-MS is small. The yield of putrescine is far below the factory mass
production level. It is likely that the protein expression is insufficient and the metabolic pathway is
small. In the future, the expression of the three proteins needs to be further optimized.