Parts

Characterization data to an existing Part from the Registry of Standard Biological Parts

BBa_J63005

Comparison of the ADH1 and TEF promoters activity

Background

In order to analyze the activity of ADH1 and TEF promoters [1] and their usefulness to express sugar transporters from Yarrowia lipolytica, S. cerevisiae EBY.VW4000 (strain deleted for all hexose transporters, kindly provided by Prof. Eckhard Boles[2]) was used as a host organism. The genes encoding sugar transporters (YALI0C06424g, YALI0C08943g, YALI0F19184g) were cloned into two types of pRS426 plasmid: with S. cerevisiae ADH1 promoter or TEF promoter. The replicative plasmids were cloned into S. cerevisiae.

The transformants were cultivated in YNB medium with maltose as carbon source. After 48 h of culture, cells were centrifuged, washed twice with distilled water and its OD600 was standardized to 10. Several decimal dilutions were prepared: 100, 10^-1, 10^-2, 10^-3, 10^-4, 10^-5. After the dilutions, cells were spoted in drop test on YNB agar plated with 2% glucose.

Results

^{Figure 1. Drop test analysis of S. cerevisiae EBY.VW4000 transformed with pRS426 plasmid containing YALI0C06424g, YALI08943g and YALI0F19184g hexose transporters from Yarrowia lipolytica under the control of ADH1 (pA) or TEF (pT) promoter.}

The analysis of promoter strength and its usefulness for expression of hexose transporters from Yarrowia lipolytica in S. cerevisiae EBY.VW4000 was analyzed. The 2µ plasmid (pRS426) was used with ADH1 or TEF promoter. After incubation of S. cerevisiae transformants for 48 h at 30°C growth of transformants expressing each of the three transporters under TEF promoter was observed. In turn, expression of only YALI0C06424g transporter was observed when ADH1 promoter was used. The results clearly indicated that AHD1 promoter is not strong enough and thus not appropriate for the analysis of heterologous hexose transporters in the S. cerevisiae EBY.VW4000.

References

[1] Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955–964. doi:10.1002/yea.1806

[2] Wieczorke, R., Krampe, S., Weierstall, T., Freidel, K., Hollenberg, C. P., & Boles, E. (1999). Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Letters, 464(3), 123–128. https://doi.org/10.1016/S0014-5793(99)01698-1

New BioBrick Part - Metallothionein-IV promoter Uniprot Q9HFC9-1

BBa_K3138000

Characterization

The promoter region was used to induce lycopene biosynthesis by Yarrowia lipolytica after exposure of the cells to heavy metals (Cd, Cu, Pb) in the growth medium

Verification of metallothionein response to heavy metals using real-time PCR

The gene responding to heavy metals was identified using real-time PCR followed by its promoter region sequence being used for further analysis. The cells were grown in Erlenmeyer flasks containing 50 ml of YPD medium. To identify genes reposnding to heavy metals three different ions in different concentrations were used: Cd (10 mM) Cu (28 mM), Pb (50 mM). The control medium in this experiment was YPD medium without any heavy metals. The concentration of metal ions were taken from the available literature [3]. Actine gene was used as a reference gene for qRT-PCR experiment. Five different genes were analyzed: YALI0A02416g, YALI0E08387g, YALI0F11275g, YALI0C18481g, YALI0C22394g.

The RNA was extracted from the cells harvested at 72h of the culture.

^{Figure 2. Relative expression of Y. lipolytica genes after exposure of cells to heavy metals (Cu, Cd, Pb). Click here to see the full version}

Results

BBa_K3138000 part shoved the highest expression level after exposure to Cd, Cu, Pb ions. The expression was 20-35 times higher comparing to the control experiment.

Lycopene biosynthesis experiment

The JMP62 series plasmid for Y. lipolytica was used as a basic vector for lycopene biosynthesis. Four genes from the lycopene biosynthesis pathway: phytoene synthase, phytoene dehydrogenase, isopentenyl-diphosphate isomerase, geranylgeranyl diphosphate synthase were assembled as one expression cassette, with the expression of each of the genes being controlled by the promoter of YALI0C18481 gene. The expression cassette was inserted into the genome of Y. lipolytica A-101 strain. The transformants of Y. lipolytica were cultured in YPG medium with and without (control) metal ions (Cd, Cu, Pb). The results are shown on Fig. 3.

^{Figure 3. The biomass of Y. lipolytica transformant with lycopene biosynthesis pathway cloned under the control of pYALI0C18481. A) Control, B) Cu, C) Cd, D) Pb.}

Results

The Y. lipolytica transformants with lycopene biosynthesis pathway controlled by pYALI0C18481 promoter proved the concept, that biosynthesis of this carotenoid may be induced by the presence of heavy metals in the growth medium (Fig. 3 B,C,D). In the control experiment, cells did not show any color after the culture (Fig. 3A).

Source

This part was isolated from chromosome C on Yarrowia lipolityca E150 genome.

References