Difference between revisions of "Part:BBa K1907001"

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−	-	+	<p><b>Introduction</b></p><p>
−	-	+	This part can be used to introduce microcystinase (MlrA) from <i>Sphingomonas</i> sp. ACM-3962 to yeast. MlrA is an enzyme degrades some cyanobacterial toxins, most notably microcystin-LR (MC-LR). To facilitate expression detection and purification of the enzyme, a 6X Histidine tag is fused on the C-terminus of MlrA, with a short linker in between. The full protein-coding sequence has been codon-optimized for expression in <i>Saccharomyces cerevisiae </i> by GeneArt.
−	-	+
−	-	+	<!-- -->
−	-	+	<span class='h3bb'><b>Sequence and Features</b></span>
−	-	+	<partinfo>BBa_K1907001 SequenceAndFeatures</partinfo>
−	-	+
−	-	+
−	-	+	<p><b>Background</b></p><p>
		+	Microcystinase, or MlrA, is a member of a set of enzymes that degrade microcystins. These enzymes - MlrA, MlrB, MlrC and MlrD - are found naturally in some gram-negative bacteria, for example <i>Sphingomonas</i> sp. They each have a different function in the overall process of microcystin degradation. MlrA is first in the sequence to act. It hydrolyzes the peptide bond between the Adda and arginine amino acids in microcystin-LR (MC-LR). The breakage of this bond linearizes the toxin (Figure 1). Following this, MlrB (a serine protease) and MlrC (a metalloprotease) are able to further degrade the linearized product into individual amino acids, which are then used in the cell’s metabolism. Therefore, the toxin is likely used as a carbon source. MlrD’s function is not fully understood at this point, but it may have a role in importing the toxins. (Bourne et al., 2001)
		+
		+	<p>
		+	Figure 1: Linearization of microcystin-LR occurs when MlrA hydrolyzes the bond between two of the toxin’s amino acids, Adda and arginine. It has been reported that the linearization of MC-LR renders the toxin 160 times less toxic (Bourne et al., 1996).
		+	</p>
		+
		+	<p>
		+	Based on a homologous structure model (Figure 2) obtained from the Phyre2 tool (Kelley et al., 2015), it seems that the enzyme is a transmembrane protein consisting of eight hydrophobic α-helices that form a channel, with the presumed active site (Bourne et al., 2012) residing in the middle. It is worth pointing out, however, that the model does not cover the whole length of the MlrA sequence, as only very few homologs were found for the sequence - the homology model is mostly based on two homologs that didn’t cover the very ends of either terminus. Other than this evidence, there are previous studies pointing to the direction of MlrA being a membrane protein (Pei et al., 2011). However, when the enzyme was heterologously produced in <i> E. coli </i>, it was noticed that it had localised mostly in the soluble fraction of the cytosol, in an active state (Dziga et al., 2012).
		+	</p>
		+
		+	<p>
		+	Figure 2: A homology-based structure model of MlrA made with Phyre2 and visualised with UCSF Chimera. Based on the model, it seems that MlrA has a typical membrane protein structure consisting of set of hydrophobic transmembrane helices.. The presumed active site (Bourne et al., 2001) resides in the middle of a channel formed by the helices.
		+	</p>
		+
		+	<p>The mechanism of MlrA’s function is not yet fully understood. The homologous structure model gives some suggestions as to how the enzyme might work: it seems that one end of the channel is wider than the other, and that the active site resides in the middle of it. Consequently, if the enzyme is located on the outer membrane of the cells, it might actually be a transporter which the cells use to import the toxin and simultaneously linearize it.
		+	</p>
		+
		+	<p><b>Expression</p></b>
		+	20 mL SD-leu + 2% glucose cultures of <i> S. cerevisiae </i> SS328-leu containing MlrAY, MlrAYa or MlrAY3a were grown overnight at 30 °C. The culture was diluted to 500 mL with OD600 value of 0.2, and refreshed in SD-leu + 0.5% glucose for 6 h. Culture was induced by adding galactose to a final amount of 2%  of the total volume and grown overnight. Culture was divided into 50 mL volumes and centrifuged, after which the pellets were stored at -20 °C.
		+
		+	<p>
		+	Figure 10a: Western blot of MlrA expression in yeast. S1 - the growth medium, CL - cell lysate, S2 - supernatant from CL and P2 - pellet from CL.
		+	</p>
		+
		+	<p>
		+	Figure 10b. S1 - growth medium, S2 - supernatant after cell lysis, P2 - pellet after cell lysis, S3 - re-folded proteins, P3 - plasma membrane + cell wall, S4 - soluble protein, P4 - inner membranes.
		+	</p>
		+
		+	<p><b>Activity measurements</p></b>
		+
		+	<p>
		+	The activity of MlrA was measured by incubating the enzyme in different dilutions with microcystin extract. The final concentration of microcystin-LR was estimated to be 1 μg/L. Samples were taken at different time points and they were analysed with liquid chromatography/mass spectrometry (LC-MS). This gives data of the relative abundance of microcystin-LR in each sample. The results were then plotted against time, together with a negative control which consisted of microcystin extract and cell lysate from <i> S. cerevisiae </i> SS328-leu strain which did not have an MlrA construct.
		+	</p>
		+
		+	<p>
		+	The results we acquired from this construct show that the amount of microcystin decreases, which means that the enzyme is active (Figure 13). The results also show that for example the 1:10 dilution degrades the microcystin-LR about ten times faster than the 1:100 dilution.
		+	</p>
		+
		+	<p>
		+	Figure 13: Enzymatic activity of MlrA produced in <i>S. cerevisiae </i>. Results show that the 1:10 dilution has the highest activity whereas 1:1000 has the lowest.
		+	</p>
		+	</p>
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here

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Revision as of 18:45, 19 October 2016

Microcystinase (MlrA) for S. cerevisiae

Introduction

This part can be used to introduce microcystinase (MlrA) from Sphingomonas sp. ACM-3962 to yeast. MlrA is an enzyme degrades some cyanobacterial toxins, most notably microcystin-LR (MC-LR). To facilitate expression detection and purification of the enzyme, a 6X Histidine tag is fused on the C-terminus of MlrA, with a short linker in between. The full protein-coding sequence has been codon-optimized for expression in Saccharomyces cerevisiae by GeneArt.

Sequence and Features

<p>Background

Microcystinase, or MlrA, is a member of a set of enzymes that degrade microcystins. These enzymes - MlrA, MlrB, MlrC and MlrD - are found naturally in some gram-negative bacteria, for example Sphingomonas sp. They each have a different function in the overall process of microcystin degradation. MlrA is first in the sequence to act. It hydrolyzes the peptide bond between the Adda and arginine amino acids in microcystin-LR (MC-LR). The breakage of this bond linearizes the toxin (Figure 1). Following this, MlrB (a serine protease) and MlrC (a metalloprotease) are able to further degrade the linearized product into individual amino acids, which are then used in the cell’s metabolism. Therefore, the toxin is likely used as a carbon source. MlrD’s function is not fully understood at this point, but it may have a role in importing the toxins. (Bourne et al., 2001)

<p> Figure 1: Linearization of microcystin-LR occurs when MlrA hydrolyzes the bond between two of the toxin’s amino acids, Adda and arginine. It has been reported that the linearization of MC-LR renders the toxin 160 times less toxic (Bourne et al., 1996).

Based on a homologous structure model (Figure 2) obtained from the Phyre2 tool (Kelley et al., 2015), it seems that the enzyme is a transmembrane protein consisting of eight hydrophobic α-helices that form a channel, with the presumed active site (Bourne et al., 2012) residing in the middle. It is worth pointing out, however, that the model does not cover the whole length of the MlrA sequence, as only very few homologs were found for the sequence - the homology model is mostly based on two homologs that didn’t cover the very ends of either terminus. Other than this evidence, there are previous studies pointing to the direction of MlrA being a membrane protein (Pei et al., 2011). However, when the enzyme was heterologously produced in E. coli , it was noticed that it had localised mostly in the soluble fraction of the cytosol, in an active state (Dziga et al., 2012).

Figure 2: A homology-based structure model of MlrA made with Phyre2 and visualised with UCSF Chimera. Based on the model, it seems that MlrA has a typical membrane protein structure consisting of set of hydrophobic transmembrane helices.. The presumed active site (Bourne et al., 2001) resides in the middle of a channel formed by the helices.

The mechanism of MlrA’s function is not yet fully understood. The homologous structure model gives some suggestions as to how the enzyme might work: it seems that one end of the channel is wider than the other, and that the active site resides in the middle of it. Consequently, if the enzyme is located on the outer membrane of the cells, it might actually be a transporter which the cells use to import the toxin and simultaneously linearize it.

Expression</p> 20 mL SD-leu + 2% glucose cultures of S. cerevisiae SS328-leu containing MlrAY, MlrAYa or MlrAY3a were grown overnight at 30 °C. The culture was diluted to 500 mL with OD600 value of 0.2, and refreshed in SD-leu + 0.5% glucose for 6 h. Culture was induced by adding galactose to a final amount of 2% of the total volume and grown overnight. Culture was divided into 50 mL volumes and centrifuged, after which the pellets were stored at -20 °C. <p> Figure 10a: Western blot of MlrA expression in yeast. S1 - the growth medium, CL - cell lysate, S2 - supernatant from CL and P2 - pellet from CL.

Figure 10b. S1 - growth medium, S2 - supernatant after cell lysis, P2 - pellet after cell lysis, S3 - re-folded proteins, P3 - plasma membrane + cell wall, S4 - soluble protein, P4 - inner membranes.

Activity measurements</p> <p> The activity of MlrA was measured by incubating the enzyme in different dilutions with microcystin extract. The final concentration of microcystin-LR was estimated to be 1 μg/L. Samples were taken at different time points and they were analysed with liquid chromatography/mass spectrometry (LC-MS). This gives data of the relative abundance of microcystin-LR in each sample. The results were then plotted against time, together with a negative control which consisted of microcystin extract and cell lysate from S. cerevisiae SS328-leu strain which did not have an MlrA construct.

The results we acquired from this construct show that the amount of microcystin decreases, which means that the enzyme is active (Figure 13). The results also show that for example the 1:10 dilution degrades the microcystin-LR about ten times faster than the 1:100 dilution.

Figure 13: Enzymatic activity of MlrA produced in S. cerevisiae . Results show that the 1:10 dilution has the highest activity whereas 1:1000 has the lowest.

</p>

Sequence and Features