Improving promoters

Getting inspired by and improving the work of iGEM teams from previous years is a central aspect of the iGEM competition.

The objective of our project is to produce medically-relevant Conjugated Linolenic Acids (CLnAs), which is a class of rare fatty acids with three conjugated bonds. In this project, we developed a launchpad for their bioproduction using the oleaginous yeast Yarrowia lipolytica, a powerful chassis organism, whose metabolism is naturally poised for lipid production. CLnAs are synthetized by bifunctional fatty acid conjugase / desaturase (FadX) enzymes from linoleic acid, a natural metabolite for our chassis. Thus, to convert it into a CLnA, only the presence of a FadX enzyme is necessary.

To drive the expression of FadX in Yarrowia lipolytica, the choice of the promoter is an important step: constitutive or inducible ? weak, medium or strong ? Several natural and synthetic promoters were characterized for Yarrowia lipolytica that allow tunable heterologous gene expression in this chassis (for a review see [1]). As producing a CLnA may lead to perturbations of vital cellular lipids (like the membrane glycerophospholipids) and thus reduce cell viability, we decided not to use an extremely strong promoter (like hp4d for instance [2]), nor a medium one (to put all odds on our side).

We started our research from iGEM’s part registry and we quickly found in the database the pTef1 promoter (BBa_K2117000), a constitutive promoter native for the oleaginous yeast Y. lipolytica. It is a strong promoter that controls the expression of the translation elongation factor-1 alpha [3], a protein that is one of the most expressed in most cells (between 3-10% of the soluble proteins [4]). We will refer to BBa_K2117000 as pTef1a.

This promoter seemed suitable for our project, but had a major disadvantage: the presence of a BsaI site that makes it incompatible with the Type IIS RFC[1000]-compatible Loop assembly system that we designed for Yarrowia lipolytica. To circumvent this incompatibility with the RFC[1000] standard, we mutated the BsaI site (GGTCTC) to GGTCTg and thus created a new compatible part, BBa_K2983050, that we’ll refer to as pTef1c.

A quick sequence analysis of BBa_K2117000 revealed several differences compared to wild-type pTef1 promoter (nucleotides 1227374 to 1226969) of Yarrowia lipolytica W29 chromosome C (GenBank Acc. n° CP028450.1). Three of the four mutations were introduced by the iGEM16_DTU-Denmark in order to remove two illegal restriction sites for BioBrick RFC[10]-compatibility (SpeI, PstI). As these sites are accepted in the Type IIS RFC[1000] standard, we created a closer to wild-type version of pTef1 promoter, BBa_K2983051, that has also a mutated BsaI site (GGTCTC to GGTCTg) which makes this part compatible with the iGEM Type IIS RFC[1000] standard. We will refer to BBa_K2983051 as pTef1d.

Continuing our research, we discovered another version of the pTef1 promoter, which is shorter and Type IIS RFC[1000] compatible [5,6]: BBa_K2983052, that we’ll refer to as pTef1e.

Unpublished observations of our PI, Jean-Marc Nicaud, suggest that the presence of a 4 nucleotide sequence CACA just upstream the ATG of the gene may lead to increased gene expression. Thus, we added BBa_K2983053 to the list of pTef1 variants to test. We will refer to BBa_K2983053 as pTef1f.

A sequence comparison of all pTef1 variants is presented in figure 1. Apart from the above mentioned promoters, we included BBa_K2117000+scar, as this promoter was used to build BBa_K2117005 by standard 3A assembly which leaves TACTAG as a scar between the promoter and the ATG of the downstream gene. We will refer to BBa_K2117000+scar as pTef1b.

One of the main questions related to the modification of a promoter sequence is related to the impact it may have on its activity. To estimate if the pTef1 promoter activity is impaired by the modifications highlighted in Figure 1, we used a fluorescent reporter gene. For this, we decided to use RedStar2, as it is the “brightest and most yeast-optimized version of the red fluorescent protein” [7], but also hrGFP, the humanized form of Renilla reniformis GFP [8, 9] (BBa_K2117001).

We placed both RedStar2 and hrGFP under the control of a pTef1 promoter variant and of the Lip2 terminator (BBa_K2983055). The resulting transcriptional units (BBa_K2983072, BBa_K2983073, BBa_K2983075, BBa_K2983076, BBa_K2983077, BBa_K2983078) were assembled into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to our chassis, the oleaginous yeast Y. lipolytica (for further details on this system, visit the dedicated page on this wiki ). Thus, we generated four RedStar2 expression plasmids (BBa_K2983175, BBa_K2983176, BBa_K2983177, BBa_K2983178) and two hrGFP expression plasmids (BBa_K2983172BBa_K2983173) able to integrate upon transformation, into a Y. lipolytica Po1d stain.

To be able to make comparisons with the expression driven by the pTef1 variant already in the registry (pTef1a, BBa_K2117000), the BBa_K2983172 and BBa_K2983175 were subject to site directed mutagenesis to restore the BsaI site and thus generate BBa_K2983171 and BBa_K2983174, respectively.

In parallel, we equipped the hrGFP expression device already present in the iGEM Registry (BBa_K2117005) with the Lip2 terminator (BBa_K2983055) and assembled the resulting transcriptional unit BBa_K2983070 into our YL-pOdd1 plasmid (BBa_K2983030) and thus we generated BBa_K2983170 able to integrate upon transformation, into an Y. lipolytica Po1d stain.

Experimental setup

For pTef1 promoter characterization we decided to use the auxotrophic wild-type Y. lipolytica strain JMY195 [10], but also JMY2033 [11]. JMY195 is a Po1d strain, thus, by the means of the Zeta sequences [12], the Y. lipolytica genome integration cassettes (table 1) will be inserted randomly. JMY2033 is a derivative of JMY195 that contains a zeta docking platform at the ura3-302 locus. In this strain, the insertion is not random, but site specific which limits the risks of multiple insertion of plasmidic constructions in the genome.

These two Y. lipolytica strains were transformed with the NotI digested RedStar2 and hrGFP expression plasmids (BBa_K2983170, BBa_K2983171, BBa_K2983172, BBa_K2983173, BBa_K2983174, BBa_K2983175, BBa_K2983176, BBa_K2983177, BBa_K2983178). As a negative control, we also transformed them with the NotI digested empty YL-pOdd1 vector (BBa_K2983030). As positive control we used the JMY7621 stain [13] which contains a single genome copy of a RedStar2 expression cassette.

For fluorescence measurements, yeast cells were first grown overnight in rich YPD medium then diluted by 100x in YNB-glucose medium (containing 1.7 g/L yeast nitrogen base with amino acids and ammonium sulfate, 1.5 g/L NH₄Cl, 50 mM KH₂PO₄-Na₂HPO₄ buffer pH 6.8, 10 g/L glucose and 0.1 g/L leucine) in an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). The plate was incubated at 28°C at 200 rpm and the RedStar2 fluorescence (λ_excitation 558 nm and λ_emission 586 nm) and OD_600nm were measured every 15 min during 300 cycles in a SynergyMx (BioTek) plate reader. The hrGFP fluorescence (λ_excitation 483 nm and λ_emission 530 nm) and OD_600nm were measured every 10 min during 500 cycles in a CLARIOstar (BMGLabtech) plate reader.

To compare the expression between each promoter, we rely on specific fluorescence [13]. We measure the turbidity of the culture at 600 nm and the fluorescence of RedStar2 or hrGFP, and determine the mean rate of fluorescence/OD_600nm (SFU/h) increase during the exponential growth phase. This method allows quantifying fluorescent protein expression in a manner independent of the length of the lag phase. Using the calibration curves presented on the dedicated page of this wiki, we converted the arbitrary units into Molecules of Equivalent Resorufin (MEResorufin) / particle, Molecules of Equivalent Rhodamine B (MERhB) / particle or Molecules of Equivalent FLuorescein (MEFL) / particle.

Results

The results of the different pTef1 promoter strength quantifications are presented in Figures 2, 3, 4 and 5 using different units of measure.

For nearly each construct, we obtained and analysed twice at least 3 clones (and up to 32), the majority of each displaying comparable specific fluorescence values similar to the value obtained for the positive control. However, a minority of clones were constantly displaying twice more SFU/h suggesting they have integrated 2 copies of the RedStar2 expression cassette.

Indeed, the difficulty in using insertion of plasmid construction in the genomic approaches is to estimate and control the insertion site and the number of copies inserted into the genome. In the auxotrophic wild-type Y. lipolytica strain JMY195, our expression cassettes integrate randomly and the protein expression may vary depending on the insertion site. In the JMY2033 strain, the insertion occurs mainly at the zeta docking platform at the ura3-302 locus.

As can be easily observed from the results presented in Figures 2, 3, 4 and 5, the different modifications of the pTef1 promoter sequence highlighted in Figure 1 do not have a drastic impact on its activity. The specific fluorescence values are similar between the different variants of pTef1 in both Y. lipolytica strains and independent of copy number / genome.

To confirm these observations, we performed a two-tailed student test with an error threshold of 5% in order to compare the specific fluorescence averages in each strain and each construct. No statistically significant difference was observed between the different constructs in both strains and at both copy number / genome.