Difference between revisions of "Part:BBa K2117001"

Latest revision as of 16:05, 21 October 2019

hrGFP optimized for Y. lipolytica

This part encodes the human proinsulin peptide codon-optimized for the yeast Yarrowia lipolytica for recombinant expression.

Usage and Biology

Characterization by Evry Paris-Saclay 2019

In the context of iGEM 2019 competition, we developed a metabolic engineering projet for the production of medically-relevant Conjugated Linolenic Acids (CLnAs) and used as a chassis the oleaginous yeast Yarrowia lipolytica, an organism whose metabolism is naturally poised for lipid production. For the expression of our enzymes, we choose the pTef1 promoter, a constitutive promoter native for Y. lipolytica. It is a strong promoter that controls the expression of the translation elongation factor-1 alpha [1], a protein that is one of the most expressed in most cells (between 3-10% of the soluble proteins [2]).

Our bibliographic research led us to designing and characterisation of several versions of the pTef1 promoter (Figure 1 and Table 1).

We started our research from iGEM’s part registry and we quickly found in the database the pTef1 promoter (BBa_K2117000), that we’ll refer to as pTef1a.

This promoter has been used to drive the expression of this part (BBa_K2117001) in the composite part BBa_K2117005 which was built by standard 3A assembly which leaves TACTAG (BBa_G0000) as a scar between the promoter and the ATG of the downstream gene. We will refer to BBa_K2117000+scar as pTef1b.

pTef1 (BBa_K2117000) 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 Y. lipolytica. To circumvent this incompatibility with the RFC[10] 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 Y. 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 [3,4]: 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.

Figure 1. Sequence comparisons of the six pTef1 promoter variants. The alignment was generated using the MUSCLE algorithm implemented in SnapGene.

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: either hrGFP (this part BBa_K2117001) or RedStar2 (BBa_K2983060).

It's in this context that we characterized the expression of this part driven by different variants of the pTef1 promoter.

To allow its expression in Y. lipolytica, we placed hrGFP (this part, BBa_K2117001) under the control of pTef1c (BBa_K2983050) and of pTef1e (BBa_K2983052) and of the Lip2 terminator (BBa_K2983055). The resulting transcriptional units (BBa_K2983072 and BBa_K2983073) were assembled into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to Y. lipolytica. Thus we generated BBa_K2983172 and BBa_K2983173 able to integrate upon transformation, into an 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 was subject to site directed mutagenesis to restore the BsaI site and thus generate BBa_K2983171.

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) which is part of our Loop assembly system dedicated to Y. lipolytica. Thus we generated BBa_K2983170 able to integrate upon transformation, into an Y. lipolytica Po1d stain.

All these parts are summarized in Table 1.

For this part characterization we decided to use the auxotrophic wild-type Y. lipolytica strain JMY195 [5], but also JMY2033 [6]. JMY195 is a Po1d strain, thus, by the means of the Zeta sequences [7], 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 hrGFP expression plasmids (BBa_K2983170, BBa_K2983171, BBa_K2983172 and BBa_K2983173). As a negative control, we also transformed them with the NotI digested empty YL-pOdd1 vector (BBa_K2983030). 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 fluorescence (λexcitation 483 nm and λemission 530 nm) and OD600nm 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 [8]. We measure the turbidity of the culture at 600 nm and the fluorescence of hrGFP (BBa_K2117001), and determine the mean rate of fluorescence/OD600nm (SFU/h) increase during the exponential growth phase. This method allows quantifying hrGFP (BBa_K2117001) expression in a manner independent of the length of the lag phase.

Using the calibration curves presented in Figures 2 and 3 (and as an excel file too), we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle (Figure 4).

Figure 2. Particle standard curve.

Figure 3. Fluorescein standard curve.

The results of this part characterization are presented in Figure 4.

As can be easily observed, the specific fluorescence values are similar between the different variants of pTef1 in both Y. lipolytica strains. The different modifications of the pTef1 promoter sequence highlighted in Figure 1 do not have a drastic impact on its activity.

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.

Figure 4. In vivo characterisation of hrGFP expression driven by different pTef1 promoter variants in two Y. lipolytica strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates)

Thus, we that this part, hrGFP, the humanized form of Renilla reniformis GFP [9,10] is a reporter fluorescent protein well adapted for Y. lipolytica.

References

[1] Müller S, Sandal T, Kamp-Hansen P, Dalbøge H. Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast (1998) 14, 1267-1283.

[2] Cavallius J, Zoll W, Chakraburtty K, Merrick WC. Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications. Biochim Biophys Acta (1993) 1163, 75-80.

[3] Celińska E, Ledesma-Amaro R, Larroude M, Rossignol T, Pauthenier C, Nicaud JM. Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica. Microb Biotechnol (2017) 10, 450-455.

[4] Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T. A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology. Microb Biotechnol (2019) 12, 1249– 1259.

[5] Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology. Springer, Berlin (1996) 1, 314-388.

[6] Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M. Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications. J Ind Microbiol Biotechnol (2013) 40, 1273-1283.

[7] Pignède G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud JM. Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol (2000) 66, 3283-3289.

[8] Park YK, Korpys P, Kubiak M, Celinska E, Soudier P, Trébulle P, Larroude M, Rossignol T, Nicaud JM. Engineering the architecture of erythritol-inducible promoters for regulated and enhanced gene expression in Yarrowia lipolytica. FEMS Yeast Res (2019) 19, foy105.

[9] Ward WW, Cormier MJ. An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J Biol Chem (1979) 254, 781-788.

[10] Felts K, Rogers B, Chen K, Ji H, Sorge J, Vaillancourt P. Recombinant Renilla reniformis GFP displays low toxicity. Strategies newsletter (2000) 13, 85–87.

Sequence and Features