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Heterologous Expression of Human SoLute Carrier in Yeast: History
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Contributor: Lorena Pochini , Michele Galluccio

For more than 20 years, yeast has been a widely used system for the expression of human membrane transporters. Among them, more than 400 are members of the largest transporter family, the SLC superfamily. SLCs play critical roles in maintaining cellular homeostasis by transporting nutrients, ions, and waste products. Based on their involvement in drug absorption and in several human diseases, they are considered emerging therapeutic targets.

  • yeast
  • expression
  • SLC transporter

1. Introduction

Transporters account for 10% of the human genome. They regulate the flux of different molecules such as sugars, amino acids, ions, lipids, vitamins, and catabolites through plasma and subcellular membranes [1]. In addition to their physiological roles, many transporters are involved in drug absorption, distribution, metabolism, and excretion (ADME) processes. Moreover, many drugs can interact with membrane transporters, leading to side effects [2]. Indeed, about 60% of FDA-approved drug targets are membrane proteins, including a growing number of transporters [3][4]. A well-established functional classification categorizes transport proteins in channels and permeases. Channels catalyze the transport of ions down their electrochemical gradient, with a turnover rate up to 108 s−1. Permeases are involved in the transport of many different molecules with a lower turnover rate. On the basis of the transport driving force, permeases can be subdivided in primary and secondary active transporters [5]. The first use ATP hydrolysis to drive the transport of molecules against their concentration gradient. The secondary active transporters retrieve the driving force from the concentration gradient of the transported substrates and are grouped into symporters, antiporters, and uniporters. The uniporters catalyze the substrate movement in a thermodynamically favorable direction; the symporters or antiporters catalyze co-transport of more molecules in the same or in the opposite direction, respectively [5][6]. Among the transporters, the SoLute Carrier (SLC) superfamily currently includes 458 proteins grouped into 65 families [7][8]. Member proteins within each family share at least 20–25% sequence similarity with at least one other member of the family [7][9]. The number of members within the SLC families is heterogeneous, ranging from only one (SLC32, SLC40, SLC48, SLC50, SLC53, SLC61, SLC62, and SLC64) to 53 members for the largest family: SLC25, the mitochondrial carrier family [7]. The members of this superfamily act as secondary active transporters [10]. Regarding the molecular mechanism of transport, the secondary active transporters use the “alternating access” transport mechanism in which the ligand binding site is available on only one side of the membrane at a time; then, as a consequence of the substrate interaction, a protein conformational change does occur and the substrate is transported to the other side of the membrane [11]. Three major types of alternating access mechanisms have been described for SLC transporters: the rocker-switch, the gated pore and the elevator mechanism [10]. Analyzing hydropathy plots, the SLC transporters are predicted to present between 1–16 transmembrane domains (TMD), although in more than 80% of the members the number of TMD is between 7 and 12 [12]. Despite their role in human pathophysiology and the efforts employed in fulfilling knowledge of them, about 30% of the SLC members are still “orphans”, with no structural and functional data available [7][13]. The reason for this gap in information lies in the difficulty in expressing, purifying, and assaying the function of this kind of proteins [14]. These difficulties arise from the hydrophobic nature of the TMD and, in some cases, from very large (about 100 amino acids) hydrophilic moieties which connect TMDs [15][16]. Different expression hosts, either prokaryotic or eukaryotic, have been adopted with success allowing structure/function studies on SLC transporters such as, E. coli [17], L. lactis [18], S. cerevisiae [19], P. pastoris [20], S. frugiperda [21], HEK293F [22]. However, less than 10% of the human SLCs structure has been solved [23]. Due to very low cultivation costs, fast growth, easy handiness, and absence of toxicity, Escherichia coli is the most used host for protein over-expression. In particular, BL21(DE3) and its derivative strains are the most commonly used for heterologous membrane protein production [23]. Even though widely exploited for protein over-expression, in the case of human membrane transporters, E. coli could be ineffective, leading to the inclusion of a bodies formation from which it is difficult to recover the target protein in an active folded form [24][25]. On the other side, expression in insect or mammalian cell systems is characterized by high cultivation costs, slow growth, and needs special equipment, often resulting in low yield. A good middle ground is represented by the yeast expression system [26] which combines the advantages of ease of manipulation, fast growth, low cost of production, eukaryotic protein processing [27] (which can facilitate the expression of correctly-folded eukaryotic proteins) [28], heterodimer formation, and prosthetic group association to the recombinant proteins [29]. Moreover, the localization of an expressed membrane transporter in the yeast membrane can facilitate its recovery in a folded state [30]. Indeed, in this milieu, the human membrane proteins can find a more similar phospholipid/sterol composition with respect to the bacterial hosts [31]; furthermore, the possibility of increasing culture volume to obtain large quantities of folded protein [31][32] can be exploited either for functional or structural studies [20][33][34]. Indeed, yeast and especially P. pastoris can reach extremely high biomass, thus obviating the need to achieve extremely high levels of overexpression that could result in the aggregation and denaturation of membrane proteins.

2. Yeasts as a System for Heterologous Expression of Human SLC Transporters

A similar cellular architecture with respect to higher eukaryotes, sharing most of the metabolic and cellular pathways, and its simplicity in molecular and genetic manipulations make yeast particularly suited to produce human proteins for structure-function studies [35][36][37]. Indeed, yeast can perform many eukaryote-specific post-translational modifications such as proteolytic digestion, disulfide bridge formation, and some type of glycosylation [38]. However, differently from other eukaryotes, no trimming of mannose residues occurs in yeast [39]. Several yeast strains have been developed in which glycan remodeling has been induced, specifically deleting genes coding for glycosyltransferases and expressing the genes that carry out mammalian-specific glycosylation [40][41][42][43][44]. Indeed, DNA can be introduced or removed into/from the yeast chromosomes, enabling the creation of unique protein expression strains [45][46][47]. This “genetic” approach is largely used in yeast by exploiting homologous recombination: yeast transformation triggers the recombination between yeast sequences carried on the plasmid and homologous sequences in the yeast genome. This approach can serve different aims (Figure 1).
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Figure 1. Uses of the homologous recombination in yeast.

2.1. The Homologous Recombination

The observation that linear DNA fragments can efficiently promote recombination in S. cerevisiae led to the development of several methods for DNA manipulation in yeast [48]. Transformation has been used to clone genes by genetic complementation [49], to clone functional chromosomal components such as origins of replication [50], centromeres [51], and telomeres [52], and to clone functional suppressors acting as dominant negative mutants [53]. Recombination-based DNA manipulation methods also include integrative DNA transformation, which is used to induce gene deletion [54], and allele rescue which involves the transplacement of a mutation from the chromosome onto a plasmid-derived copy of that gene (Table 1) [55]. Indeed, homologous recombination in Saccharomyces cerevisiae can be considered as an easy and highly efficient cloning alternative. Yeast recombination cloning allows in a single step the assembly of multiple DNA fragments, deriving for example from many PCR reactions [56] (Figure 1). It is extremely efficient, requiring only 29 nucleotides of overlapping sequences that can be added to the synthesized oligonucleotides. Starting from these observations, by exploiting homologous recombination a yeast cloning cassette was used to point out a versatile method called any-gene-any-plasmid (AGAP) allowing the cloning of any gene (or combination of DNA fragments) into any vector in a single step without the need for ligase or a molecular cloning kit [57]. Towards the aim of obtaining the expression of a membrane transporter different aspects must be taken into account to transform yeast with the gene of interest (Figure 2).
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Figure 2. Workflow of a SLC production in yeast for structural and functional studies.
Table 1. Yeast strains and genotypes.
Host Strain Genotype Feature References
P. pastoris KM71H aox1::ARG4, arg4 Strain with MutS phenotype [58]
P. pastoris SMD1168H pep4 Strain without protease A activity [59]
S. cerevisiae PAP1500 MATα ura3-52 trp1::GAL10-GAL4 lys2-801 leu2Δ1 his3Δ200pep4::HIS3prb1Δ1.6Rcan1GAL Overexpression of the Gal4 transcription factor [60]
S. cerevisiae AB11c ena1-4Δnhx1Δnha1Δ Deletion of endogenous cation/proton antiporters and pumps [61]
S. cerevisiae MSY6210 MAT α leu2-3,112 ura3-52 his3200 trp1-901lys2-801suc2-9 smf1::HIS3,smf2::KANR Deletion of Mg2+ transporters [62]
S. cerevisiae MSY6211 MAT a leu2-3,112 ura3-52 his3200 trp1-901 ade2-101 suc2-9 smf3::LEU2 Deletion of Mg2+ transporters [62]
P. pastoris GS115 his4 Deletion of histidinol dehydrogenase [63]
S. cerevisiae EBY.S7 MATα hxt1-17Δgal2Δagt1Δstl1Δleu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2–8c SUC2 hxtΔfgy1 Deletion of hexose transporters [64]
S. cerevisiae EBY.F4–1 MATα hxt1-17Δgal2Δagt1Δstl1Δleu2-3,112 ura3-52 trp1-289 his3-Δ1 MAL2–8c SUC2 hxtΔfgy1 fgy41 Deletion of hexose transporters [64]
S. cerevisiae EBY.VW4000 MATa leu2-3,112 ura3-52 trp1-289 his3-1 MAL2-8c SUC2 Δhxt1-17 Δgal2 Δstl1::loxP Δagt1::loxP Δmph2::loxP Δmph3::loxP Deletion of hexose transporters [65]
S. cerevisiae SDY.022 MATa leu2-3,112 ura3-52 trp1-289 his3-∆1 MAL2-8C SUC2 ∆hxt1-17 ∆gal2 ∆agt1 ∆stl1 fgy1-1 erg4::kanMX Deletion of hexose transporters [66]
S. cerevisiae BJ5457 MATα ura3-52 trp1 lys2-801 leu2-Δ1 his3-Δ200 pep4:HIS3 prb1-delta1.6R can1 GAL Protease deficient [67]
S. cerevisiae RE700A MATa hxt1::HIS3::hxt4 hxt5::LEU2 hxt2::HIS3hxt3::LEU2::hxt6 hxt7::HIS3 Deletion of hexose transporters [68]
S. cerevisiae BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 Minimize homologous recombination [59]
S. cerevisiae BY4742 GEV MATa, (PGAL10+gal1)Δ::loxP, leu2Δ0::PACT1-GEV-NatMX, gal4Δ::LEU2, HAP1+ Minimize homologous recombination [69]
P. pastoris SMD1168H pep4 Protease A deficiency [59]
S. cerevisiae FAB158 MATa his3- Δ200 leu2- Δ1 lys2-801 trp1- Δ1 ade2-101 ura3-52 tat2 Δ::HIS3 Deletion of tryptophan transporter [70]
S. cerevisiae TMY203 MATa his3- Δ200 leu2- Δ1 lys2-801 trp1- Δ1 ade2-101 ura3-52 tat1 Δ::kanMX4 tat2Δ::LEU2 Deletion of tryptophan transporters [70]
S. cerevisiae FAY18A MATa his3- Δ200 leu2- Δ1 lys2-801 trp1- Δ1 ade2-101 ura3-52 HPG1-1, Rsp5P514T Deletion of Rsp5 ubiquitin ligase [70]
S. cerevisiae XPY1263a MATa thi3Δ::natMX thi7D::kanMX Deletion of thiamine transporter [71]
S. cerevisiae BY4741mp MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; mir1Δ; pic2Δ Deletion of phosphate and copper transporter [72]
S. cerevisiae BY4741 pic2Δ MATa, leu2,met15, ura3, his3, PIC2::KANMX Deletion of copper transporter [73]
S. cerevisiae WB-12 MATα ade2-1 trp1-1 ura3-1 can1-100 aac1::LEU2 aac2::HIS3 Deletion of adenine nucleotide carriers [74]
S. cerevisiae JL1-3Δ2 Matα leu2-3,112 his3-11,15 ade2-1 trp1-1 ura3-1can1-100 anc1::LEU2 Δ anc2::HIS3 anc3::URA3 Deletion of adenine nucleotide carriers [75]
S. cerevisiae W303-B1 Mata; ade2-1; his3-11, -15; leu2-3, -112; ura3-1; canR; cyr+ Poor leucine uptake [76]
S. cerevisiae W303-1A MATa: ade2-2; trp1-1; can1-100; leu2-3, 112; his 3-11, 15; ura3-1 Poor leucine uptake [77]
S. cerevisiae W303 MATa/MATα (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) [phi+] Poor leucine uptake [78]
S. cerevisiae W303 Wagc1Δ Mat a/Mat a, ura3-1/ura3-1, trp1- Δ2/trp1- Δ2, leu2-3,112/leu2-3,112, his3-11/his3-11, ade2-1/ade2-1, can1-100/can1-100 Poor leucine uptake; deletion of AGC1 carrier [79]
S. cerevisiae PW001 BY4741 agc1∆::URA3 Deletion of AGC1 carrier [80]
S. cerevisiae PW002 BY4741 agc1∆::HIS3 Deletion of AGC1 carrier [80]
S. cerevisiae ΔArg11 Y02386 MATa; his3Δ1; leu2Δ0; met15Δ0; ura3D0; YOR130c::KanMX4 Deletion of ornithine transporter 1 [81]
S. cerevisiae ΔAnt1 BJ1991 MATa, leu2, trp1, ura3-251, prb1-1122, and pep4-3 Deletion of ANT1 carrier [82]
S. cerevisiae TCY119 MAT α ura3–52 leu2–3, 112 trp1-Δ1 ade2 his3-Δ1::hisG aac1-Δ1::hisG aac2-Δ1::kanMX6 aac3-Δ1::hisG [r+, TRP1] Deletion of AAC1, AAC2 and AAC3 carriers [83]
S. cerevisiae W303 his3-11,15; ade2-1; leu2-3,112; ura3-1; trp1-1; can1-100; RIM2/RIM2::kanMX Poor leucine uptake; deletion of pyrimidine nucleotide carrier [84]
S. cerevisiae ST9352 MATa, aro10Δ, pdc5Δ, pTEF1->ARO7, pPGK1->ARO4, pTEF1->FjTAL, pTEF1->HsSLC25A44 Alteration of aromatic amino acid metabolism [85]
S. cerevisiae CEN.PK113-7D ndt1Δndt2Δ MATa MAL2-8c SUC2 ndt1Δndt2Δ Deletion of NAD transporter [86]
S. cerevisiae BY4742 ndt1Δndt2Δ MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0, ndt1Δndt2Δ Deletion of NAD transporter [87]
S. cerevisiae FOH33 MATα MAL2-8c SUC2 his3Δ ura3-52 hfd1Δ pox1Δ faa1Δ faa4Δ adh6Δ::kanMX gal80Δ gal1/10/7Δ::(GAL7p-MmCAR-ADH1t)+(GAL3p-npgA-FBA1t); pAOH9 Deletion of alcohol dehydrogenase [88]
S. cerevisiae KY114 MATα, gal, ura3-52, trp1, lys2, ade2, his d2000 Deletion of thymidine transport [89]
S. cerevisiae BY4742-YBR021W MATα, his3, leu2, lys2, ura3, ΔFUR4 Deletion of uridine permease [90]
S. cerevisiae DY150 Mata ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc) Deletion of Fe2+/Mn2+ transporter [91]
S. cerevisiae YPL1 MATα fui1 Δ::
HIS3, ura3-52, lys2-801, HIS3 Δ		 Deletion of uridine permease [92]
S. cerevisiae W303-Δpep4 leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 hwas3-11,15 Δ pep4 MATα Deletion of vacuolar endopeptidase Pep4 [93]
S. cerevisiae W303-1A MATa ade2-1, can1-100, cyh2, his3-11,15, leu1-c, leu2-3,112, trp1-1, ura3-1 No growth in absence of adenine [94]
S. cerevisiae FGY217 MATα, ura3-52, lys2 Δ 201 and pep4 Δ Deletion of vacuolar endopeptidase Pep4 [95]
S. cerevisiae BY4743 Mata/α his3Δ1/ his3Δ1 leu2Δ0/ leu2Δ0 lys2Δ0/+ met15Δ0/+ ura3Δ0/ ura3Δ0 Essential genes deletion [96]
S. cerevisiae BY4743 Δpmr1::KanMX Mata/α his3Δ1/ his3Δ1 leu2Δ0/ leu2Δ0 lys2Δ0/+ met15Δ0/+ ura3Δ0/ ura3Δ0, Δpmr1::KanMX Deletion of essential genes and Pmr1 Ca2+ and Mn2+ transporter [97]
S. cerevisiae ctr1 MATa ura3 lys2 ade2 trp1 his3 leu2 Dctr1::LEU2 Deletion of CTR1 transporter [98]
S. cerevisiae YPH499 MATa ura3-52 lys2-801ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1   [99]
S. cerevisiae YPH500 MAT α/ura3–52/lys2–801/ade2–101/trp1-Δ63/his3-Δ200/leu2-Δ1   [100]
S. cerevisiae STY50 MATa,his4-401,leu2-3,-112,trp1-1,ura3-52,HOL1-1,suc2::LEU2 Deletion of invertase 2 [101]
P. pastoris CBS7435 Wild type   [102]
S. cerevisiae YPH501 MATa/MATα ura3-52/ura3-52 lys2-801/lys2-801 ade2-101/ade2-101 trp1-Δ63/ trp1-Δ63 his3- Δ200/his3- Δ200 Ieu2- Δ1/leu2- Δ1   [100]
S. cerevisiae CB001L MATα, leu2, trp1, ura3, prb−, pep4::LEU2 Deletion of vacuolar proteinase A and B [103]
S. cerevisiae JRY472 Δmpc1/2/3 Deletion of pyruvate carriers [104]
S. cerevisiae BY4741 gdt1Δpmr1Δ MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 gdt1::KanMX4 pmr1::KanMX4 Deletion of Ca2+ and Mg2+ transporters [105]

2.2. Vector Choice

A yeast vector may exist as a self-replicating particle, independently of the yeast genome, or be stably integrated into the yeast genome. This last case should be considered to reduce expression fluctuations. Moreover, a chromosomally borne transgene confers the advantage of a uniform cell population containing the transgene. In contrast, plasmid numbers vary from cell-to-cell, with as many as 50% of cells in a culture under selection lacking the plasmid. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. Except for the YLp plasmids (yeast linear plasmids), all these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. Indeed, they contain two types of selectable genes: plasmid-encoded drug resistance genes for bacterial selection and a yeast gene which acts as dominant selectable marker only when the recipient yeast cell has a recessive mutation in the corresponding chromosomal copy of the cloned gene [106]. Yeast integrating plasmids (YIp) contain selectable yeast genes but lack sequences that allow autonomous replication of the plasmid in yeast. The frequency of transformation of YIp plasmids is only 1 to 10 transformants/μg DNA, but transformation frequency can be increased 10- to 1000-fold by linearizing the plasmid within yeast sequences that are homologous to the intended site of integration on the yeast chromosome [106]. Besides integrating vectors, there are three classes of circular yeast plasmids characterized by extrachromosomal autonomous replication sequences (ARS) which confer the ability to replicate autonomously: YRp (yeast replicating plasmids), YCp (yeast centromeric plasmids), and YEp (yeast episomal plasmids). YRp plasmids have high frequencies of transformation (103 to 104 transformants/μg DNA), but transformants are very unstable both mitotically and meiotically. YRp plasmids can be present in high copy number (up to 100 copies per plasmid-bearing cell, although the average copy number per cell is 1 to 10). Introducing DNA portion from yeast centromeres into YRp-generated YCp plasmids allows an increase in plasmid stability during mitosis and meiosis. These plasmids (present in 1 to 2 copies per cell), have a loss rate of approximately 1% per generation. The increase in plasmid stability is fundamental for allowing protein production. The YEp vectors contain sequences from a naturally occurring yeast plasmid called the 2 μ circle and confer high transformation frequencies (∼104 to 105 transformants/μg DNA). Since they are propagated quite stably through mitosis and meiosis in high copy number, these plasmids are commonly used for high-level gene expression in yeast [106]. YLp (yeast linear plasmids) contain G-rich repeated sequences at their termini which act as telomeres and allow the plasmid to replicate as a linear molecule. They are highly unstable due to random segregation during mitosis. A crucial factor that drastically influences transcription and consequently translation rate is the promoter region. Commonly used promoters can be divided in two main classes: constitutive and inducible promoters (Table 2). Constitutive promoters lead to stable expression levels across varying culture conditions, while ‘dynamic’ or inducible promoters drive huge changes in expression level in response to specific stimuli [107] (Table 2).
Table 2. Plasmids: promoters and regulation.
Plasmid Promoter Type Gene Product Regulation References
pPICZB AOX Inducible Alcohol oxydase Methanol (+) [108]
pYES2 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
YEp4H7 HXT7 Constitutive hexose transporter HXT7 Low glucose (+) [110]
p426H7 HXT7 Constitutive hexose transporter HXT7 Low glucose (+) [110]
pVT102 ADH1 Constitutive Alcohol dehydrogenase I Ethanol (+) [111]
P426-GPD GPD Constitutive Glyceraldehyde—3 -phosphate dehydrogenase Glucose [112][113]
pEMBLyex4 CYC-GAL Hybrid/inducible Cytochrome C1, GAL1-GAL10 intergenic promoter Galactose (+) [114]
p426MET25 MET25 Inducible methionine and cysteine synthase methionine (−) [115]
YEpM Pma1 Constitutive Plasma membrane H+-ATPase   [67]
pPGK PGK1 Constitutive 3-phosphoglycerate kinase Glucose [61][116]
pYES2.1 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pYES GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pPICHOLI AOX Inducible Alcohol oxydase Methanol (+) [108]
pSM1052 PGK1 Constitutive Phosphatidyl glycerol kinase—1 Glucose [61][116]
pPIC3L AOX Inducible Alcohol oxydase Methanol (+) [108]
pPIC3.5K AOX Inducible Alcohol oxydase Methanol (+) [108]
pRS315 TDH3 Constitutive Glyceraldehyde—3-phosphate dehydrogenase Glucose [112][113]
pXP951 THI7   Thiamine transporter   [71]
pCM188 CYC1 Constitutive Cytochrome c oxidase glucose (−) [117]
YEp352 Lac Inducible Β-galactosidase Lactose (+) [72]
pRS314-YA2P yAAC2 Intrinsic Yeast ADP/ATP translocase Glucose (−) [74]
pRS424 yAAC2 Intrinsic Yeast ADP/ATP translocase Glucose (−) [74]
pYeDP-1/8-10 GAL10-CYC1 Hybrid/inducible Cytochrome C1, GAL10 promoter Galactose (+) [114]
pCGS110 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pYeDP60 GAL10-CYC1 Hybrid/inducible Cytochrome C1, GAL10 promoter Galactose (+) [114]
pGal110 GAL1/GAL10 Inducible Galactokinase/epimerase glucose (−)/galactose (+) [109]
pCGS110 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pYX142 TPI Constitutive Triose-phosphateisomerase   [118]
pEL30 CTA1 Inducible Catalase Oleate (+) [119]
pESC-Leu2d GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pRS42H RIM2   Yeast mitochondrial pyrimidine nucleotide carrier   [84]
pcfB9056 TEF Constitutive Translational elongation factor EF-1 α   [120]
YCplac33-NAT_MCART1 TEF Constitutive Translational elongation factor EF-1 α   [120]
pRS415 TEF Constitutive Translational elongation factor EF-1 α   [120]
pIYH01 HXT6, HXT7 Constitutive Hexose transporter 6 and 7 Low glucose (+) [88]
pYPGE15 PGK1 Constitutive 3-phosphoglycerate kinase Glucose [61][116]
pDDGFP-2 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pPICZ AOX Inducible Alcohol oxydase Methanol (+) [108]
pYES-DEST52 GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pDB20 ADH1 Constitutive Alcohol dehydrogenase I Ethanol (+) [121]
pYEX-BX CUP1 Inducible metallothionein Cu(II) (+) [122]
pPIC9K AOX Inducible Alcohol oxydase Methanol (+) [108]
pKTΔATG GPD Constitutive Glyceraldehyde—3-phosphate dehydrogenase Glucose [113]
p426GFP GPD Constitutive Glyceraldehyde—3-phosphate dehydrogenase Glucose [112][113]
pYEScupFLAGK or pYEScupFLAGE CUP1 Inducible Metallothionein Cu(II) (+) [122]
pYEX-BESN CUP1 Inducible Metallothionein Cu(II) (+) [122]
pBEVY-GU GAL1 Inducible Galactokinase Galactose (+)/Glucose (−) [109]
pRS416 TPI1 Constitutive Triose-phosphate isomerase,   [105]
pPICZA AOX Inducible Alcohol oxydase Methanol (+) [108]
Normally, constitutive strong promoters are used for biotechnological application to produce high levels of non-toxic target. Sometimes, the constitutive expression of certain proteins can be detrimental to cell growth due to product toxicity and the imposed metabolic burden [123]. To overcome this problem, several attempts at promoter engineering have been made aimed at tuning gene expression for biotechnological application [124].

2.3. Selection Strategies

The original and most commonly used strategy for the selection of transformed yeast cells exploits the auxotrophic markers: TRP1 [125], HIS3 [126], LEU2, URA3 [127], MET15, and ADE2 [128]. Expression plasmids are paired with available S. cerevisiae strains that are auxotrophic for tryptophan, histidine, leucine, uracil, methionine, and adenosine, respectively, by carrying full or functional knock-outs of these auxotrophic genes [129]. Besides auxotrophic markers, antibiotics can also be used as an alternative selection tool in a rich growth medium. However, since antibiotics affect ribosome function, expression studies should be performed in liquid media lacking antibiotics [129]. In the case of P. pastoris, the transformant selection is mostly conducted by using the antibiotic Zeocin, a member of the bleomycin/phleomycin family isolated from Streptomyces. Thus, clones deriving from multiple integration events can be selected increasing Zeocin concentration.

This entry is adapted from the peer-reviewed paper 10.3390/life12081206

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