Weinhandl et al. Microbial Cell Factories 2014, 13:5
`http://www.microbialcellfactories.com/content/13/1/5
`
`R E V I E W
`Open Access
`Carbon source dependent promoters in yeasts
`Katrin Weinhandl1, Margit Winkler1, Anton Glieder1 and Andrea Camattari2*
`
`Abstract
`
`Budding yeasts are important expression hosts for the production of recombinant proteins.
`The choice of the right promoter is a crucial point for efficient gene expression, as most regulations take place at
`the transcriptional level. A wide and constantly increasing range of inducible, derepressed and constitutive
`promoters have been applied for gene expression in yeasts in the past; their different behaviours were a reflection
`of the different needs of individual processes.
`Within this review we summarize the majority of the large available set of carbon source dependent promoters for
`protein expression in yeasts, either induced or derepressed by the particular carbon source provided. We examined
`the most common derepressed promoters for Saccharomyces cerevisiae and other yeasts, and described carbon
`source inducible promoters and promoters induced by non-sugar carbon sources. A special focus is given to
`promoters that are activated as soon as glucose is depleted, since such promoters can be very effective and offer
`an uncomplicated and scalable cultivation procedure.
`
`Introduction
`Recombinant protein production in yeast has repre-
`sented, in the last thirty years, one of the most import-
`ant tools of modern biotechnology. The possibility to
`express a high amount of a single protein, separated
`from its original context, allowed major leaps forward in
`the understanding of many cellular functions and en-
`zymes. However, since every host has its specific genetic
`system, species-specific tools have been established for
`each individual host/vector combination. In particular,
`promoters drive the transcription of the gene of interest
`and therefore are key parts of efficient expression sys-
`tems to produce recombinant proteins. Furthermore ex-
`pression of enzyme cascades and whole heterologous or
`synthetic pathways fully relies on a tool box of pro-
`moters with different sequence and properties.
`Typically, there are two major choices concerning tran-
`scription of a gene of interest: inducible or constitutive pro-
`moters. The decision for one of these alternatives depends
`on the specific requirements of a bioprocess and the prop-
`erties of the target protein to be produced. Constitutive ex-
`pression, performed by a range of very strong promoters
`like PGAP (glycerinaldehyde-3-phosphate dehydrogenase)
`[1], PPGK1 (3-Phosphoglyceratekinase) [2] or PTEF1 (transla-
`tion elongation factor) [3] from Saccharomyces cerevisiae is
`
`* Correspondence: andrea.camattari@tugraz.at
`2Institute of Molecular Biotechnology, Technical University Graz, Graz, Austria
`Full list of author information is available at the end of the article
`
`not always preferable, since recombinant proteins can have
`a toxic effect on their host organism at constantly high ex-
`pression level.
`Controllable gene expression can be achieved with indu-
`cible and derepressed promoters. Most of these inducible
`promoters are responsive to catabolite repression or react
`to other environmental conditions, such as stress, lack or
`accumulation of essential amino acids, ion concentrations
`inside the cell and others [4-6]. For practical applications,
`carbon source dependent promoters have the main advan-
`tage in the segregation of the host growth phase from the
`protein production phase, allowing maximizing growth
`before inducing a potentially burdening expression phase.
`Very recently, Da Silva & Srikrishnan have summarized
`important tools for controlled gene expression and meta-
`bolic engineering in S. cerevisiae, such as useful vectors,
`promoters and the procedure of chromosomal integration
`of recombinant genes [7].
`In order to categorize a large amount of information,
`and due to its practical importance, in this review we de-
`scribe the various promoters according to their basic be-
`havior in relation to carbon sources. This includes the
`most essential regulatory elements and mechanisms of
`carbon source regulation as described by the main chap-
`ters of this review: glucose repression in yeast and pro-
`moters which are either induced by simple de-repression
`or induced by carbohydrates or other non sugar carbon
`sources.
`
`© 2014 Weinhandl et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
`Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
`distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public
`Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
`article, unless otherwise stated.
`
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`Wherever possible, special emphasis is given on the
`applicability of individual promoters in different hosts
`and application spectra for industrial protein synthesis.
`Figure 1 gives an overview of the particular target pro-
`moters described within this work and their localization
`in the yeast cell metabolism.
`
`Glucose repression in yeasts
`Glucose is a favored carbon and energy source in yeast.
`Glucose repression and derepression essentially concern
`genes involved in oxidative metabolism and TCA (tri-
`carboxylic acid) cycle, genes encoding for the metabolism
`of alternative carbon sources (e.g. sucrose, maltose, gal-
`actose), or genes for gluconeogenesis [8-10]. In presence
`of glucose, decrease in transcription or translation at the
`gene level or increase in protein degradation at the pro-
`tein level are the most common mechanism to regulate
`the gene products involved [11].
`In an early attempt to clarify carbon source dependence
`in S. cerevisiae, Gancedo has listed the elements of catab-
`olite repression in yeast, focusing on regulatory elements
`on transcriptional level (Table 1), which was extended to
`additional proteins such as Oaf1 or Mig2 and Mig3.
`
`The current understanding of the mechanism of glucose
`derepression suggests that first of all the presence of glu-
`cose has to be signaled to the related genes. This signal
`transduction is likely performed by hexose transporters
`(HXT-gene products, Rgt2, Snf3) and hexokinases (HXK
`gene products). In yeast cells, a fully functional hexose
`transport is essential to provide functional glucose repres-
`sion events, since repression is prompted by uptake and
`metabolism of glucose [13]. This is consistent with the
`phenotype of a HXT deletion strain [14], and also with the
`observation that the AMP/ATP ratio reflects the glucose
`level inside the cell (a high AMP/ATP ratio leads to activa-
`tion of Snf1 [9], a kinase directly involved in gene regula-
`tion by carbon sources). However, most
`likely the
`processed metabolite of monosaccharides in the cell–glu-
`cose-6-phosphate–is the main signal that activates glucose
`repression [15].
`The event of glucose repression usually follows glucose
`level recognition, by repressors belonging to the Mig fam-
`ily comprising a group of C2H2-zinc-finger DNA-binding
`proteins. This family takes the name after Mig1, the most
`important repressor protein in this context, regulating the
`majority of glucose repressed genes (Figure 2).
`
`Figure 1 Target genes for inducible (orange) and derepressed (blue) carbon source dependent promoters in yeasts and their
`localization in the metabolism.
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`Table 1 Promoter interacting elements of catabolite
`repression in Saccharomyces cerevisiae (as reviewed
`in [10], [11], [12])
`Element
`Designation Function
`Activator (DNA-
`Hap2/3/4/5
`Activates transcription of proteins for
`binding proteins)
`complex
`respiratory functions
`
`Gal4
`
`Mal63
`
`Adr1, Cat8,
`Sip4
`
`Oaf1
`
`Activates transcription of proteins for
`galactose and melobiose metabolism
`
`Activates transcription of proteins for
`maltose utilization
`
`Activates transcription of proteins for
`ethanol, glycerol and lactate
`utilization, as well as for
`gluconeogenic proteins
`
`Activates transcription of proteins for
`oleate utilization
`
`Repressor (DNA-
`binding proteins)
`
`Mig1 (Mig2,
`Mig3)
`
`Recruits Ssn6-Tup1 complex (repressor
`complex) in glucose repressed genes
`
`Intermediate
`elements
`
`Snf1
`
`Glc7
`
`Protein kinase (in complex with Snf4);
`derepression of glucose-repressed
`genes by phosphorylation of Mig1
`
`Protein phosphatase;
`dephosphorylation of Snf1
`
`Glucose signaling Hxt-proteins Hexose transporter
`
`Snf3
`
`Rgt2
`
`Glucose transporter
`
`Glucose transporter
`
`Hxk-proteins Hexokinase
`
`Phosphorylation of glucose
`
`At high glucose level, Mig1 is transferred from the
`cytoplasm into the nucleus, where it binds a GC-rich
`recognition site in the promoter sequence (for consensus
`sequences see Table 2), and recruits a repressor complex
`consisting of Ssn6-Tup1 [17-19]. Using SUC2 promoter
`as a reporter system, it has been observed that the bind-
`ing of Mig1 leads to a conformational change of the
`chromatin structure, further reinforced by Tup1 inter-
`action with histones H3 and H4. Consequently, tran-
`scription initiating factors (such as Sip4) have no access
`to their binding sites [20].
`
`Many glucose repressed genes, for example hexose trans-
`porters (e.g. MTH1, HXT4, HXK1), are solely affected by
`Mig1-repression. However,
`two more Mig repressors
`(Mig2 and Mig3) are reported to be involved in glucose
`repression, by partly assisting Mig1 in a synergistic way
`(e.g. ICL1, ICL2, GAL3, HXT2, MAL11, MAL31, MAL32,
`MAL33, MRK1, SUC2 are repressed by Mig1 and Mig2) or
`taking over complete repression events in some genes
`without the intervention of Mig1 activity (SIR2 is repressed
`by Mig3). The involvement of a particular Mig repressor
`in gene expression is strongly correlated to glucose con-
`centrations inside the cell, as has been observed for HXT
`genes [10].
`Generally, MIG1 from several yeast species are highly
`conserved, but there are some differences in regulation
`of homologous genes in different yeasts. One example is
`GAL4 of Saccharomyces cerevisiae, which is regulated by
`Mig1 as described above, although GAL4 homologue
`LAC9 in Kluyveromyces lactis is triggered by a regulatory
`function of KlGAL1 and has no Mig1 binding site [18].
`As soon as glucose is depleted, the protein kinase Snf1
`is activated, mediating the release of Mig1 and the repres-
`sor complex by phosphorylation. Subsequently, Mig1 is
`exported from the nucleus, the promoter is derepressed
`and the gene expression gets activated [8]. Again, in the
`SUC2 expression model, the ATPase activity of the com-
`plex Swi/Snf triggers an ATP-dependent change of nucleo-
`somal structure (chromatin remodeling) and facilitates the
`binding of transcription factors [20,23]. Consequently,
`activator proteins are binding to particular consensus
`sequences (Table 2) and initiate transcription [21,22,24].
`
`Promoters derepressed by carbon source depletion
`The peculiarity of all these promoters (Table 3), all in-
`duced at low glucose levels, lays in the lack of a proper
`induction for their activity. Such a behavior, in fact, rep-
`resents also a reason for interest in potential applica-
`tions, as the expression of the protein of interest does
`not start during cell growth, when the carbon source is
`typically abundant, but only at
`the late exponential
`
`Figure 2 Mechanism of glucose repression in yeast; modified from [16].
`
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`Table 2 DNA-motifs for regulator protein binding in
`natural promoter sequences of carbon source dependent
`S. cerevisiae promoters
`DNA-binding protein
`Mig1
`
`Consensus sequence
`SYGGGG
`
`Reference
`[11]
`
`Gal4
`
`Mal63
`
`Cat8
`
`Sip4
`
`Adr1
`
`Oaf1
`
`Hap2
`
`CGGASGACAGTCSTCCG
`
`GAAAWTTTCGC
`
`YCCNYTNRKCCG
`
`TCCATTSRTCCGR
`
`TTGGRG
`
`CGGN3TNAN9-12CCG
`TNATTGGT
`
`[11]
`
`[11]
`
`[21]
`
`[21]
`
`[22]
`
`[22]
`
`[22]
`
`phase, allowing de facto a regulated gene expression
`without external induction step. The advantage of these
`promoters is even more promising moving from batch
`cultivations to fed-batch processes: during the feeding
`phase, a strict control on growth rate (and, in turns, on
`carbon source concentration in the fermenter) can be
`easily achieved, hence having a tight control on recom-
`binant protein production with relatively simple fermen-
`tation procedures.
`These promoter regions attract the binding of special
`transcription factors (e.g. Adr1), but as long as the car-
`bon source is available, the chromatin structure is orga-
`nized in such a way that the promoter is inaccessible to
`the activator protein. In the case of glucose, when its
`concentrations decreases, dephosphorylation of DNA-
`binding domains (as well as acetylation of histones H3
`and H4) occurs, leading to a conformational change of
`the DNA region. Subsequently, the promoter region is
`accessible and gene expression can be activated by the
`activator protein without any induction signal [38].
`Recently, Thierfelder and colleagues presented a new
`set of plasmids for Saccharomyces cerevisiae, containing
`several glucose dependent promoters induced at a low
`level of glucose (PHXK1, PYGR243, PHXT4, PHXT7; [39]). In
`Pichia pastoris, a set of 6 novel glucose dependent pro-
`moters was described; promoters of hexose transporters,
`of a mitochondrial aldehyde dehydrogenase and of some
`proteins with unknown function were represented in this
`list. Generally, all of them were also activated during
`glucose starvation [40].
`
`Hexose transporter genes in S. cerevisiae and other yeasts
`Hexose transporters in S. cerevisiae are encoded by 17
`HXT genes. Some of them are induced (e.g. HXT1),
`whereas others are repressed by high levels of glucose (e.
`g. HXT2, HXT4, HXT7) [41]. In this section we will
`focus on the glucose-repressed fraction of HXT genes,
`that includes all high-affinity glucose transporters. In
`addition, high-affinity hexose transporters from other
`
`yeasts, that may have the potential of good promoter ac-
`tivity, will be discussed.
`Hexose transporter proteins Hxt2, 4, 6 and 7 in S. cerevi-
`siae are repressed by high glucose concentration, and
`induced when glucose concentration decreases below a
`certain level [39]. Two independent transcription repres-
`sion mechanisms apply, mediated respectively by Mig re-
`pressor (high glucose level) or by Rgt1, a C6-zinc cluster
`(no glucose). Both proteins are responsible for recruiting
`the Ssn6-Tup1 complex [29]. While derepression upon
`Mig1 release is dependent by Snf1, Rgt1 dissociation re-
`quires Grr1-mediated phosphorylation, which is dependent
`from Mth1 and Std1 activities [42]. Interestingly, another
`regulatory complex, depending on pH and the correspond-
`ing altered calcineurin pathway, was hypothesized. This as-
`sumption is based on observations on HXT2 regulation:
`after shifting the media pH to 8, the expression of HXT2
`reaches a plateau, while in snf1 mutant strains the expres-
`sion was not completely inhibited. It was suggested that
`HXT2 promoter might be a target for the transcription fac-
`tor Crz1, which is active at high pH and activates the cal-
`cineurin pathway, a response to environmental stress in
`yeast. Also related to pH shift, although to a lesser extent,
`is the induction of HXT7 and other glucose dependent
`proteins like Hxk1, Tps1, and Ald4. Overall, the response
`to alkaline stress of genes involved in glucose utilization
`suggests an impairment of glucose metabolism, probably
`due to a disturbed electrochemical gradient and subse-
`quent uptake of nutrient through the cell wall: a sudden
`increase of pH value is a signal for the activation of stress
`responsive enzymes (e.g. superoxide dismutase, SOD) in
`order to maintain an appropriate pH for a functioning
`electrochemical gradient [27].
`Many hexose transporter genes are not well described
`yet. Greatrix and colleagues compared the expression levels
`of HXT1-17. HXT13, for example, showed similar induc-
`tion characteristics as HXT2 (i.e. induction at 0.2% w/v glu-
`cose). Furthermore, HXT6, closely related to HXT7,
`is
`induced at low glucose concentrations [43], but its expres-
`sion is more dependent on the Mig2 repressor [10].
`HXT7 seems to bind glucose with the highest affinity
`among all glucose transporters, and this fact is associ-
`ated to a strong induction at low glucose level. The
`HXT7 promoter region turned out to be suitable for re-
`combinant protein production in yeast and was com-
`pared to other yeast promoters (PTEF1, PADH1, PTPI1,
`PPGK1, PTDH3 and PPYK1) using lacZ as a reporter gene.
`Among them, PHXT7 was stated as the strongest pro-
`moter in continuous culture with limited glucose level
`[44]. Also in comparison with PADH1 for SUC2- and
`GFP-expression, respectively, PHXT7 produced promising
`results [25].
`A variant of PHXT7 (PHXT7-391, 5′ deletion [26]), show-
`ing strong constitutive expression, was applied for
`
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`Table 3 Yeast promoters derepressed by gradual glucose consumption (repressed by glucose), and respective known
`regulator elements and binding sites
`Promoter
`Protein function
`Organism Derepressed by: (strength)
`
`Ref.
`
`DNA-binding target
`Regulating
`protein
`sequence
`No information available
`
`−590 to −579
`−430 to −424
`−393 to −387
`−504 to −494
`−427 to −415
`−291 to −218
`−226 to −218
`−645 to −639
`
`No information
`available
`
`Rgt1
`
`Mig1
`
`UAS
`
`Activator protein?
`
`Rgt1
`
`Mig2
`
`[25]
`[26]
`
`[27]
`[28]
`
`[27]
`
`[29]
`
`[29]
`
`[28]
`
`[10]
`
`[30]
`
`[31]
`
`[20]
`
`High affinity hexose
`transporter
`
`High affinity hexose
`transporter
`
`S. cerevisiae
`
`Low glucose level (10-15×)
`
`S. cerevisiae
`
`Low glucose level (10-15×)
`
`HXT7
`
`HXT2
`
`HXT4
`
`HXT6
`
`KHT2
`
`High affinity hexose
`transporter
`
`High affinity hexose
`transporter
`
`High affinity hexose
`transporter
`
`HGT9, 10, 12,
`17
`
`High affinity hexose
`transporter
`
`SUC2
`
`Invertase
`
`S. cerevisiae
`
`Low glucose level
`
`S. cerevisiae
`
`Low glucose level (10×)
`
`K. lactis
`
`Low glucose level (2×)
`
`No information available
`
`C. albicans
`
`Low glucose level
`
`No information available
`
`S. cerevisiae
`
`Sucrose low glucose level
`(200×)
`
`Mig1/2
`
`Sko1
`
`Low glucose level (100×)
`
`−499 to −480
`−442 to −425
`−627 to −617
`−650 to −418
`−133
`−319 to −292
`−291 to ??
`Low glucose level (10×), lactate −651 to −632
`−1321 to −1302
`−660 to −649
`−1447 to −1436
`−739 to −727
`−245 to −112
`−507 to −430
`
`Low glucose level, glycerol
`
`UAS
`
`RNA-Pol II
`
`Cat8
`
`Adr1
`
`Cat8
`
`Mig1
`
`Abf1
`
`Adr1
`
`UAS
`
`ADH2
`
`Alcohol dehyrogenase
`
`S. cerevisiae
`
`JEN1
`
`Lactate permease
`
`S. cerevisiae
`
`MOX
`
`Methanol oxidase
`
`H.
`polymorpha
`
`AOX delta 6
`
`Alcohol oxidase
`
`P. pastoris
`
`Low glucose level, glycerol
`
`deleted GCR1-site
`
`GLK1
`
`Glucokinase
`
`S. cerevisiae
`
`Low glucose level (6×), ethanol
`(25×)
`
`HXK1
`
`ALG2
`
`Hexokinase
`
`Isocitrate lyase
`
`S. cerevisiae
`
`Low glucose level (10×),
`ethanol
`
`H.
`polymorpha
`
`Low glucose level
`
`No information available
`
`Gcr1
`
`URS
`
`−881 to −702
`−572 to −409
`−408 to −104
`No information available
`
`Msn2/4
`
`[24]
`
`[21]
`[32]
`
`[32]
`
`[32]
`
`[33]
`
`[34]
`
`[33]
`
`[35]
`
`[36]
`
`[37]
`
`overexpression of phosphoglucomutase 2 to improve an-
`aerobic galactose metabolism [45].
`PHXT2 was successfully used for the recombinant pro-
`duction of squalene synthase (ERG9), which plays an im-
`portant role in synthesis of compounds for perfumes
`and pharmaceuticals [46].
`
`the characterization of hexose trans-
`As expected,
`porters, and relative promoters, is poorly characterized
`in less conventional yeasts. Nevertheless, KHT1 and
`KHT2 from K. lactis, GHT1-6 from Schizosaccharomyces
`pombe, orHGT -genes from C. albicans have been de-
`scribed [47,48].
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`KHT1 and 2 represent a sort of genetic anomaly, as
`both are located in a polymorphic gene locus of RAG1
`[30], which encodes either a low (Kht1, Rag1) or a mod-
`erate affinity hexose transporter (Kht2). Therefore, PKHT2
`is more interesting for application where a more sensitive
`glucose dependent promoter element is required. KHT2
`turned out to be, sequence-wise, a close relative of HXT7
`and is similarly regulated. It has to be considered that
`KHT2 is only weakly repressed by high glucose level and
`about 2-fold induced at concentrations below 0.1% (w/v)
`[49]. To our knowledge, the KHT2 promoter has not yet
`been applied for recombinant protein production so far.
`The GHT genes from S. pombe not only encode glucose
`transporters (GHT1, 2 and 5) but also gluconate trans-
`porters (GHT3 and 4). GHT2 and 5 are not repressed by
`glucose, in contrast to GHT1, GHT3 and 4. Nevertheless,
`GHT5 is expected to be a high affinity glucose trans-
`porter, but so far no promoter studies about any of the
`GHT gene group of fission yeast is available [50].
`Expression of another set of hexose transporters–the
`HGT genes–was studied in Candida albicans. In con-
`junction with derepressed genes (and promoters) HGT9,
`HGT10, HGT12 and HGT17 are most interesting for this
`review, since they are strongly induced at low glucose
`concentrations (0.2% w/v) [31].
`Not surprisingly, also hexose transporters in the indus-
`trial workhorse Pichia pastoris attracted interest in the
`context of natural promoters and strain engineering aim-
`ing at methanol-free alcohol oxidase (AOX1)-promoter
`controlled expression. The only two known hexose trans-
`porters are PpHxt1 and PpHxt2. PpHxt1 is related to the
`S. cerevisiae HXT genes, is induced at high glucose con-
`centrations and seems to play a minor role in P. pastoris.
`PpHxt2 is more species specific, has characteristics of a
`high-affinity glucose transporter, but is also responsible
`for main glucose transport during high glucose concen-
`trations. Interestingly, a deletion of PpHXT1 leads to a
`hexose mediated induction of PAOX1 [14], most probably
`due to the resulting low intracellular glucose concentra-
`tion in such deletion variants.
`Additionally, Prielhofer and colleagues described the
`use of several Pichia species’ hexose transporters as new
`promoter targets with green fluorescent protein (GFP)
`as reporter and, therefore, provided a potential alterna-
`tive to methanol induced promoters [40] or engineered
`synthetic promoters, which also do not need methanol
`for induction [33].
`
`SUC2 promoter
`The SUC2 gene of S. cerevisiae encodes an invertase
`(beta-fructofuranosidase) and is inducible by sucrose. As
`for other glucose repressed genes, also the promoter of
`SUC2 enables expression to a high level without any ex-
`ternal inducer. Similarly to HXT genes, derepression of
`
`SUC2 promoter takes place when the level of glucose (or
`fructose as well) is decreasing below a certain level
`(0.1% w/v); SUC2 promoter, interestingly, gets repressed
`again when glucose concentration drops to zero. In cul-
`tivations with glycerol as only (non-repressing) carbon
`source, the expression of SUC2 was shown to be 8-fold
`lower than expression in media with low glucose con-
`centration [51]. The regulation of the SUC2-promoter is
`subjected to Mig1 and Mig2 binding sites on one hand
`(repression at high glucose level, [52]) and to Rgt1 re-
`pressor on the other hand (repression at lack of glucose,
`basal SUC2 transcription). At low glucose concentra-
`tions, Mig1/2, as well as Rgt1, are phosphorylated by the
`Snf1/Snf4 complex and thus transcription of SUC2 is
`initiated [53]. Additionally, the promoter activity can be
`further enhanced by sucrose induction but this is not es-
`sential for good promoter activity [51].
`PSUC2 is a very suitable promoter for heterologous pro-
`tein expression in yeast, and processes have been opti-
`mized for several applications, also above laboratory
`scale. For example, significant results for α-amylase ex-
`pression by PSUC2 have been obtained using lactic acid
`as carbon source, a substrate supporting recombinant
`gene expression as well as cell growth by providing a fast
`way of energy production (lactate is converted to pyru-
`vate and enters the TCA cycle). The advantage of an ex-
`tended cell growth phase driven by a non repressing
`carbon source opened the possibility for the use of PSUC2
`also in large scale applications [54].
`In analogy, inv1 from Schizosaccharomyces pombe was
`subject of the development of a regulated expression
`system in S. pombe, since also the Pinv1 is repressed by
`glucose (Scr1 mediated, which is another DNA binding
`protein recognizing GC-rich motifs within the promoter)
`and is further inducible by sucrose [55].
`In Kluyveromyces marxianus, INU1, which is a closely
`related gene to SUC2 and encodes an inulase enzyme, re-
`sponsible for fructose hydrolyzation, also carries two puta-
`tive Mig1-recognition sites [18]. The promoter is activated
`by addition of sucrose or inulin, the derepression is con-
`trolled in a similar way to SUC2 [56]. PINU1 was applied to
`several protein synthesis approaches in K. marxianus and
`S. cerevisiae, such as expression of inulase (inuE) or glu-
`cose oxidase (GOX) from Aspergillus niger [57,58].
`
`JEN1 promoter
`JEN1 encodes a transporter for carboxylic acids (e.g. lac-
`tate, pyruvate) in S. cerevisiae. JEN1 expression is re-
`pressed by glucose and derepressed when glucose level
`falls below 0.3 % (w/v), reaching a peak of activity at 0.1 %
`(w/v) glucose. Additionally, a weak PJEN1 activation by lac-
`tic acid was observed, using GFP as reporter gene [59].
`The regulation of PJEN1 by the transcription factor Adr1
`and the alternative carbon source responsive activator
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`Cat8 was confirmed [60]. Two Mig1 binding sites in the
`upstream sequence of JEN1 were identified [32,61]. Subse-
`quently, however, Andrade and colleagues published an
`alternative mechanism of regulation, proposing that Jen1
`is post-transcriptionally regulated by mRNA degradation,
`rather than by Mig1 mediated repression [62].
`JEN1 promoter has been successfully applied to Flo1
`expression, a protein involved in flocculation processes
`[59].
`
`ADH2 promoter
`A very popular promoter, used in several yeasts, is the pro-
`moter of the alcohol dehydrogenase II gene from S. cerevi-
`siae [63]. In contrast to the widely used constitutive yeast
`ADH1 promoter, PADH2 is strongly repressed in presence
`of glucose, and derepressed as soon as the transcription
`factor Adr1 binds to the upstream activating sequence
`UAS1 of PADH2. Adr1 is dephosphorylated when glucose is
`depleting, and the cell switches to growth on ethanol
`(Adr1 dephosphorylation appears to be Snf1-dependent).
`There is also a second glucose dependent UAS (namely
`UAS2), less characterized but likely activated by Cat8 in a
`synergistic way with Adr1, and thus identified as a CSRE
`sequence (carbon source responsive element) [24,64]. Fur-
`thermore, some other protein kinases, such as Sch9, Tpk1
`and Ccr1, that also derepress PADH2, influence the expres-
`sion level of ADH2. Interestingly, there is no typical Mig1-
`binding site in the ADH2 promoter sequence; glucose
`repression is mainly mediated by the Glc7/Reg1 complex
`[11].
`The potential of PADH2 was evaluated and compared to
`the inducible S. cerevisiae promoters PCUP1 and PGAL1
`and turned out to yield the highest level of expression
`after 48 hours [65].
`S. cerevisiae ADH2 promoter is not the only alcohol
`dehydrogenase promoter used in expression studies. Padh
`from S. pombe, (adh shows high homology with S. cerevi-
`siae Adh2 at the protein level) is a frequently used
`promoter in fission yeast, but is described as being con-
`stitutively expressed [66].
`The related ADH4 gene from K. lactis is characterized
`by a strong ethanol induction, and is therefore separately
`described in Section Induction by non-sugar carbon
`sources.
`A Pichia-specific ADH2 promoter was isolated from
`Pichia stipitis and is–in contrast
`to ScADH2–not
`glucose- but oxygen-dependent (induction at low O2
`level). This PsADH2 promoter was used in the heterol-
`ogous host Pichia pastoris for the expression of Vitreos-
`cilla hemoglobin (VHb) [67].
`
`HXK1, GLK1 promoter
`Hexokinase (HXK1) and Glucokinase (GLK1) in S. cerevi-
`siae are involved in the first reaction of glycolysis, the
`
`phosphorylation of glucose, and are activated when the
`cell is entering a starvation phase or when switched to an-
`other carbon source [36]. Both enzymes are not expressed
`in presence of high glucose levels (subjected by a classical
`Mig1 repression; [10]), but become derepressed as soon as
`glucose is depleting. In case of GLK1, a 6-fold increase of
`expression level by derepression and further 25-fold in-
`duction by ethanol was reported [35]. HXK1, in compari-
`son,
`is 10-fold repressed by glucose in dependence of
`Hxk2 protein [36] and was listed as one of Thierfelder’s
`glucose dependent promoters with average strength, when
`it is induced at low glucose concentration [39].
`PHXK1, for instance, was successfully applied to the ex-
`pression of a GST-cry11A fusion protein in S. cerevisiae
`[68] or, in more recent years, to the expression of bovine
`β-casein [69]. In case of PGLK1, no application in terms
`of recombinant protein production was reported.
`
`Carbon source dependent inducible promoters
`Other promoters are derepressed in absence of glucose
`and additionally need to be induced by an alternative car-
`bon source to obtain full expression efficiency (Table 4).
`The inducer is either produced by the cell in course of
`time or has to be provided in the medium.
`Galactose, maltose, sucrose, and some other ferment-
`able carbon sources, as well as oleate, glycerol, acetate
`or ethanol, as non-fermentable carbon sources, can be
`considered as alternative inducers for regulated gene ex-
`pression, since the genes that are involved in the particu-
`lar metabolism are repressed, as long as the preferred
`carbon source glucose is available.
`
`Induction by carbohydrates
`Induction by galactose
`The promoters of the S. cerevisiae GAL genes are the most
`typical and most characterized examples of galactose-
`inducible promoters. They are strongly regulated by cis-
`acting elements, depending on glucose level, whereupon
`galactose is acting as the main inducer [70].
`Gal6 and Gal80 are negative regulators of Gal4, which is
`classified as the activator of the main proteins of galactose
`utilization pathway GAL1 (galactokinase), GAL7 (α-D-gal-
`actose-1- phosphate uridyltransferase) and GAL10 (uri-
`dine diphosphoglucose 4-epimerase) [89], as shown in
`Figure 3. Negative regulators for GAL genes have been
`shown to work in synergy with Mig1 [71]. Gal3 is ex-
`pected to act as a signal transducer that forms a complex
`with galactose and Gal80, further releasing Gal4 inside the
`nucleus and activating GAL1, 7 and 10 expression [90,91].
`PGAL1 and PGAL10 are widely used in S. cerevisiae for
`recombinant protein production, for which different cul-
`tivation protocols have been developed. The crucial point
`is the maintenance of a low glucose level, which is im-
`portant for efficient induction [92]. Since also galactose
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`Table 4 Yeast promoters induced in dependence of carbon sources and their regulator elements
`Promoter Protein function Organism
`Induced by (strength)
`Repressed by Regulating
`sequence
`−390 to −255
`−201 to −187
`−264 to −161
`No information available
`−324 to −216
`
`GAL1
`
`GAL7
`
`GAL10
`
`Galactose
`metabolism
`
`Galactose
`metabolism
`
`Galactose
`metabolism
`
`S. cerevisiae
`
`Galactose (1000×)
`
`Glucose
`
`S. cerevisiae
`
`K. lactis
`
`S. cerevisiae
`
`Galactose (1000×)
`
`Glucose
`
`Galactose
`
`Galactose (1000×)
`
`Glucose
`
`DNA-bindingtarget
`protein
`Gal4
`
`Mig1
`
`Gal4
`
`Gal4
`
`Galactose
`metabolism
`
`Phosphoinositol
`synthase
`
`PIS1
`
`C. maltosa
`
`Galactose
`
`Glucose
`
`No information available
`
`S. cerevisiae
`
`Galactose, hypoxia (2×),
`zinc depletion (2×)
`
`(glycerol)
`
`−149 to −138
`
`−224 to −205
`
`Rox1
`
`Gcr1
`
`Ste12
`
`Pho2
`
`LAC4
`
`Lactose
`metabolism
`
`K. lactis
`
`Lactose, galactose (100×)
`
`-
`
`Maltase
`
`Maltase
`
`H. polymorpha
`
`S. cerevisiae
`
`Maltose sucrose
`
`Maltose sucrose
`
`Glucose
`
`Glucose
`
`MAL1
`
`MAL62
`
`AGT1
`
`Alpha-glucoside
`transporter
`
`Brewing strains S.
`cerevisiae, S. pastorianus
`
`Ma