Carbon source dependent promoters in yeasts.

Carbon source dependent promoters in yeasts

Katrin Weinhandl 1,Margit Winkler 1,Anton Glieder 1and Andrea Camattari 2*

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 P GAP (glycerinaldehyde-3-phosphate dehydrogenase)[1],P PGK1(3-Phosphoglyceratekinase)[2]or P TEF1(transla-tion elongation factor)[3]from Saccharomyces cerevisiae is

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.

*Correspondence:andrea.camattari@tugraz.at 2

Institute of Molecular Biotechnology,Technical University Graz,Graz,Austria Full list of author information is available at the end of the

article

?2014Weinhandl et al.;licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://www.sodocs.net/doc/9711612961.html,/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 (https://www.sodocs.net/doc/9711612961.html,/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.

Weinhandl et al.Microbial Cell Factories 2014,13:5https://www.sodocs.net/doc/9711612961.html,/content/13/1/5

Wherever possible,special emphasis is given on the applicability of individual promoters in different hosts and application spectra for industrial protein synthesis. Figure1gives 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(Table1),which was extended to additional proteins such as Oaf1or Mig2and 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).

Figure1Target genes for inducible(orange)and derepressed(blue)carbon source dependent promoters in yeasts and their localization in the metabolism.

At high glucose level,Mig1is transferred from the cytoplasm into the nucleus,where it binds a GC-rich recognition site in the promoter sequence(for consensus sequences see Table2),and recruits a repressor complex consisting of Ssn6-Tup1[17-19].Using SUC2promoter as a reporter system,it has been observed that the bind-ing of Mig1leads to a conformational change of the chromatin structure,further reinforced by Tup1inter-action with histones H3and 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 (Mig2and Mig3)are reported to be involved in glucose repression,by partly assisting Mig1in a synergistic way (e.g.ICL1,ICL2,GAL3,HXT2,MAL11,MAL31,MAL32, MAL33,MRK1,SUC2are repressed by Mig1and Mig2)or taking over complete repression events in some genes without the intervention of Mig1activity(SIR2is 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,MIG1from several yeast species are highly conserved,but there are some differences in regulation of homologous genes in different yeasts.One example is GAL4of Saccharomyces cerevisiae,which is regulated by Mig1as described above,although GAL4homologue LAC9in Kluyveromyces lactis is triggered by a regulatory function of KlGAL1and has no Mig1binding site[18]. As soon as glucose is depleted,the protein kinase Snf1 is activated,mediating the release of Mig1and the repres-sor complex by phosphorylation.Subsequently,Mig1is exported from the nucleus,the promoter is derepressed and the gene expression gets activated[8].Again,in the SUC2expression 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(Table2)and initiate transcription[21,22,24]. Promoters derepressed by carbon source depletion

The peculiarity of all these promoters(Table3),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

Table1Promoter interacting elements of catabolite repression in Saccharomyces cerevisiae(as reviewed in[10],[11],[12])

Element Designation Function

Activator(DNA-binding proteins)Hap2/3/4/5

complex

Activates transcription of proteins for

respiratory functions

Gal4Activates transcription of proteins for

galactose and melobiose metabolism Mal63Activates transcription of proteins for

maltose utilization

Adr1,Cat8,

Sip4

Activates transcription of proteins for

ethanol,glycerol and lactate

utilization,as well as for

gluconeogenic proteins

Oaf1Activates transcription of proteins for

oleate utilization

Repressor(DNA-binding proteins)Mig1(Mig2,

Mig3)

Recruits Ssn6-Tup1complex(repressor

complex)in glucose repressed genes

Intermediate elements Snf1Protein kinase(in complex with Snf4);

derepression of glucose-repressed

genes by phosphorylation of Mig1 Glc7Protein phosphatase;

dephosphorylation of Snf1

Glucose signaling Hxt-proteins Hexose transporter

Snf3Glucose transporter

Rgt2Glucose transporter

Hxk-proteins Hexokinase

Phosphorylation of glucose

glucose repression in yeast;modified from[16].

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(P HXK1,P YGR243,P HXT4,P HXT7;[39]).In Pichia pastoris,a set of6novel 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 by17 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 Hxt2,4,6and7in 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-Tup1complex[29].While derepression upon Mig1release is dependent by Snf1,Rgt1dissociation re-quires Grr1-mediated phosphorylation,which is dependent from Mth1and Std1activities[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 HXT2regulation: after shifting the media pH to8,the expression of HXT2 reaches a plateau,while in snf1mutant strains the expres-sion was not completely inhibited.It was suggested that HXT2promoter 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 HXT7and 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 at0.2%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 Mig2repressor[10].

HXT7seems 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 HXT7promoter region turned out to be suitable for re-combinant protein production in yeast and was com-pared to other yeast promoters(P TEF1,P ADH1,P TPI1, P PGK1,P TDH3and P PYK1)using lacZ as a reporter gene. Among them,P HXT7was stated as the strongest pro-moter in continuous culture with limited glucose level [44].Also in comparison with P ADH1for SUC2-and GFP-expression,respectively,P HXT7produced promising results[25].

A variant of P HXT7(P HXT7-391,5′deletion[26]),show-ing strong constitutive expression,was applied for

Table2DNA-motifs for regulator protein binding in natural promoter sequences of carbon source dependent S.cerevisiae promoters

DNA-binding protein Consensus sequence Reference Mig1SYGGGG[11]

Gal4CGGASGACAGTCSTCCG[11]

Mal63GAAAWTTTCGC[11]

Cat8YCCNYTNRKCCG[21]

Sip4TCCATTSRTCCGR[21]

Adr1TTGGRG[22]

Oaf1CGGN3TNAN9-12CCG[22]

Hap2TNATTGGT[22]

overexpression of phosphoglucomutase2to improve an-aerobic galactose metabolism[45].

P HXT2was 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].

As expected,the characterization of hexose trans-porters,and relative promoters,is poorly characterized in less conventional yeasts.Nevertheless,KHT1and KHT2from https://www.sodocs.net/doc/9711612961.html,ctis,GHT1-6from Schizosaccharomyces pombe,or HGT-genes from C.albicans have been de-scribed[47,48].

Table3Yeast promoters derepressed by gradual glucose consumption(repressed by glucose),and respective known regulator elements and binding sites

Promoter Protein function Organism Derepressed by:(strength)Regulating

sequence DNA-binding target

protein

Ref.

HXT7High affinity hexose

transporter S.cerevisiae Low glucose level(10-15×)No information available[25]

[26]

HXT2High affinity hexose

transporter S.cerevisiae Low glucose level(10-15×)?590to?579Rgt1[27]

[28]

?430to?424

?393to?387

?504to?494Mig1[27]

?427to?415

?291to?218UAS[29]

?226to?218Activator protein?[29]

HXT4High affinity hexose

transporter

S.cerevisiae Low glucose level?645to?639Rgt1[28]

HXT6High affinity hexose

transporter S.cerevisiae Low glucose level(10×)No information

available

Mig2[10]

KHT2High affinity hexose

transporter

https://www.sodocs.net/doc/9711612961.html,ctis Low glucose level(2×)No information available[30]

HGT9,10,12, 17High affinity hexose

transporter

C.albicans Low glucose level No information available[31]

SUC2Invertase S.cerevisiae Sucrose low glucose level

(200×)?499to?480Mig1/2[20]?442to?425

?627to?617Sko1

?650to?418UAS

?133RNA-Pol II

ADH2Alcohol dehyrogenase S.cerevisiae Low glucose level(100×)?319to?292Cat8[24]

?291to??Adr1

JEN1Lactate permease S.cerevisiae Low glucose level(10×),lactate?651to?632Cat8[21]

[32]

?1321to?1302

?660to?649Mig1[32]

?1447to?1436

?739to?727Abf1[32]

MOX Methanol oxidase H.

polymorpha Low glucose level,glycerol?245to?112Adr1[33]

?507to?430UAS[34]

AOX delta6Alcohol oxidase P.pastoris Low glucose level,glycerol deleted GCR1-site[33]

GLK1Glucokinase S.cerevisiae Low glucose level(6×),ethanol

(25×)?881to?702Gcr1[35]?572to?409URS

?408to?104Msn2/4

HXK1Hexokinase S.cerevisiae Low glucose level(10×),

ethanol

No information available[36]

ALG2Isocitrate lyase H.

polymorpha

Low glucose level No information available[37]

KHT1and2represent 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,P KHT2 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 KHT2is only weakly repressed by high glucose level and about2-fold induced at concentrations below0.1%(w/v) [49].To our knowledge,the KHT2promoter has not yet been applied for recombinant protein production so far. The GHT genes from S.pombe not only encode glucose transporters(GHT1,2and5)but also gluconate trans-porters(GHT3and4).GHT2and5are not repressed by glucose,in contrast to GHT1,GHT3and4.Nevertheless, GHT5is 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,HGT12and HGT17are 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 Pp Hxt1and Pp Hxt2.Pp Hxt1is related to the S.cerevisiae HXT genes,is induced at high glucose con-centrations and seems to play a minor role in P.pastoris. Pp Hxt2is 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 Pp HXT1leads to a hexose mediated induction of P AOX1[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].

SUC2promoter

The SUC2gene of S.cerevisiae encodes an invertase (beta-fructofuranosidase)and is inducible by sucrose.As for other glucose repressed genes,also the promoter of SUC2enables expression to a high level without any ex-ternal inducer.Similarly to HXT genes,derepression of SUC2promoter takes place when the level of glucose(or fructose as well)is decreasing below a certain level (0.1%w/v);SUC2promoter,interestingly,gets repressed again when glucose concentration drops to zero.In cul-tivations with glycerol as only(non-repressing)carbon source,the expression of SUC2was shown to be8-fold lower than expression in media with low glucose con-centration[51].The regulation of the SUC2-promoter is subjected to Mig1and Mig2binding sites on one hand (repression at high glucose level,[52])and to Rgt1re-pressor on the other hand(repression at lack of glucose, basal SUC2transcription).At low glucose concentra-tions,Mig1/2,as well as Rgt1,are phosphorylated by the Snf1/Snf4complex and thus transcription of SUC2is initiated[53].Additionally,the promoter activity can be further enhanced by sucrose induction but this is not es-sential for good promoter activity[51].

P SUC2is 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 forα-amylase ex-pression by P SUC2have 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 P SUC2 also in large scale applications[54].

In analogy,inv1from Schizosaccharomyces pombe was subject of the development of a regulated expression system in S.pombe,since also the P inv1is repressed by glucose(Scr1mediated,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 SUC2and 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].P INU1was 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].

JEN1promoter

JEN1encodes a transporter for carboxylic acids(https://www.sodocs.net/doc/9711612961.html,c-tate,pyruvate)in S.cerevisiae.JEN1expression is re-pressed by glucose and derepressed when glucose level falls below0.3%(w/v),reaching a peak of activity at0.1% (w/v)glucose.Additionally,a weak P JEN1activation by lac-tic acid was observed,using GFP as reporter gene[59]. The regulation of P JEN1by the transcription factor Adr1 and the alternative carbon source responsive activator

Cat8was confirmed[60].Two Mig1binding sites in the upstream sequence of JEN1were 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 Mig1mediated repression[62].

JEN1promoter has been successfully applied to Flo1 expression,a protein involved in flocculation processes [59].

ADH2promoter

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 ADH1promoter,P ADH2is strongly repressed in presence of glucose,and derepressed as soon as the transcription factor Adr1binds to the upstream activating sequence UAS1of P ADH2.Adr1is dephosphorylated when glucose is depleting,and the cell switches to growth on ethanol (Adr1dephosphorylation appears to be Snf1-dependent). There is also a second glucose dependent UAS(namely UAS2),less characterized but likely activated by Cat8in 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 P ADH2,influence the expres-sion level of ADH2.Interestingly,there is no typical Mig1-binding site in the ADH2promoter sequence;glucose repression is mainly mediated by the Glc7/Reg1complex [11].

The potential of P ADH2was evaluated and compared to the inducible S.cerevisiae promoters P CUP1and P GAL1 and turned out to yield the highest level of expression after48hours[65].

S.cerevisiae ADH2promoter is not the only alcohol dehydrogenase promoter used in expression studies.P adh from S.pombe,(adh shows high homology with S.cerevi-siae Adh2at the protein level)is a frequently used promoter in fission yeast,but is described as being con-stitutively expressed[66].

The related ADH4gene from https://www.sodocs.net/doc/9711612961.html,ctis is characterized by a strong ethanol induction,and is therefore separately described in Section Induction by non-sugar carbon sources.

A Pichia-specific ADH2promoter was isolated from Pichia stipitis and is–in contrast to ScADH2–not glucose-but oxygen-dependent(induction at low O2 level).This PsADH2promoter was used in the heterol-ogous host Pichia pastoris for the expression of Vitreos-cilla hemoglobin(VHb)[67].

HXK1,GLK1promoter

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 Mig1repression;[10]),but become derepressed as soon as glucose is depleting.In case of GLK1,a6-fold increase of expression level by derepression and further25-fold in-duction by ethanol was reported[35].HXK1,in compari-son,is10-fold repressed by glucose in dependence of Hxk2protein[36]and was listed as one of Thierfelder’s glucose dependent promoters with average strength,when it is induced at low glucose concentration[39].

P HXK1,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 P GLK1,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(Table4). 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].

Gal6and Gal80are 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 diphosphoglucose4-epimerase)[89],as shown in Figure3.Negative regulators for GAL genes have been shown to work in synergy with Mig1[71].Gal3is ex-pected to act as a signal transducer that forms a complex with galactose and Gal80,further releasing Gal4inside the nucleus and activating GAL1,7and10expression[90,91]. P GAL1and P GAL10are 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

Table4Yeast promoters induced in dependence of carbon sources and their regulator elements

Promoter Protein function Organism Induced by(strength)Repressed by Regulating

sequence DNA-bindingtarget protein

Ref.

GAL1Galactose

metabolism S.cerevisiae Galactose(1000×)Glucose?390to?255Gal4[70]

?201to?187Mig1[71]

GAL7Galactose

metabolism S.cerevisiae Galactose(1000×)Glucose?264to?161Gal4[70] https://www.sodocs.net/doc/9711612961.html,ctis Galactose No information available

GAL10Galactose

metabolism

S.cerevisiae Galactose(1000×)Glucose?324to?216Gal4[70]

Galactose

metabolism

C.maltosa Galactose Glucose No information available

PIS1Phosphoinositol

synthase S.cerevisiae Galactose,hypoxia(2×),

zinc depletion(2×)

(glycerol)?149to?138Rox1[72]

Gcr1

?224to?205Ste12

Pho2

?184to?149Mcm1(2×)

LAC4Lactose

metabolism https://www.sodocs.net/doc/9711612961.html,ctis Lactose,galactose(100×)-?173,?235RNA-Pol II[73]

?437to?420Lac9

?673to?656

?1088to?1072

MAL1Maltase H.polymorpha Maltose sucrose Glucose No information available[74] MAL62Maltase S.cerevisiae Maltose sucrose Glucose?759to?743Mal63[75]

[11]

AGT1Alpha-glucoside

transporter Brewing strains S.

cerevisiae,S.pastorianus

Maltose sucrose Glucose Divergent(strain

dependent)

Mig1[76]

Malx3

ICL1Isocitrat lyase P.pastoris Ethanol(200×)Glucose No information available[77]

C.tropicalis Ethanol Glucose No information available[78]

S.cerevisiae Ethanol(200×)Glucose?397to?388Cat8,Sip4[21]

[79]

?261to?242URS[79]

?96RNA-Pol II[77]

FBP1Fructose-1,6-

bisphosphatase S.cerevisiae Glycerol,acetate,ethanol

(10×)

Glucose?248to?231Hap2/3/4(2×)[80]

No information

available

Cat8,Sip4[21]

PCK1PEP carboxykinase S.cerevisiae Glycerol,acetate,ethanol

(10×)Glucose?480to?438Cat8,Sip4[21]

[80]

PEP carboxykinase C.albicans Succinate,casaminoacids Glucose?320to?123Hap2/3/4(2×)[80]

?444to?108Mig1(3×)[80]

GUT1Glycerol kinase S.cerevisiae Glycerol,acetate,ethanol,

oleate Glucose?221to?189Adr1[81]

?319to?309Ino2/4[81]

CYC1Cytochrome c S.cerevisiae O2(200×),lactate(5-10×)Glucose No information available[82] ADH4Alcohol

dehyrogenase

https://www.sodocs.net/doc/9711612961.html,ctis Ethanol-?953to?741UAS[83]

AOX1,2Alcohol oxidase P.pastoris Methanol Glucose?414to?171Mxr1[84]

[85] AUG1,2Alcohol oxidase P.methanolica Methanol Glucose No information available[84] DAS1Dihydroxy-

acetone-synthase

P.pastoris Methanol Glucose?980to?1Mxr1[84]

FDH Formate

dehydrogenase

H.polymorpha Methanol Glucose No information available[84]

concentration decreases during activation of the galact-ose utilizing pathway,the inducing effect diminishes over time.The high cost of galactose feeding demands a strat-egy to overcome this problem[93,94].Several authors have generated Saccharomyces cerevisiae gal1mutant strains that lack the ability to use galactose as a carbon source.Furthermore,MIG1and HXK2were disrupted to circumvent glucose repression[91,92,94].Consequently, P GAL10is induced even at low galactose concentrations, while presence of glucose does not affect promoter activ-ity.In this case,the optimum concentration of galactose for induction was reported to be0.05%(w/v)for express-ing human serum albumin[92].Interestingly,Ahn and colleagues found out that P GAL10works under anaerobic conditions as well,and easily keeps up with other pro-moters(P PGK,P PDC or P ADH1)for fermentative applica-tion.Therefore,P GAL10is another strong promoter suitable,for example,for microaerobic or anaerobic pro-cesses like bioethanol production[95].

Other yeast genera than Saccharomyces,like Kluyvero-myces or Candida,present homologous protein functions for galactose utilization.In https://www.sodocs.net/doc/9711612961.html,ctis,Lac9resembles the function of Gal4and is blocked by Kl Gal80,which is very similar to Gal80from S.cerevisiae.In contrast to S.cerevi-siae,there is no Gal3equivalent in https://www.sodocs.net/doc/9711612961.html,ctis.Galactose me-tabolism is mediated by Kl Gal1,Kl Gal7and Kl Gal10[11] [96].Besides,Kl Lac9additionally activates Kl Lac4,aβ-galactosidase,responsible for lactose-utilization(see also Section Induction by lactose).In contrast to S.cerevisiae, the regulatory genes of the https://www.sodocs.net/doc/9711612961.html,ctis GAL expression are not strongly repressed by glucose.

Gonzalez and colleagues took advantage of the resem-bling lac-gal-regulon in https://www.sodocs.net/doc/9711612961.html,ctis and applied the S.cere-visiae promoter P GAL1to express Trigonopsis variabilis D-aminoacid oxidase(DAO1)in https://www.sodocs.net/doc/9711612961.html,ctis[97].

GAL1and GAL10promoters of Candida maltosa have been successfully isolated,with the intention to create a functional expression system in this species,and were tested with https://www.sodocs.net/doc/9711612961.html,ctis LAC4as a reporter gene.Both pro-moters were applied to high level expression of several cytochrome P450s,encoded by the ALK gene cluster. P GAL1and P GAL10of C.maltosa were integrated into a low-copy and a high-copy plasmid,respectively,and CO-spectra were measured to prove the P450expression.In the low-copy plasmid the authors obtained an expression level of0.96–1.21nmol/mg wet cell weight,whereas quite

Table4Yeast promoters induced in dependence of carbon sources and their regulator elements(Continued)

FLD1Formaldehyde

dehydrogenase P.pastoris Methanol,methylamine,

choline

Glucose No information available[84]

POX2Peroxisomal

protein

Y.lipolytica Oleate Glucose No information available[86]

PEX8Peroxisomal

protein P.pastoris Oleate methanol(3-5×)Glucose?1000to?1Mxr1[87]

[88]

INU1Inulase K.marxianus Fructose,Inulin,Sucrose Glucose?271to?266RNA-Pol II[56]

?163to?153Mig1[58] Figure3Regulation and function of the GAL genes.

notably the high-copy plasmid enabled a3-fold amount of expressed protein[98].Other important expression hosts, such as P.pastoris,lack a functional pathway and the re-spective promoters for galactose metabolism.

PIS1(phosphatidylinositol-synthase,protein involved in the synthesis of phospholipids)presents an unusual behavior,since it is not subjected to conventional glucose repression in S.cerevisiae,but is known for increased transcription as soon as galactose is present in the medium.Interestingly,the presence of glycerol leads to a significant decrease of expression,while the expression level was not affected in glucose-containing medium. The regulatory mechanism is mainly mediated by Mcm1, a DNA-binding protein,which further interacts with an-other modulating protein,Sln1[99].Additionally,PIS1is repressed at anaerobic conditions[72]and is responsive to zinc(increased PIS1expression after zinc depletion was reported;[100]).Hence,PIS1provides a range of possibilities for regulated gene expression with one single promoter.

There are no PIS1promoter applications reported yet, but Stadlmayr and colleagues described the Pichia pas-toris P PIS1in a comparative Pichia promoter study,where the promoter activity was accounted for being rather low, when different carbon sources(glucose,glycerol and methanol)were tested[101,102].

Induction by lactose

A distinctive feature of https://www.sodocs.net/doc/9711612961.html,ctis is the ability to use lactose as a carbon source.Primarily the proteins of lactose and galactose metabolism are co-regulated by the lac-gal-regu-lon.A lactose permease(Lac12)is transporting lactose into the cell,where it is cleaved to glucose and galactose by aβ-galactosidase(Lac4).Subsequently,the galactose metabolism is activated,involving genes(Kl Gal1,Kl Gal7 and Kl Gal10)corresponding to the S.cerevisiae counter-parts described above[103].

The gene products of lactose and galactose metabolisms are controlled by an activator protein Lac9(=Kl Gal4)and all of them are induced by lactose or galactose. Interestingly,the lactose utilization genes are not re-pressed by glucose in https://www.sodocs.net/doc/9711612961.html,ctis,while the galactose me-tabolism is weakly repressed[11,104].The low catabolite repression,along with strong induction potential,is one of the advantages of many https://www.sodocs.net/doc/9711612961.html,ctis promoters.

The promoter P LAC4has been successfully used for re-combinant protein production in https://www.sodocs.net/doc/9711612961.html,ctis[105].Signifi-cant applications consisted,for example,in the controlled expression of prochymosin,important in the context of cheese production[106],or in the expression of Rhizopus oryzaeα-amylase[107].The consumption of the inducer is a problem in practical applications of P LAC4also in this organism:https://www.sodocs.net/doc/9711612961.html,ctis strains with disrupted KlGAL1were generated to prevent an early consumption of the inducer,following a similar strategy as the one showed for S.cere-visiae[108,109].

An interesting side effect is also the occurrence of Prib-now box-like sequences in the native promoter,which en-ables P LAC4to constitutively express heterologous proteins in E.coli.However,this feature is rather unwelcome for a typical protein expression process,since a prior correct as-sembling of the constructs in E.coli as an intermediate host can be problematic.With the goal to circumvent this inconvenience,a set of P LAC4-variants,mutated in Prib-now box-like sequence,has been developed.Such a pro-moter modification allowed to successfully express bovine enterokinase,whose expression had been problematic before[110].

Induction by maltose

Maltose utilization is a feature of several yeasts,among which S.cerevisiae,Hansenula polymorpha(the only methylotrophic species for which this phenotype was re-ported)and https://www.sodocs.net/doc/9711612961.html,ctis.The MAL gene group is repressed by glucose and induced by maltose and sucrose.There are up to5unlinked MAL loci in yeast(MAL1,MAL2, MAL3,MAL4,MAL6),and each of them consists of a permease(MALx1),a maltase(MALx2)and an activator protein(MALx3)[74].The promoter region for MALx1 and MALx2is a bidirectionally active intergenic region, consisting of an UAS,2symmetrically organized TATA-boxes,2Mig1binding sites and intermediate tandem repeats,which are assumed to regulate the expression level of MALx1and MALx2.This bidirectional pro-moter was applied to the simultaneous expression of re-porter genes MEL1and lacZ[111].Several authors highlighted the potential of MAL-promoters in expres-sion vectors for regulated protein synthesis,by using maltose as an inducer[112].For example,using P MAL62 from S.cerevisiae provided similar expression results as P GAL1,when LexA was expressed as a reporter gene. Notably,background expression driven by P MAL62was definitely higher(compared to P GAL1)under non-repressing and non-inducing conditions.Nevertheless, the expression of a very toxic protein,cyclin A from Drosophila was efficient for the maltose regulated pro-tein synthesis by P MAL62compared to its constitutive expression with P ADH1[113].

AGT1,which encodes aα-glucoside transporter,is highly homologous to the S.cerevisiae MAL61.P AGT1 sequence from several beer yeast strains(S.cerevisiae,S. pastorianus),was recently analyzed.AGT1is repressed by glucose in a similar manner in all tested strains(it showed to be Mig1-dependent),while derepression and maltose induction strength are strain-dependent,probably due to a certain divergence in AGT1promoter sequences.The regulation of maltose induction is dependent by the MAL activator proteins,[76].

In H.polymorpha,the native P MAL1performed very well–also in comparison to the commonly used P MOX, especially when the promoter was induced by sucrose. Furthermore,H.polymorpha P MAL1was transferable to another maltose utilizing yeast species–S.cerevisiae-, for recombinant expression of native maltase[75]. Induction by non-sugar carbon sources

Induction and derepression by ethanol,glycerol or acetate Glycerol is a very relevant inducer of many promoters; interestingly,glycerol is often used to“derepress”a pro-moter,prior to actively induce transcription activation by another inducer,such as ethanol,methanol or acetate. Most of related genes are involved in gluconeogenesis. Below,the most important promoter sequences belong-ing to this group will be described.

One meaningful promoter in this category is the pro-moter of ICL1,which encodes for isocitrate lyase,a key enzyme of the TCA and glyoxylate cycle,enabling the cell to grow on non-fermentable carbon sources.It is re-pressed by glucose,derepressed by depletion of glucose and strongly induced by ethanol or acetate.P ICL1is mainly regulated by the two C6-zinc finger proteins Cat8and Sip4 (see Table1),which bind to a UAS as soon as glucose is depleted and ethanol or acetate are available[79].

ICL1promoter sequences from several yeasts such as S. cerevisiae,P.pastoris,Yarrowia lipolytica or Candida tro-picalis are well established and frequently applied to pro-tein expression[77,78,114].In https://www.sodocs.net/doc/9711612961.html,ctis,ICL1is assumed to be regulated without a Mig1repressor,even if the dere-pression and induction are mediated by the Snf1/Snf4 complex[115];it is still unclear if other repressor proteins are involved with URS regulation of ICL1.

In S.cerevisiae,the5′upstream region of ICL from Candida tropicalis is often used as an inducible pro-moter;its optimum glucose concentration for derepres-sion was measured at0.5%(w/v),when Rhizopus oryzae lipase was expressed[116].While expressing secreted β-galactosidase,the induction with acetate leads to a 300-fold enhancement of product activity.It needs to be mentioned that this level of expression is protein-dependent(e.g.expression of lipase yielded only a frac-tion of the protein amount after induction compared to its expression under derepressed promoter condition [116]).Therefore,the volumetric activity of an expressed enzyme does not necessarily correlate with the strength of transcription.This might also be a reason why in Pichia pastoris the native P ICL1was praised as a good al-ternative for methanol free protein production[77],while on the other hand,according to a recent review,the tran-scription levels of this promoter in Pichia pastoris appear to be lower than with the classic P AOX1or P GAP[117]. The P ICL1of Y.lipolytica is a standard promoter used for this host and was reported to be induced about10-fold by ethanol,whenβ-galactosidase was expressed [114].Besides,it has been reported to be inducible by fatty acids and alkanes[118].

A special case is represented by ALG2in H.polymorpha, which encodes another isocitrate lyase with50–60%se-quence homology to ICL of other yeasts.The promoter of ALG2is activated by derepression at low glucose level (0.2%w/v)rather than by ethanol induction[37].

The promoter region of FBP1,encoding fructose-1,6-bisphosphatase,was analyzed in several occasions,con-cerning upstream regulating sequences[80,119].P FBP1is repressed by sugars like glucose,shows a Mig1binding site in the upstream sequence from?200to?184[11] and carries a Cat8and Sip4recognition site(UAS2)for activation of transcription when non-fermentable carbon sources(ethanol,acetate,glycerol)are available[21,22]. Additionally,P FBP1was reported to have another regula-tory sequence(UAS1),showing a different sensitivity to glucose than UAS2[119],a genetic arrangement unique within this group of presented promoters.

Within the group of budding yeast,no practical applica-tions of P FBP1have been found:only the fbp1+promoter from fission yeast was mentioned several times as an op-portunity for controlled gene expression in S.pombe [120].However,this might also be explained by the fact that Fbp1activity in glycolysis is also strongly regulated on the protein level,and not mainly by transcription.

The PEP carboxykinase(PCK1)promoter,which is in-ducible in absence of glucose by glycerol,ethanol,acet-ate or lactate as well,was already isolated from several yeasts,like S.cerevisiae[80]or C.albicans:in particular, the P CaPCK1gained popularity within Candida commu-nity.By means of the S.cerevisiae PCK1promoter,Cat8 and Sip4have been identified as responsible activator proteins for transcription as well[21].It has however to be mentioned,that,at least in the case of CaPCK1pro-moter,other inducers,such as casamino acids or succin-ate,have been proved to be more efficient regarding expression of LAC4in C.albicans[121].This observa-tion was confirmed by an example,where the CaPCK1 promoter was applied to Ca Cse4-expression in C.albi-cans by succinate induction[122]and furthermore by Ca Cdc42-expression,which was driven by casamino acid induction[123].

Technically speaking,also the promoters of the gluco-neogenetic proteins Acs1(acetyl-CoA-synthase)or Mls1 (malate synthase)belong to this group,and have been characterized regarding their upstream regulatory se-quences[124,125],but to the best of our knowledge they have not been applied for protein production yet.

The S.cerevisiae glycerol kinase(GUT1)is another ex-ample of a gene whose expression is mainly induced by glycerol,but also by ethanol,lactate,acetate or oleate. Complete depletion of glucose is necessary to derepress

the promoter.The regulation mechanism is subjected to Adr1and Ino2/4activation,and repression by Opi1ac-tivity.Even if there might be a Mig1-binding site,this re-pressor seems to play a minor role[81].The use of the P. pastoris P GUT1promoter was proposed quite recently and was successfully applied to expression ofβ-lactamase as a model protein[126].

The CYC1(cytochrome c)gene product is an import-ant element of the electron transport in S.cerevisiae and is repressed under anaerobic conditions and in presence of glucose.The intracellular heme level mediates the O2-dependent activation of UAS1element in the CYC1pro-moter region by binding of Hap1.UAS2binds the Hap2/ 3/4/5complex,and is activated by any non-fermentable carbon source[82].Induction with O2increases expres-sion about200-fold,whereas lactate-induction is not as effective(5–10fold)[127].

In the respiratory yeast https://www.sodocs.net/doc/9711612961.html,ctis,CYC1is expressed to a high level too,but glucose repression is also in this case almost irrelevant because the major part of expres-sion is fulfilled by O2-induction and UAS1activation [128].

Cytochrome c is a highly conserved protein in several eukaryotes,and is therefore easy to transfer between dif-ferent yeast species.In many S.cerevisiae vectors,the terminator of CYC1gene is used for termination of tran-scription.Nonetheless,the promoter region of CYC1is not particularly exploited,in any case often evaluated as hybrid promoter with GAL10(UAS G-GAL10/CYC1). This construct consists of365bp of GAL10,including a UAS sequence,and the core promoter of CYC1(TATA-Box,transcription start site and first four basepairs of CYC1gene).Da Silva&Bailey have applied such hybrid promoter,among others,in order to determine the influ-ence of different promoter strengths on fermentative pro-tein expression in yeast,and as a result UAS G-GAL10/ CYC1promoter showed moderate strength compared to P GAL1,when it was induced with galactose[129].Never-theless,one example of successful application of the hy-brid promoter is the expression of HbsAg and preS2-S in S.cerevisiae for HBV vaccine preparation[130].

The use of the https://www.sodocs.net/doc/9711612961.html,ctis ADH4promoter was patented by Falcone and colleagues[131].It is located in the mito-chondria,is not repressed by glucose but strongly induced by ethanol.The important control region for regulation of ethanol induction was found to be located between?953 and?741[83].

For the sake of completeness,it has to be men-tioned that also the S.cerevisiae ADH2promoter is induced by ethanol,but due to its efficient repres-sion/derepression mechanism this promoter was de-scribed in Section Promoters derepressed by carbon source depletion.The same applies to the hexokinase genes HXK1and GLK1.Induction by methanol

This promoter type has been sufficiently reviewed in the past by several authors,and will therefore be men-tioned only briefly.For further detailed information,we refer the reader to the corresponding literature(see below).

The use of methanol as an inducer is confined to methy-lotrophic yeasts,like Pichia pastoris,Pichia methanolica, Hansenula polymorpha or Candida boidinii,which are able to metabolize methanol as a carbon source[132].The most established promoters comprise those from genes encoding alcohol oxidases(namely P AOX1and?2in P.pas-toris,P AUG1and?2in P.methanolica,P MOX in H.polymor-pha,P AOD1in C.boidinii),dihydroxyacetone synthases (P DAS1and P DAS2in P.pastoris,P DAS in H.polymorpha, P DAS1in C.boidinii)and formate dehydrogenases(P FDH in H.polymorpha,P FDH1in C.boidinii).All of them are ele-ments of the methanol utilization(MUT)pathway,and are repressed by glucose and strongly induced by addition of methanol(importantly,they are also derepressed by a non-fermentable carbon source,e.g.glycerol).Especially H.polymorpha P MOX shows a significant derepression ef-fect in presence of glycerol,since protein activity is already 80%of the methanol induced status.A special case in this context is the group of formaldehyde dehydrogenases (P FLD1in P.pastoris,P FLD in P.methanolica,P FLD in H. polymorpha),which are not only negatively regulated by glucose,but additionally are responsive to methylamine or choline induction[84,101,133].

At present,a set of engineered promoter variants based on these natural sequences of the MUT pathway genes have been developed.Such modified promoters (e.g.P MOX in H.polymorpha and P AOX1in P.pastoris) are no longer methanol inducible,showing in most cases either an inducible phenotype from molecules other than methanol,or a more pronounced derepressed phenotype[134,135].

In case of P FLD,Resina and colleagues exploited an ad-vantageous characteristic of the promoter(P FLD is indu-cible by methylamine)thereby circumventing methanol induction[136].

PEX8is a peroxisomal protein(formerly PER3)in P. pastoris,whose promoter leads to a moderate expression level on glucose.A weak induction by methanol or ole-ate(3–5fold)has been reported[87,118].The main regulator protein in P PEX8is Mxr1,which is characteris-tic for all methanol inducible genes in Pichia and binds the promoter in a5′-CYCCNY-3′motif[88].It remains to be demonstrated if multiple Mxr1binding sites such as in the P DAS and P AOX promoters would increase P PEX8 strength.

P PEX8was chosen for instance in the framework of Pex14characterization,and was applied under methanol-and oleate-inducing conditions,respectively[137].

Induction by oleate

Oaf1and Pip2are important DNA-binding proteins for the transcriptional activation of oleate responsive pro-teins in yeast.In many cases(e.g.CTA1;peroxisomal catalase,POX1;peroxisomal acyl CoA oxidase,FOX3;3-ketoacyl CoA thiolase,PEX1;peroxisomal biogenesis factor1)also Adr1is involved in initiating gene tran-scription[138].Most of these proteins are functionally connected to the peroxisomes and are mainly involved inβ-oxidation.For example POX1,FOX3(=POT1), ECI1and PEX11are strongly induced by oleate and re-pressed by glucose,whereupon a significant derepression already occurs in presence of glycerol.Besides,PEX5, CRC1,CTA1and QDR1are also induced by oleate,al-though at a lower level[12].

In terms of industrial applications,no relevant oleate inducible promoters have been reported for S.cerevisiae so far.Up to now,especially,POX2and POT1promoters from Y.lipolytica,which are also activated by oleate, have been validated for recombinant protein synthesis of lipase in Y.lipolytica[138].In the meantime P POX2has been frequently used,especially,when hydrophobic sub-strate conditions were required.The performance of P POX2was further improved,testing human interferone alpha2b expression,by co-feeding glucose at a limited rate during induction with oleate[139].

Conclusions

This review describes the current state of art for a set of potential promoters for controlled protein synthesis,out of several yeasts.Especially in case of inducible pro-moters,the presented genetic tools are already well established,with several examples now summarized within this work.Nevertheless,also some less popular promoters show interesting features,which might be en-hanced by promoter engineering:such a technique,des-pite its potential,is not yet very common for promoter improvements.

Generally,any gene subjected to derepression at low glucose concentrations,opens up the potential of carrying a strong promoter sequence.Referring to transcriptome analysis covering31%of the genome[140,141],about163 genes from S.cerevisiae were upregulated at glucose-limited conditions.Many of these genes are still poorly characterized,and their function is not known yet.For in-stance,YGR243promoter from S.cerevisiae was already introduced as an interesting promoter tool[39],where-upon P YGR243could easily keep up with P HXK1.

A comprehensive knowledge of promoter elements is also helpful in terms of the development of synthetic promoters,since this field of research is relatively new, but gained increased popularity within the last ten years. Sequences of strong natural promoters are combined, and transcription factor binding sites are deleted or amplified with the objective of obtaining a new,more convenient promoter sequence[142].

Very recently,Blazeck and colleagues presented a set of synthetic yeast promoters by assembling very strong transcriptional enhancing elements(coming from CLB2, CIT1,GAL1,respectively)with the core of a particular promoter.The essential finding was a direct proportion between the number of additional UAS and promoter activity[143].Interestingly,most yeast promoter studies are still focused on endogenous promoters and rarely on heterologous applications or fully orthogonal systems.

A broad knowledge of different potentials of promoter elements paves the way for creating a comprehensive promoter tool box and facilitates protein synthesis for appropriate applications.

Competing interests

The authors declare that they have no competing interests.

Authors’contributions

KW and AC collected all the relevant publications,arranged the general structure of the review and drafted the text;AC,AG and MW revised and amended the general flow.KW produced tables and figures.All authors read and approved the final manuscript.

Acknowledgements

This work has been supported by the Federal Ministry of Economy,Family and Youth(BMWFJ),the Federal Ministry of Traffic,Innovation and Technology(bmvit),the Styrian Business Promotion Agency SFG,the Standortagentur Tirol and ZIT–Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG and European Union Seventh Framework Programme(FP7/2007-2013)under grant agreement n°289646.

Author details

1Austrian Centre of Industrial Biotechnology,Graz,Austria.2Institute of Molecular Biotechnology,Technical University Graz,Graz,Austria. Received:1October2013Accepted:16December2013

Published:9January2014

References

1.Waterham HR,Digan ME,Koutz PJ,Lair SV,Cregg JM:Isolation of the Pichia

pastoris glyceraldehyde-3-phosphate dehydrogenase gene and

regulation and use of its promoter.Gene1997,186:37–44.

2.Tuite M,Dobson MJ,Roberts NA,King RM,Burke DC,Kingsman SM,

Kingsman AJ:Regulated high efficiency expression of human interferon-alpha in Saccharomyces cerevisiae.EMBO J1982,1(5):603–608.

3.Gatignol A,Dassain M,Tiraby G:Cloning of Saccharomyces cerevisiae

promoters using a probe vector based on phleomycin resistance.Gene 1990,91(1):35–41.

4.Li N,Zhang LM,Zhang KQ,Deng JS,Pr?ndl R,Sch?ffl F:Effects of heat

stress on yeast heat shock factor-promoter binding in vivo.Acta Biochim Biophys Sin2006,38(5):356–362.

5.Koller A,Valesco J,Subramani S:The CUP1promoter of Saccharomyces

cerevisiae is inducible by copper in Pichia pastoris.Yeast2000,16:651–656.

6.Solow SP,Sengbusch J,Laird MW:Heterologous protein production from

the inducible MET25promoter in Saccharomyces cerevisiae.Biotechnol

Prog2005,21:617–620.

7.Da Silva NA,Srikrishnan S:Introduction and expression of genes for

metabolic engineering applications in Saccharomyces cerevisiae.FEMS

Yeast Res2012,12:197–214.

8.Carlson M:Glucose repression in yeast.Curr Opin Microbiol1999,

2:202–207.

9.Wilson WA,Hawley SA,Hardie DG:Glucose repression/derepression in

budding yeast:SNF1protein kinase is activated by phosphorylation

under derepressing conditions,and this correlates with a high AMP:ATP ratio.Curr Biol1996,6(11):1426–1434.

10.Westholm J,Nordberg N,Murén E,Ameur A,Komorowski J,Ronne H:

Combinatorial control of gene expression by the three yeast repressors Mig1,Mig2and Mig3.BMC Genomics2008,9:601–614.

11.Gancedo JM:Yeast carbon catabolite repression.Microbiol Mol Biol Rev

1998,62(2):334–361.

12.Karpichev IV,Durand-Heredia JM,Luo Y,Small GM:Binding characteristics

and regulatory mechanisms of the transcription factors controlling

oleate-responsive genes in Saccharomyces cerevisiae.J Biol Chem2008,

283(16):10264–10275.

13.?zcan S:Two different signals regulate expression and induction of gene

expression by glucose.J Biol Chem2002,277(49):46993–46997.

14.Zhang P,Zhang W,Zhou X,Bai P,Cregg JM,Zhang Y:Catabolite

repression of Aox in Pichia pastoris is dependent on hexose transporter Pp Hxt1and pexophagy.Appl Environ Microbiol2010,76(18):6108–6118. 15.Suppi S,Michelson T,Viigand K,Alam?e T:Repression vs.activation of

MOX,FMD,MPP1and MAL1promoters by sugars in Hansenula

polymorpha:the outcome depends on cell’s ability to phosphorylate

sugar.FEMS Yeast Res2012,13(2):219–232.

16.Hedbacker K,Carlson M:SNF1/AMPK pathways in yeast.Front Biosci2008,

13:2408–2420.

17.Treitel MA,Carlson M:Repression by SSN6-TUP1is directed by MIG1,a

repressor/activator protein.Genetics1995,92:3132–3136.

18.Cassart JP,Georis I,Ostling J,Ronne H,Vandenhaute J:The MIG1repressor

from Kluyveromyces lactis:cloning,sequencing and functional analysis in Saccharomyes cerevisiae.FEBS Lett1995,371:191–194.

19.Erickson JR,Johnston M:Suppressors reveal two classes of glucose

repression genes in the yeast Saccharomyces cerevisiae.Genetics1994,

136:1271–1278.

20.Bu Y,Schmidt MC:Identification of cis-acting elements in the SUC2

promoter of Saccharomyces cerevisiae required for activation of

transcription.Nucleic Acids Res1998,26(4):1002–1009.

21.Roth S,Kumme J,Schüller HJ:Transcriptional activators Cat8and Sip4

discriminate between sequence variants of the carbon source-

responsive promoter element in the yeast Saccharomyces cerevisiae.

Curr Genet2004,45(3):121–128.

22.Schüller HJ:Transcriptional control of nonfermentative metabolism in the

yeast Saccharomyces cerevisiae.Curr Genet2003,43:139–160.

23.Fleming AB:Pennings pp.Tup1–Ssn6and Swi-Snf remodelling activities

influence long-range chromatin organization upstream of the yeast

SUC2gene.Nucleic Acids Res2007,35(16):5520–5531.

24.Walther K,Schüller HJ:Adr1and Cat8synergistically activate the

glucose-regulated alcohol dehydrogenase gene ADH2of the yeast

Saccharomyces cerevisiae.Microbiology2001,147:2037–2044.

https://www.sodocs.net/doc/9711612961.html,i MT,Liu DYT,Hseu TH:Cell growth restoration and high level protein

expression by the promoter of hexose transporter,HXT7,from

Saccharomyces cerevisiae.Biotechnol Lett2007,29(8):1287–1292.

26.Hauf J,Zimmermann FK,Müller S:Simultaneous genomic overexpression

of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae.

Enzyme Microb Tech2000,26:688–698.

27.Ruiz A,Serrano R,Arino J:Direct regulation of genes involved in glucose

utilization by the calcium/calcineurin pathway.J Biol Chem2008,

283(20):13923–13933.

28.Kim JH:DNA-binding properties of the yeast Rgt1repressor.Biochimie

2009,91(2):300–303.

29.Ozcan S,Johnston M:Two different repressors collaborate to restrict

expression of the yeast glucose transporter genes HXT2and HXT4to

low levels of glucose.Mol Cell Biol1996,16(10):5536–5545.

30.Weirich J,Goffrinil P,Kuger P,Ferrero I,Breunig KD:Influence of mutations

in hexose-transporter genes on glucose repression in Kluyveromyces

lactis.Eur J Biochem1997,249:248–257.

31.Fan J,Chaturvedi V,Shen SH:Identification and phylogenetic analysis of a

glucose transporter gene family from the human pathogenic yeast

Candida albicans.J Mol Evol2002,55(3):336–346.

32.Bojunga N,Entian K:Cat8p,the activator of gluconeogenic genes in

Saccharomyces cerevisiae,regulates carbon source-dependent expression of NADP-dependent cytosolic isocitrate dehydrogenase(Idp2p)and

lactate permease(Jen1p).Mol Gen Genet1999,262:869–875.

33.Hartner FS,Ruth C,Langenegger D,Johnson SN,Hyka P,Lin-Cereghino GP,

Lin-Cereghino J,Kovar K,Cregg JM,Glieder A:Promoter library designed

for fine-tuned gene expression in Pichia pastoris.Nucleic Acids Res2008, 36(12):e76.

34.G?decke S,Eckart M,Janowicz ZA,Hollenberg CP:Identification of

sequences responsible for transcriptional regulation of the strongly

expressed methanol oxidase-encoding gene in Hansenula polymorpha.

Gene1994,139:35–42.

35.Herrero P,Flores L,Cera T,Moreno F:Functional characterization of

transcriptional regulatory elements in the upstream region of the yeast GLK1gene.Biochem J1999,343:319–325.

36.Rodriguez A,Cera T,Herrero P,Moreno F:The hexokinase2protein

regulates the expression of the GLK1,HXK1and HXK2genes of

Saccharomyces cerevisiae.Biochem J2001,355:625–631.

37.Berardi E,Gambini A,Bellu AR:ALG2,the Hansenula polymorpha isocitrate

lyase gene.Yeast2003,20(9):803–811.

38.Infante JJ,Law GL,Wang IT,Chang HWE,Young ET:Activator-independent

transcription of Snf1-dependent genes in mutants lacking histone tails.

Mol Microbiol2011,80(2):407–422.

39.Thierfelder S,Ostermann K,G?bel A,R?del G:Vectors for glucose-

dependent protein expression in Saccharomyces cerevisiae.Appl Biochem Biotech2011,163(8):954–964.

40.Prielhofer R,Maurer M,Klein J,Wenger J,Kiziak C,Gasser B,Mattanovich D:

Induction without methanol:novel regulated promoters enable high-

level expression in Pichia pastoris.Microb Cell Fact2013,12(1):5.

41.Diderich JA,Schepper M,van Hoek P,Luttik MA,van Dijken JP,Pronk JT,

Klaassen P,Boelens HF,de Mattos MJ,van Dam K,et al:Glucose uptake

kinetics and transcription of HXT genes in chemostat cultures of

Saccharomyces cerevisiae.J Biol Chem1999,274(22):15350–15359.

42.Flick KM,Spielewoy N,Kalashnikova TI,Guaderrama M,Zhu Q,Chang HC,

Wittenberg C:Grr1-dependent inactivation of Mth1mediates glucose-

induced dissociation of Rgt1from HXT gene promoters.Mol Biol Cell

2003,14:3230–3241.

43.Greatrix BW,Vuuren HJJ:Expression of the HXT13,HXT15and HXT17

genes in Saccharomyces cerevisiae and stabilization of the HXT1gene

transcript by sugar-induced osmotic stress.Curr Genet2006,

49(4):205–217.

44.Partow S,Siewers V,Bj?rn S,Nielsen J,Maury J:Characterization of

different promoters for designing a new expression vector in

Saccharomyces cerevisiae.Yeast2010,27(11):955–964.

45.Sanchez RG,Hahn-Hagerdal B,Gorwa-Grauslund MF:PGM2overexpression

improves anaerobic galactose fermentation in Saccharomyces cerevisiae.

Microb Cell Fact2010,9:40–47.

46.Scalcinati G,Knuf C,Partow S,Chen Y,Maury J,Schalk M,Daviet L,Nielsen J,

Siewers V:Dynamic control of gene expression in Saccharomyces

cerevisiae engineered for the production of plant sesquitepeneα-

santalene in a fed-batch mode.Metab Eng2012,14(2):91–103.

47.Leandro MJ,Fonseca C,Goncalves P:Hexose and pentose transport in

ascomycetous yeasts:an overview.FEMS Yeast Res2009,9(4):511–525. 48.Lin Z,Li WH:Expansion of hexose transporter genes was associated with

the evolution of aerobic fermentation in yeasts.Mol Biol Evol2011,

28(1):131–142.

https://www.sodocs.net/doc/9711612961.html,kowski C,Krampe S,Weirich J,Hasse V,Boles E,Breunig KD:Feedback

regulation of glucose transporter gene transcription in Kluyveromyces

lactis by glucose uptake.J Bacteriol2001,183(18):5223–5229.

50.Heiland S,Radovanovic N,Fer MH,Winderickx J,Lichtenberg AH:Multiple

Hexose Transporters of Schizosaccharomyces pombe.J Bacteriol2000,

182(8):2153–2162.

51.?zcan S,Vallier LG,Flick JS,Carlson M,Johnston M:Expression of the SUC2

gene of Saccharomyces cerevisiae is induced by low levels of glucose.

Yeast1997,13:127–137.

52.Lutfiyya LL,Johnston M:Two zinc-finger-containing repressors are

responsible for glucose repression of SUC2expression.Mol Cell Biol1996, 16(9):4790–4797.

53.Belinchon MM,Gancedo JM:Different signalling pathways mediate

glucose induction of SUC2,HXT1and pyruvate decarboxylase in yeast.

FEMS Yeast Res2007,7(1):40–47.

54.Zhang X,Xia Z,Zhao B,Cen P:Enhancement of production of cloned

α-amylase by lactic acid feeding from recombinant Saccharomyces

cerevisiae using a SUC2promoter.Biotechnol Lett2001,23:259–262.

55.Iacovoni JS,Russell P,Gaits F:A new inducible protein expression

system in fission yeast based on the glucose-repressed inv1promoter.

Gene1999,232:53–58.

56.Bergkamp RJ,Bootsman TC,Toschka HY,Mooren AT,Kox L,Verbakel JM,

Geerse R,Planta RJ:Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus.

Appl Microbiol Biotechnol1993,40:309–317.

57.Kim HE,Qin R,Chae KS:Increased production of exoinulinase in

Saccharomyces cerevisiae by expressing the Kluyveromyces marxianus

INU1gene under the control of the INU1promoter.J Microbiol Biotechn 2005,15(2):447–450.

58.Rocha SN,Abrah?o-Neto J,Cerdán ME,González-Siso MI,Gombert AK:

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.Microb Cell Fact2010,9:4–15.

59.Chambers P,Issaka A,Palecek SP:Saccharomyces cerevisiae JEN1

promoter activity is inversely related to concentration of repressing

sugar.Appl Environ Microbiol2004,70(1):8–17.

60.Tachibana C,Yoo JY,Tagne JB,Kacherovsky N,Lee TI,Young ET:Combined

global localization analysis and transcriptome data identify genes that

are directly coregulated by Adr1and Cat8.Mol Cell Biol2005,

25(6):2138–2146.

61.Kaniak A,Xue Z,Macool D,Kim JH,Johnston M:Regulatory network

connecting two glucose signal transduction pathways in Saccharomyces cerevisiae.Eucaryotic Cell2004,3(1):221–231.

62.Andrade R,K?tter P,Entian KD,Casal M:Multiple transcripts regulate

glucose-triggered mRNA decay of the lactate transporter JEN1

from Saccharomyces cerevisiae.Biochem Bioph Res Co2005,

332:254–262.

63.Badziong W,Habermann P,M?ller J,Aretz W:Process for using the yeast

ADH2promoter system for the production of heterologous proteins in high yield(US5866371).US Patent Office2012:1–16.

64.Donoviel MS,Young ET:Isolation and identification of genes activating

UAS2-dependent ADH2expression in Saccharomyces cerevisiae.Genetics 1996,143:1137–1148.

65.Shen MWY,Fang F,Sandmeyer S,Da Silva NA:Development and

characterization of a vector set with regulated promoters for systematic metabolic engineering in Saccharomyces cerevisiae.Yeast2012,

29(12):495–503.

66.Russell PR,Hall B:The primary structure of the alcohol dehydrogenase

gene from the fission yeast Schizosaccharomyces pombe.J Biol Chem

1983,258(1):143–149.

67.Chien LJ,Lee CK:Expression of bacterial hemoglobin in the yeast,Pichia

pastoris,with a low O2-induced promoter.Biotechnol Lett2005,

27(19):1491–1497.

68.Quintana-Castro R,Ramirez-Suero M,Moreno-Sanz F,Ramirez-Lepe M:

Expression of the cry11A gene of Bacillus thuringiensis ssp.

israelensis in Saccharomyces cerevisiae.Can J Microbiol2005,

51:165–170.

69.Jimenez-Flores R,Richardson T,Bisson LF:Expression of bovine&casein in

Saccharomyces cerevisiae and characterization of the protein produced in vivo.J Agr Food Chem1990,38:1134–1141.

70.Johnston M:A model fungal gene regulatory mechanism:the GAL genes

of Saccharomyces cerevisiae.Microbiol Rev1987,51(4):458–476.

71.Nehlin JO,Carlberg M,Ronne H:Control of yeast GAL genes by MIG1

repressor:a transcriptional cascade in the glucose response.EMBO J

1991,10(11):3373–3377.

72.Gardocki ME,Lopes JM:Expression of the yeast PIS1gene requires

multiple regulatory elements including a Rox1p binding site.J Biol Chem 2003,278(40):38646–38652.

73.Leonardo JM,Bhairi SM,Dickson RC:Identification of upstream activator

sequences that regulate induction of the3-Galactosidase gene in

Kluyveromyces lactis.Mol Cell Biol1987,7(12):4369–4376.

74.Vanoni M,Sollitti P,Goldenthal M,Marmur J:Structure and regulation of

the multigene family controlling maltose fermentation in budding yeast.

Prog Nucleic Acid Res Mol Biol1989,37:281–322.

75.Alamae T,Parn P,Viigand K,Karp H:Regulation of the maltase gene

promoter in H.polymorpha and Saccharomyces cerevisiae.FEMS Yeast Res 2003,4(2):165–173.

76.Vidgren V,Kankainen M,Londesborough J,Ruohonen L:Identification of

regulatory elements in the AGTI promoter of ale and lager strains of

brewer’s yeast.Yeast2011,28:579–594.

77.Menendez J,Valdes I,Cabrera N:The ICL1gene of Pichia pastoris,

transcriptional regulation and use of its promoter.Yeast2003,

20(13):1097–1108.78.Umemura K,Atomi H,Kanai T,Teranishi Y,Ueda M,Tanaka A:A novel

promoter,derived from the isocitrate lyase gene of Candida tropicalis,

inducible with acetate in Saccharomyces cerevisiae.Appl Microbiol

Biotechnol1995,43:489–492.

79.Ordiz I,Herrero P,Rodicio R,Gancedo JM,Moreno F:A27kDa protein

binds to a positive and a negative regulatory sequence in the promoter of the ICL1gene from Saccharomyces cerevisiae.Biochem J1998,

329:383–388.

80.Mercado JJ,Gancedo JM:Regulatory regions in the yeast FRPl and PCKl

genes.FEBS J1992,311(2):110–114.

81.Grauslund M,Lopes JM,Ronnow B:Expression of GUT1,which encodes

glycerol kinase in Saccharomyces cerevisiae,is controlled by the

positive regulators Adr1p,Ino2p and Ino4p and the negative regulator Opi1p in a carbon source-dependent fashion.Nucleic Acids Res1999,

27(22):4391–4398.

82.Martens C,Krett B,Laybourn PJ:RNA polymerase II and TBP occupy the

repressed CYC1promoter.Mol Microbiol2001,40(4):1009–1019.

83.Saliola M,Mazzoni C,Solimando N,Crisa A,Falcone C,Jung G,Fleer R:Use

of the Kl ADH4promoter for ethanol-dependent production of recombin-ant human serum albumin in Kluyveromyces lactis.Appl Environ Microbiol 1999,65(1):53–60.

84.Hartner FS,Glieder A:Regulation of methanol utilisation pathway genes

in yeasts.Microb Cell Fact2006,5(1):39–59.

85.Xuan Y,Zhou X,Zhang W,Zhang X,Song Z,Zhang Y:An upstream

activation sequence controls the expression of AOX1gene in Pichia

pastoris.FEMS Yeast Res2009,9(8):1271–1282.

86.Juretzek T,Wang HJ,Nicaud JM,Mauersberger S,Barth G:Comparison of

promoters suitable for regulated overexpression ofβ-galactosidase in

the alkane-utilizing yeast Yarrowia lipolytica.Biotechnol Bioproc E2000,

5:320–326.

87.Liu H,Tan X,Russell KA,Veenhuis M,Cregg JM:PER3,a gene required for

peroxisome biogenesis in Pichia pastoris,encodes a peroxisomal

membrane protein involved in protein import.J Biol Chem1995,

270(18):10940–10951.

88.Kranthi BV,Vinod Kumar HR,Rangarajan PN:Identification of Mxr1p-

binding sites in the promoters of genes encoding dihydroxyacetone

synthase and peroxin8of the methylotrophic yeast Pichia pastoris.

Yeast2010,27(9):705–711.

89.Ostergaard S,Olsson L,Johnston M,Nielsen J:Increasing galactose

consumption by Saccharomyces cerevisiae through metabolic

engineering of the GAL gene regulatory network.Nat Biotechnol2000,

18:1283–1286.

90.Li Y,Chen G,Liu W:Multiple metabolic signals influence GAL gene

activiation by modulating the interaction of Gal80p with the

transcriptional activator Gal4p.Mol Microbiol2010,78(2):414–428.

91.Matsuyama T,Yamanishi M,Takahashi H:Improvement of galactose

induction system in Saccharomyces cerevisiae.J Biosci Bioeng2011,

111(2):175–177.

92.Ah Kang H,Kyu Kang W,Go SM,Rezaee A,Hari Krishna S,Ki Rhee S,Kim JY:

Characteristics of Saccharomyces cerevisiae gal1Δand gal1Δhxk2Δ

mutants expressing recombinant proteins from the GAL promoter.

Biotechnol Bioeng2005,89(6):619–629.

93.Whang J,Ahn J,Chun CS,Son YJ,Lee H,Choi ES:Efficient,galactose-

free production of Candida antarctica lipase B by GAL10promoter in Dgal80mutant of Saccharomyces cerevisiae.Process Biochem2009,

44:1190–1192.

94.Hovland P,Flick J,Johnston M,Sclafani RA:Galactose as a gratuitous

inducer of GAL gene expression in yeasts growing on glucose.

Gene1989,83:57–64.

95.Ahn J,Hong J,Park M,Lee H,Lee E,Kim C,Lee J,Choi ES,Jung JK,Lee H:

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.Appl Environ Microbiol2009,75(11):3528–3534.

96.Salmeron JM,Johnston SA:Analysis of the Kluyveromyces lactis positive

regulatory gene LAC9reveals functional homology to,but sequence

divergence from,the Saccharomyces cerevisiae GAL4gene.Nucleic Acids Res 1986,14(19):7767–7781.

97.Gonzalez F,Montes J,Martin F,Lopez M,Ferminan E,Catalan J,Galan M,

Dominguez A:Molecular cloning of Tv DAO1,a gene encoding a

D-amino acid oxidase from Trigonopsis variabilis and its expression in

Saccharomyces cerevisiae and Kluyveromyces lactis.Yeast1997,

13:1399–1408.

98.Park SM,Ohkuma M,Masuda Y,Ohta A,Takagi M:Galactose-inducible

expression systems in Candida maltosa using promoters of

newly-isolated GAL1and GAL10genes.Yeast1997,13:21–29.

99.Anderson MS,Lopes JM:Carbon source regulation of PIS1gene

expression in Saccharomyces cerevisiae involves the MCM1gene

and the two-component regulatory gene,SLN1.J Biol Chem1996,

271(43):26596–26601.

100.Han SH,Han GS,Iwanyshyn WM,Carman GM:Regulation of the PIS1-encoded phosphatidylinositol synthase in Saccharomyces cerevisiae by zinc.J Biol Chem2005,280(32):29017–29024.

101.Stadlmayr G,Mecklenbr?uker A,Rothmüller M,Maurer M,Sauer M, Mattanovich D,Gasser B:Identification and characterisation of novel

Pichia pastoris promoters for heterologous protein production.

J Biotechnol2010,150(4):519–529.

102.Gasser B,Mattanovich D,Sauer M,Stadlmayr G:Expression system (US20100297738A1).US Patent Office2012:1–86.

103.Dickson R,Riley M:The lactose-galactose regulon of Kluyveromyces lactis.

Biotechnology1989,13:19–40.

104.Dong J,Dickson RC:Glucose represses the lactose–galactose regulon in Kluyveromyces lactis through a SNF1and MIG1-dependent pathway that modulates galactokinase(GAL1)gene expression.Nucleic Acids Res1997, 25(18):3657–3664.

105.Van Ooyen AJJ,Dekker P,Huang M,Olsthoorn MMA,Jacobs DI,Colussi PA, Taron CH:Heterologous protein production in the yeast Kluyveromyces lactis.FEMS Yeast Res2006,6(3):381–392.

106.van den Berg JA,van der Laken KJ,van Ooyen AJ,Renniers TC,Rietveld K, Schaap A,Brake AJ,Bishop RJ,Schultz K,Moyer D,et al:Kluvyveromyces as

a host for heterologous gene expression:Expression and secretion of

prochymosin.Biotechnology1990,8:135–139.

107.Li S,Shen W,Chen X,Shi G,Wang Z:Secretory expression of Rhizopus oryzae alpha-amylase in Kluyveromyces lactis.Afr J Biotechnol2011,

10(20):4190–4196.

108.Hsieh HB,Da Silva NA:Development of a LAC4promoter-based gratuitous induction system in Kluyveromyces lactis.Biotechnol Bioeng 2000,67(4):408–416.

109.Panuwatsuk W,Da Silva NA:Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted

proteins during fed-batch culture.Biotechnol Bioeng2003,81(6):712–718. 110.Colussi PA,Taron CH:Kluyveromyces lactis LAC4promoter variants that lack function in bacteria but retain full function in https://www.sodocs.net/doc/9711612961.html,ctis.Appl Environ Microbiol2005,71(11):7092–7098.

111.Bell PJ,Bissinger PH,Evans RJ,Dawes IW:A two-reporter gene system for the analysis of bi-directional transcription from the divergent MAL6T-

MAL6S promoter in Saccharomyces cerevisiae.Curr Genet1995,

28:441–446.

112.Yao B,Marmur J,Sollitti P:Construction of glucose-repressible yeast expression vectors.Gene1993,137:223–226.

113.Finley RL,Zhang H,Zhong J,Stanyon CA:Regulated expression of proteins in yeast using the MAL61–62promoter and a mating scheme to increase dynamic range.Gene2002,285:49–57.

114.Juretzek T,Dall MTL,Mauersberger S,Gaillardin C,Barth G,Nicaud JM: Vectors for gene expression and amplification in the yeast Yarrowia

lipolytica.Yeast2001,18:97–113.

115.Lopez M,Redruello B,Valdes E,Moreno F,Heinisch JJ,Rodicio R:Isocitrate lyase of the yeast Kluyveromyces lactis is subject to glucose repression

but not to catabolite inactivation.Curr Genet2004,44(6):305–316.

116.Matsumoto T,Takahashi S,Ueda M,Tanaka A,Fukuda H,Kondo A: Preparation of high activity yeast whole cell bioctalysts by optimization of intracellular production of recombinant Rhizopus oryzae lipase.J Mol Catal B-Enzym.2002,17:143–149.

117.Potvin G,Ahmad A,Zhang Z:Bioprocess engineering aspects of heterologous protein production in Pichia pastoris:a review.Biochem

Eng J2012,64:91–105.

118.Gellissen G,Kunze G,Gaillardin C,Cregg J,Berardi E,Veenhuis M,Vanderklei I:New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica–A comparison.FEMS Yeast Res2005,

5(11):1079–1096.

119.Zaragoza O,Vincent O,Gancedo JM:Regulatory elements in the FBP1 promoter respond differently to glucose-dependent signals in

Saccharomyces cerevisiae.Biochem J2001,359:193–201.120.Hirota K,Hoffman GS,Shibata T,Ohta K:Fission yeast Tup1-like repressors repress chromatin remodelling at the fbp1+promoter and the

ade6-M26recombination hotspot.Genetics2003,165(2):505–515.

121.Leuker CE,Sonneborn A,Delbrück S,Ernst IF:Sequence and promoter regulation of the PCK1gene encoding phosphoenolpyruvate

carboxykinase of the fungal pathogen Candida albicans.Gene1997,

192:235–240.

122.Sanyal K,Carbon J:The CENP-A homolog Ca Cse4p in the pathogenic yeast Candida albicans is a centromere protein essential for

chromosome transmission.Proc Natl Acad Sci USA2002,

99(20):12969–12974.

https://www.sodocs.net/doc/9711612961.html,hinsky SC,Harcus D,Ash J,Dignard D,Marcil A,Morchhauser J, Thomas DY,Whiteway M,Leberer E:CDC42is required for polarized growth in human pathogen Candida albicans.Eukaryot Cell2002,

1(1):95–104.

124.Kratzer S,Schüller HJ:Transcriptional control of the yeast acetyl-CoA synthetase gene,ACS1,by the positive regulators CAT8and

ADR1and the pleiotropic repressor UME6.Mol Microbiol1997,

26(4):631–641.

125.Caspary F,Hartig A,Schüller HJ:Constitutive and carbon source responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1.Mol Gen

Genet1997,255(6):619–627.

126.Cregg JM,Tolstorukov I:Novel P.pastoris promoters and the use thereof to direct expression of proteins in yeast preferably using

a haploid mating strategy(US20080108108).US Patent Office

2008:1–28.

127.Guarente L,Mason T:Heme regulates transcription of the CYCl gene of S.

cerevisiae via an upstream activation site.Cell1983,32:1279–1286.

128.Ramil E,Freire-Picos MA,Cerdan ME:Characterization of promoter regions involved in high expression of Kl CYC1.Eur J Biochem1998,256:67–74. 129.Da Silva NA,Bailey JE:Influence of plasmid origin and promoter strength in fermentations of recombinant yeast.Biotechnol Bioeng1991,

37:318–324.

130.Hadiji-Abbes N,Borchani-Chabchoub I,Triki H,Ellouz R,Gargouri A, Mokdad-Gargouri R:Expression of HBsAg and preS2-S protein in different yeast based system:a comparative analysis.Protein Expres Purif2009,

66(2):131–137.

131.Falcone C,Fleer R,Saliola M:Yeast promoter and its use(US5627046).

US Patent Office1997:1–28.

132.Stockmann C,Scheidle M,Klee D,Dittrich B,Merckelbach A,Hehmann G, Melmer G,Buchs J,Kang HA,Gellissen G:Process development in

Hansenula polymorpha and Arxula adeninivorans,a re-assessment.Microb Cell Fact2009,8(1):22–31.

133.Yurimoto H,Komeda T,Lim CR,Nakagawa T,Kondo K,Kato N,Sakai Y: Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.Biochim Biophys Acta2000,

1493:56–63.

134.Krasovska OS,Stasyk OG,Nahorny VO,Stasyk OV,Granovski N,Kordium VA, Vozianov OF,Sibirny AA:Glucose-induced production of recombinant

proteins in Hansenula polymorpha mutants deficient in catabolite

repression.Biotechnol Bioeng2006,97(4):858–870.

135.Hartner F,Glieder A:Mutant AOX1promoters(US2010/0196913A1).

US Patent Office2010:1–63.

136.Resina D,Cos O,Ferrer P,Valero F:Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia

pastoris using the nitrogen source-regulated FLD1promoter.Biotechnol Bioeng2005,91(6):760–767.

137.Johnson MA,Snyder WB,Cereghino JL,Veenhuis M,Subramani S,Cregg JM: Pichia pastoris Pex14p,a phosphorylated peroxisomal membrane

protein,is part of a PTS-receptor docking complex and interacts with

many peroxins.Yeast2001,18:621–641.

138.Gurvitz A,Rottensteiner H:The biochemistry of oleate induction: transcriptional upregulation and peroxisome proliferation.Biochim

Biophys Acta2006,1763:1392–1402.

139.Gasmi N,Ayed A,Ammar BBH,Zrigui R,Nicaud JM,Kallel H:Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast,Yarrowia lipolytica.Microb Cell

Fact2011,10:90–100.

140.Boer VM,De Winde JH,Pronk JT,Piper MDW:The Genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic

chemostat cultures limited for carbon,nitrogen,phosphorus,or

sulfur.J Biol Chem2003,278(5):3265–3274.

141.Tai SL,Boer VM,Daran-Lapujade P,Walsh MC,Winde JH,Daran JM,Pronk JT:Two-dimensional transcriptome analysis in chemostat cultures:

combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae.J Biol Chem2005,280(1):437–447.

142.Ruth C,Glieder A:Perspectives on synthetic promoters for biocatalysis and biotransformation.ChemBioChem2010,11(6):761–765.

143.Blazeck J,Garg R,Reed B,Alper HS:Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters.

Biotechnol Bioeng2012,109(11):2884–2895.