repression vs. activation of mox , fmd , mpp1 and mal1 promoters by sugars in hansenula polymorpha :...

R E S EA RCH AR T I C L E

Repression vs. activation of MOX, FMD, MPP1 and MAL1promoters by sugars in Hansenula polymorpha: the outcome

depends on cell’s ability to phosphorylate sugar

Sandra Suppi, Tiina Michelson, Katrin Viigand & Tiina Alam€ae

Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia

Correspondence: Tiina Alam€ae, Institute of

Molecular and Cell Biology, University of

Tartu, Riia 23, 51010 Tartu, Estonia.

Tel.: +372 7 375013; fax: +372 7 420286;

e-mail: [email protected]

Received 28 September 2012; accepted 8

November 2012.

Final version published online 17 December

2012.

DOI: 10.1111/1567-1364.12023

Editor: Monique Bolotin-Fukuhara

Keywords

cell permeabilization; glucokinase; glucose

repression; glucose signalling; hexokinase;

methylotrophic yeasts.

Abstract

A high-throughput approach was used to assess the effect of mono- and disac-

charides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymor-

pha. Site-specifically designed strains deficient for (1) hexokinase, (2)

hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as

hosts for reporter plasmids in which b-glucuronidase (Gus) expression was

controlled by these promoters. The reporter strains were grown on agar plates

containing varied carbon sources and Gus activity was measured in permeabi-

lized cells on microtitre plates. We report that monosaccharides (glucose,

fructose) repress studied promoters only if phosphorylated in the cell. Glucose-

6-phosphate was proposed as a sugar repression signalling metabolite for

H. polymorpha. Intriguingly, glucose and fructose strongly activated expression

from these promoters in strains lacking both hexokinase and glucokinase, indi-

cating that unphosphorylated monosaccharides have promoter-derepressing

effect. We also show that maltose and sucrose must be internalized and split

into monosaccharides to exert repression on MOX promoter. We demonstrate

that at yeast growth on glucose-containing agar medium, glucose-limitation is

rapidly created that promotes derepression of methanol-specific promoters and

that derepression is specifically enhanced in hexokinase-negative strain. We

recommend double kinase-negative and hexokinase-negative mutants as hosts

for heterologous protein production from MOX and FMD promoters.

Introduction

Yeasts live in a sugar-rich environment and prefer sugars

over other carbon sources. When a high amount of glu-

cose is available, utilization of alternative carbon sources

such as alcohols and organic acids is prevented through

transcriptional down-regulation of the respective genes.

This mechanism is called glucose repression. To trigger

the repression, cells must first detect a sugar. In Saccharo-

myces cerevisiae, two transporter-like transmembrane pro-

teins, SNF3 and RGT2, with long C-terminal cytosolic

extensions sense respectively low and high external glu-

cose concentrations. Binding of glucose to the sensor

initiates a signalling pathway that controls expression of

glucose transporter genes (reviewed in Gancedo, 2008).

Glucose reaching the cell provokes repression of target

promoters by a mechanism in which MIG1 protein and

one of hexokinase isoforms, HXK2, have specific roles in

S. cerevisiae. At glucose abundance, HXK2 and MIG1

proteins enter the nucleus, where MIG1 directs corepres-

sors TUP1 and CYC8 to target promoters. If glucose is

depleted, SNF1 protein kinase will inactivate MIG1 by

phosphorylation whereby it will be exported from the

nucleus, allowing transcriptional activation of the pro-

moters (Ahuatzi et al., 2004, 2007; Barnett & Entian,

2005; Gancedo, 2008; Pelaez et al., 2010). At high glucose,

HXK2 stabilizes the repressor complex by interfering with

MIG1 phosphorylation by SNF1 protein kinase (Ahuatzi

et al., 2007). How the presence of glucose is sensed inside

the S. cerevisiae cell is still an open question.

Glucose also affects gene expression in methylotrophic

yeasts. At growth of these yeasts on methanol, promoters

of methanol-specific genes are extremely strongly induced.

This is why respective promoters are widely used for

heterologous protein production in methylotrophic

yeasts (Gellissen, 2000; Hartner & Glieder, 2006). In the

FEMS Yeast Res 13 (2013) 219–232 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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presence of abundant glucose, methanol-specific functions

of cells are inhibited and methanol is not used (Eggeling

& Sahm, 1978; Sibirny et al., 1988; Hartner & Glieder,

2006; Yurimoto & Sakai, 2009; Yurimoto et al., 2011).

However, in the case of Hansenula polymorpha, some

methanol-specific promoters are also remarkably dere-

pressed at glucose-limited growth (Mayer et al., 1999),

which allows toxic and flammable methanol to be

excluded from the protein production scheme. Glucose

repression mutants of H. polymorpha have also been used

to produce heterologous proteins from methanol-specific

promoters (Krasovska et al., 2007).

Data on sensing, transport and metabolism of sugars in

H. polymorpha are accumulating. Stasyk et al. (2004,

2008) have described two transmembrane sensor proteins

GCR1 and HXS1 in this yeast. Whereas the GCR1 has no

cytosolic extension (Stasyk et al., 2004), the HXS1 is a

typical sensor protein with a long C-terminal signalling

domain (Stasyk et al., 2008). One hexose transporter

gene, HXT1, which encodes a permease for glucose and

fructose, has also been cloned from H. polymorpha (Sta-

syk et al., 2008). We have characterized transport of glu-

cose (Karp & Alam€ae, 1998), maltose and sucrose

(Viigand & Alam€ae, 2007) in H. polymorpha, and cloned

the genes for glucokinase (Laht et al., 2002), hexokinase

(Karp et al., 2003), maltase (Liiv et al., 2001) and maltose

(a-glucoside) permease (Viigand & Alam€ae, 2007). Data

in the literature suggest that sugar repression in H. poly-

morpha cannot be described on the basis of the baker’s

yeast model. The first evidence of that came from enzy-

matic assay of hexokinase-negative mutants of H. poly-

morpha LR9 (Kramarenko et al., 2000). These mutants

retained glucose repression, meaning that hexokinase pro-

tein has no specific regulatory role in glucose repression.

Thereafter, Oliveira et al. (2003) showed that the TUP1

homologue of H. polymorpha had no role in glucose

repression of alcohol oxidase, dihydroxyacetone synthase

and catalase. In 2007, Stasyk et al. reported that disrup-

tion of MIG1 and MIG2 genes of H. polymorpha also had

only a minor effect on glucose repression of alcohol oxi-

dase.

In the current work we proceed with the study of

sugar repression in H. polymorpha. We will analyze

expression from four sugar-repressed promoters in

H. polymorpha strain 201 applying a simple Gus reporter

assay on a set of mutants. Aside of well-known MOX

and FMD promoters of H. polymorpha, we included in

the assay two less studied, but biotechnologically

perspective, novel promoters, those of peroxisomal acti-

vator gene MPP1 (Leao-Helder et al., 2003) and the

maltase gene MAL1 (Liiv et al., 2001). Novel and

intriguing data on promoter regulation in H. polymorpha

will be presented.

Materials and methods

Strains, plasmids and oligonucleotide primers

Escherichia coli strain DH5a [supE44 DlacU169 (ø80

lacZDM15) recA1 endA1 hsdR17 thi-1 gyrA96 relA1]

(Invitrogen) was used for DNA manipulation procedures.

Hansenula polymorpha strain 201 (HXK1 GLK1 ura3-1

leu2-2 met4-220; Lahtchev et al., 2002) isogenic to CBS

4732 was used as a wild type. In this strain, maltase-

disruption mutant mal1 and maltose permease disruption

mutant mal2 were constructed by us earlier (Alam€ae

et al., 2003; Viigand & Alam€ae, 2007). The plasmids used

in this work and their construction are described in

Table 1. The primers used in this work are presented in

Supporting Information, Table S1.

Disruption of HXK1 gene in H. polymorpha 201,

obtaining the hexokinase-negative strain hxk1

HXK1 gene was inactivated in H. polymorpha by homolo-

gous recombination with a disruption fragment. For that, a

linear 3773-bp fragment was PCR-amplified from

pRS426HXK1::URA3 using primers HK13 and HK21, puri-

fied and electroporated to wild-type cells. Uracil-positive

clones that grew on glucose, glycerol and methanol, but not

on sorbitol or fructose, were obtained. Genomic DNA of

three clones was isolated and disruption of genomic HXK1

locus was verified by PCR. Extracts of glucose-grown cells

of tested isolates did not phosphorylate fructose and

displayed no hexokinase band in native polyacrylamide gel

electrophoresis (PAGE). Complementation of H. polymor-

pha hxk1 strains with pYT3HXK1 restored fructose and

sorbitol growth to the mutants and hexokinase band was

again revealed at PAGE of extracts.

Deletion of the GLK1 gene in hexokinase-

negative H. polymorpha, obtaining the double

kinase-negative strain hxk1 glk1

We intended to use the URA3 marker once again to dis-

rupt the GLK1 gene in hxk1 H. polymorpha. For that, a

PCR-amplified DNA fragment containing the H. polymor-

pha URA3 gene bordering with GLK1 sequences was elec-

troporated to H. polymorpha hxk1 strain and the cells

were plated out onto agar medium containing 2% glyc-

erol and 200 mg L�1 of 2-deoxy-D-glucose (2DG).

Double kinase-negative H. polymorpha strains can grow

on glycerol or methanol in the presence of 2DG

(Kramarenko et al., 2000) and we hoped to promote

GLK1 disruption using selective force of 2DG. In this

selection, we isolated a strain with a 595-bp deletion in

the GLK1 open reading frame as was verified by genomic

ª 2012 Federation of European Microbiological Societies FEMS Yeast Res 13 (2013) 219–232Published by Blackwell Publishing Ltd. All rights reserved

220 S. Suppi et al.

DNA sequencing. The obtained strain designated as

hxk1glk1 did not grow on sugars and retained its leu2

selection marker for introduction of reporter plasmids.

Extract of glycerol-grown hxk1glk1 strain had no detect-

able glucose and fructose phosphorylating activity and

native electrophoresis of the extract revealed no bands of

hexokinase and glucokinase. As expected, complementation

of hxk1glk1 strain with pYT3HXK1 restored its growth on

glucose and fructose, and complementation with

pYT3GLK1 restored growth on glucose. PAGE of cell

extracts of complemented strains agreed with growth

phenotype. The hxk1 and hxk1glk1 strains and the wild

type (HXK1 GLK1) were used in this study for the assay

of sugar-related regulatory events in H. polymorpha 201.

Cultivation of yeasts and bacteria

Yeasts were grown for reporter assay on 0.67% Yeast

Nitrogen Base (YNB) medium without amino acids (Difco)

to which 2% agar and adequate auxotrophic supplements

were added. Carbon sources were used at concentrations

shown in the text. All sugars were autoclaved separately

in distilled water and then added to the medium. The

cells pregrown on 2% glycerol were streaked onto three

sectors of agar plate (see insert of Fig. 1) containing vari-

ous carbon sources. Standard-size Petri dishes with

20 mL of the agar medium were used. One Petri dish was

Table 1. Plasmids

Plasmid Description/construction Reference

pRS426HXK1 Contains a 2569-bp SpeI-SalI fragment from pJ1 with HXK1 gene of Hansenula polymorpha

(Karp et al., 2003) cloned between the same sites of pRS426 polylinker

This work

pRS426HXK1::URA3 The H. polymorpha URA3 gene was amplified from pHpURA3 (from Dr J. Siverio, Spain)

using primers HpURA3FwNheI and HpURA3RevNheI, the product was cleaved with NheI

and inserted into XmaJI (AvrII) site of HXK1 gene in pRS426HXK1

This work

pYT3HXK1 Contains a 2569-bp SpeI-SalI fragment from pJ1 with H. polymorpha hexokinase gene HXK1

between the XbaI and SalI restriction sites of pYT3 (Tan et al., 1995)

Karp et al. (2003)

pYT3GLK1 Contains the H. polymorpha glucokinase gene GLK1 in the XbaI site of the pYT3

(Tan et al., 1995)

Laht et al. (2002)

pX4-HNBESX Plasmid with H. polymorpha MOX promoter and AMO terminator, kanR and ScLEU2 From Dr. Kiel (Gr€oningen)

pHIPX8 Plasmid with H. polymorpha TEF2 promoter and AMO terminator, kanR and ScLEU2 From Dr Kiel (Gr€oningen)

pHIPMALprom A derivative of pHIPX8 in which the TEF2 promoter is replaced by H. polymorpha MAL1

promoter.

Visnapuu et al. (2008)

pHIPMALpromGus Gus reporter under the control of H. polymorpha MAL1 promoter. Promoterless gusA

gene of E. coli was excised from pIB-GusA (Sears et al., 1998) on a BamHI-PstI fragment

and inserted between the same sites in pHIPMALprom

This work

pX4GusA Gus reporter under the control of H. polymorpha MOX promoter. Promoterless gusA gene

of Escherichia coli was excised from pGUS102 (Marits et al., 2002) on an EcoRI fragment

and inserted into the EcoRI site of pX4-HNBESX

This work

pHIPFMDpromGus Gus reporter under the control of H. polymorpha FMD promoter. The promoter region

(650 bp) of the FMD gene was PCR-amplified from genomic DNA of

H. polymorpha 201 using primers FMDpromFwNotI and FMDpromRevBamHI. The obtained

fragment, cut with and NotI and BamHI, was cloned between the same sites of

pHIPMALpromGus to replace the MAL1 promoter

This work

pHIPMPP1promGus Gus reporter under the control of H. polymorpha MPP1 promoter. The promoter

region (776 bp) of MPP1 gene was PCR-amplified from genomic DNA of

H. polymorpha 201 using primers MPP1promFwNotI and MPP1promRevBglII. The obtained

fragment, cut with NotI and BglII, was cloned between the NotI and BamHI sites of

pHIPMALpromGus to replace the MAL1 promoter

This work

Fig. 1. Gus expression from MOX, FMD and MPP1 promoters in

wild-type Hansenula polymorpha grown on agar plates containing

2% glucose (glc), 2% glycerol (gly) or 1% methanol (met) as carbon

sources. Growth time of transformants (reporter strains) was 2 days

in the case of glucose and glycerol and 3 days in case of methanol.

Average Gus activities in Miller units and standard deviations for at

least three transformants are indicated. The insert shows the agar

plate with three seeded sectors of a reporter strain.


Sugar regulation of Hansenula polymorpha promoters 221

used for each transformant and at least three different

transformants of the strain were grown and analyzed in

each reporter assay. Incubation temperature was 37 °Cand the growth time was either 2 or 3 days. If the cells

were grown in liquid culture, batch cultivation in flasks

on orbital shaker was used. In tests of yeast growth on

YNB agar medium with 1% methanol + 2DG, the latter

was added at concentrations 200, 400 or 1000 mg L�1.

Escherichia coli was grown at 37 °C in Luria–Bertanimedium containing kanamycin (0.1 mg mL�1) when

required.

DNA manipulations, sequencing, PCR and

transformation

DNA manipulations were carried out using standard

methods (Sambrook et al., 1989). Plasmid DNA was puri-

fied with AxyPrep kit (Axygen Biosciences). DNA frag-

ments for cloning and gene disruption were purified

using UltraClean 15 kit from MoBio. Yeast genomic DNA

was isolated as in Liiv et al. (2001). Restriction endonu-

clease digestions and DNA ligations were performed

according to the manufacturer’s (Fermentas) recommen-

dations. In PCR-based cloning of promoter regions, the

Pfu DNA polymerase (Fermentas) was used. DNA was

sequenced using an ABI PrismTM 377 DNA sequencer

(Perkin Elmer) and DYEnamic ET terminator cycle

sequencing kit (Amersham). Hansenula polymorpha was

electrotransformed according to Faber et al. (1994) using

a BioRad Xcell GenePulser.

Preparation of yeast suspensions and assay of

b-glucuronidase (Gus) activity in permeabilized

cells on microtitre plates

The cells grown on agar plates were harvested using a

plastic inoculation loop, washed twice in K-phosphate

buffer (50 mM, pH 7.0) by centrifugation, resuspended

in the same buffer to optical density (OD)600 nm � 10

and the cell suspension was kept on ice during the experi-

ment. For Gus activity assay, 20 lL of this suspension

were pipetted in triplicate into wells of a 96-well trans-

parent flat-bottom microtitre plate (655101, Greiner

bio-one, Germany) and 80 lL of the buffer (50 mM

K-phosphate, 0.01 mM EDTA, pH 7.0) containing 0.1%

cetyl trimethylammonium bromide (CTAB) as a permea-

bilizing agent (Alam€ae & J€arviste, 1995) was added to

each well. The plate was gently rocked for 20 min for cell

permeabilization and then the b-glucuronidase reaction

was initiated by adding 10 lL of 10 mM p-nitrophenyl-

b-D-galacturonide (p-NPG). At the appearance of pale

yellow colour, the reaction was stopped by adding 50 lLof 1 M Na2CO3. Reference samples consisted of

permeabilized cells to which 50 lL of 1 M Na2CO3 and

10 lL of p-NPG were added simultaneously. Absorbance

of the samples was measured at 405 nm, using a Tecan

Sunrise™ microplate reader (Tecan Group Ltd., Switzer-

land) and Magellan™ data analysis software (Tecan).

Both the cell permeabilization and enzyme assay were

performed at room temperature.

Gus activity was calculated in Miller units according to

the formula:

1000� DOD405 min�1

OD600 � Vsusp

where DOD405 min�1 refers to the absorbance of reaction

mixture at 405 nm from which the absorbance of reference

sample was subtracted, divided by the reaction duration in

minutes. OD600 is the absorbance of the prepared yeast

suspension at 600 nm, measured after suitable dilution in

a standard 1-cm pathway-length cuvette and Vsusp is the

amount of suspension (mL) used in the reaction.

Enzyme assay in cell extracts

Preparation of cell extracts, determination of protein con-

centration and hexokinase activity are described in Karp

et al. (2003). Maltase and b-glucuronidase assays are

described in Viigand et al. (2005). For every assay, at least

three parallel samples from at least three transformants

were prepared and analyzed. Enzyme activity was

expressed in either nanomoles or micromoles of substrate

converted per min (mU and U, respectively) per mg of

protein in the reaction mixture.

Determination of glucose concentration in the

medium and inside the cells

To assess residual glucose concentration in the agar med-

ium, the cells were scraped off, the agar plate surface was

washed once with distilled water and small discs were

excised from it using sterile 1-mL pipette tips (Putrin�s

et al., 2011; see also Fig. 4c). The discs were cut from two

regions: adjacent to yeast growth area and from under-

neath of it. The disks were melted at 100 °C and cooled

to 65 °C. Glucose content in melted agar was determined

with a Glucose Liquicolor kit (Human GmbH, Germany)

according to the instructions of the producer. The Glu-

cose Liquicolor assay was also used to determine glucose

concentration in supernatants of liquid cultures and

inside the cells. The extracts for intracellular glucose assay

were prepared according to Miseta et al. (2003). In calcu-

lations, 1 mg of dry weight was taken to correspond to

2 lL of cell water (Guijarro & Lagunas, 1984). Cell dry

weight was measured as in Karp & Alam€ae (1998).


222 S. Suppi et al.

Presence of glucose in agar plates was visualized by over-

lay of agar surface with 5 mL of 50 mM Tris-buffer (pH

7.5) containing 0.1% agarose, 100 U mL�1 of glucose

oxidase and 60 lg mL�1 of both N-methylphenazonium

ethanesulphonate and nitroblue tetrazolium.

Results and discussion

Our toolbox

In earlier experiments on sugar repression in H. polymor-

pha, we used the LR9 strain and its mutants that were

derived by chemical mutagenesis. Enzymatic analysis of

these mutants suggested that hexokinase protein has no

specific role in glucose repression in this yeast and

referred to some metabolite as sugar repression triggerer

(Kramarenko et al., 2000; Karp et al., 2003). In the cur-

rent study, we used targeted mutagenesis to design stable

hxk1 and hxk1glk1 mutants of H. polymorpha 201. These

two mutants and earlier constructed mal1 (Alam€ae et al.,

2003) and mal2 (Viigand & Alam€ae, 2007) mutants of

strain 201were used as hosts for reporter plasmids. In

these plasmids, b-glucuronidase gene of E. coli was

cloned under control of MOX, FMD, MPP1 and MAL1

promoters of H. polymorpha. The transformants (reporter

strains) were grown on agar medium of varied carbon

source composition and b-glucuronidase (Gus) activity of

permeabilized cells was quantified on microtitre plates.

Notably, we have earlier used microtitre plate-based assay

on permeabilized cells for semiquantitative measurement

of alcohol oxidase and maltase activities (Alam€ae & Liiv,

1998; Kramarenko et al., 2000) and for quantitative assay

of levansucrase activity (Alam€ae et al., 2012). Due to sim-

ple cultivation mode and high-throughput enzymatic

assay we could address a large number of strains, pro-

moters and carbon sources. As will be presented further,

agar plate cultivation creates nutrient limitation condi-

tions that are crucial for promoter derepression.

MPP1 promoter is regulated by carbon sources

similarly to MOX and FMD promoters

The MPP1 gene was described as a regulator of peroxi-

somal protein levels of H. polymorpha. It encodes a puta-

tive transcriptional activator of 684 amino acids with

N-terminal zinc-finger domain. In mpp1 mutants grown

on glycerol + methanol, the amount of alcohol oxidase

and several peroxins was strongly reduced compared with

wild type, and dihydroxyacetone synthase was absent.

A MPP1-GFP fusion protein was absent from H. poly-

morpha cells grown on glucose, but was present and

detected in the nucleus in methanol-grown cells (Leao-

Helder et al., 2003). As the MPP1 gene was most induced

after the transfer of H. polymorpha from glucose to meth-

anol (van Zutphen et al., 2010), its promoter was

expected to have biotechnological potential in regulated

overexpression of proteins in H. polymorpha.

First, we compared regulation of MOX, FMD and

MPP1 promoters by carbon sources in wild-type H. poly-

morpha. Figure 1 shows that Gus expression from all

three promoters was highest at methanol growth with

strongest activation recorded for the MOX promoter

(� 700 Miller units) and about twice less for FMD and

MPP1 promoters. These results contradict the transcrip-

tomic assay data by van Zutphen et al. (2010) according

to which the MPP1 showed the highest methanol induc-

tion (394-fold), FMD the second best induction (347-

fold) and the MOX only a 17-fold induction. Our data

show that at growth on 2% glycerol or 2% glucose, the

MOX and FMD promoters had low expression, and the

MPP1 promoter-driven Gus activity was below detection

(Fig. 1). All three promoters were significantly dere-

pressed at growth of cells on 0.1% of glucose. The highest

glucose derepression (� 470 Miller units) was recorded

for the MOX promoter. Notably, methanol-induced and

glucose-derepressed Gus expression from FMD promoter

were quite the same.

In methylotrophic yeasts, aside from MPP1 protein,

three methanol-specific zinc finger activators have been

described: MXR1 in Pichia pastoris (Lin-Cereghino et al.,

2006) and TRM1 and TRM2 in Candida boidinii (Sasano

et al., 2008, 2010). TRM1 was considered a master tran-

scriptional regulator of methanol-specific gene activation

(Sasano et al., 2008). TRM2, which is homologous to

MXR1 of P. pastoris and ADR1 of S. cerevisiae, is essential

for TRM1-dependent activation. As described in Sasano

et al. (2008), the MUT3 protein of H. polymorpha that

has high sequence similarity to TRM1 of C. boidinii may

have a role in activation of methanol-specific genes in

H. polymorpha. The mut3 strain of H. polymorpha could

not grow on methanol and had reduced MOX promoter

activity (Vallini et al., 2000). Unfortunately, the MUT3

protein itself has not been studied.

Hexokinase-negative H. polymorpha is

insensitive to fructose repression and has

enhanced derepression of methanol-specific

promoters at low glucose growth

Glucose and fructose repression of MOX, FMD and

MPP1 promoters was assayed in the wild type and hexo-

kinase-negative strains of H. polymorpha 201. In both

strains, MOX, FMD and MPP1 promoters were not

induced by methanol in the presence of 2% glucose.

In hxk1 H. polymorpha 201, glucose but not fructose

repressed methanol-specific promoters, which agrees with



our earlier data on H. polymorpha LR9 (Kramarenko

et al., 2000; Karp et al., 2003). Figure 2 illustrates the

data on MOX and FMD regulation. According to the

literature, the MOX and FMD genes are significantly

derepressed when cells are growing at glucose limitation

(Egli et al., 1980; Kensy et al., 2009). We presumed that

promoter derepression may be higher for hxk1 H. poly-

morpha. Figure 3 shows that this is indeed the case. In

both strains, the studied promoters were equally repressed

at growth of cells on 2% glucose and equally slightly de-

repressed at growth on 0.5% glucose. At growth on 0.1%

glucose, the hxk1 strain exhibited much higher derepres-

sion than the wild type. When comparing the promoters,

the highest derepression was recorded for MOX and the

lowest for MPP1 promoter. According to our results, the

MOX promoter was more strongly activated at methanol

growth and more highly derepressed at glucose limitation

compared with the FMD promoter.

We next asked whether addition of methanol to low

glucose (0.1%) medium would further enhance expres-

sion from these promoters. It turned out that in the wild

type, addition of methanol almost doubled the expression

from all three promoters, whereas in the hxk1 strain, no

further increase of reporter expression was detected (data

not shown). Significant derepression of methanol-specific

promoters in media lacking methanol is a specific feature

of H. polymorpha (Hartner & Glieder, 2006) that can be

implemented in H. polymorpha-based heterologous pro-

tein production systems. So, a very high expression level

(13.5 mg L�1) was achieved in production of secreted

phytase in H. polymorpha from FMD promoter by shift-

ing glycerol-pregrown cells to glucose limitation (Mayer

et al., 1999). Our results show that hexokinase-negative

mutant of H. polymorpha should be superior to the wild

type in a similar type of expression system using, for

example, the MOX or FMD promoter.

Glucose limitation is created at growth of

yeast on glucose-containing agar plates

Figure 3 shows derepression of methanol-specific promot-

ers when H. polymorpha is grown on low glucose agar

medium. In contrast to Eggeling & Sahm (1978), we

could not detect derepression of methanol-specific

enzymes at liquid batch cultivation of H. polymorpha on

either 0.1% or 0.2% of glucose, even in late stationary

growth phase (our unpublished data). As derepression

was disclosed at growth on agar plates, we suggested that

this cultivation mode created glucose limitation condi-

tions. Figure 4 indicates that this is indeed the case. Panel

D of Fig. 4 demonstrates rapid exhaustion of glucose

from the agar medium under the cells, becoming very

low (� 9 mM) already on second day of growth, whereas

in the periphery of the agar plate it still remains high at

that time. Thus, yeasts growing on glucose-containing

agar plate certainly face glucose limitation. Glucose limi-

tation has been created by cultivation of H. polymorpha

in a chemostat run at a low dilution rate (Egli et al.,

1980). Recently, a highly interesting application to create

glucose limitation at batch cultivation was described for

H. polymorpha. In that application, liquid cultures were

supplied with silicon discs from which glucose was

released slowly (Kensy et al., 2009; Scheidle et al., 2010).

Fig. 2. Gus expression from MOX and FMD promoters in wild-type

Hansenula polymorpha (WT) and hexokinase-negative mutant (hxk1)

grown for 3 days on agar plates containing either 2% glucose (glc) or

2% fructose (fru) and 1% methanol (met) as carbon sources. Average

Gus activities in Miller units and standard deviations of at least three

transformants are indicated.

Fig. 3. Gus expression from MOX, FMD and MPP1 promoters in wild-type Hansenula polymorpha (WT) and hexokinase-negative mutant (hxk1)

grown on agar plates containing varied concentrations of glucose (glc). The growth time was 2 days. Average Gus activities in Miller units of at

least three transformants and standard deviations are presented.


224 S. Suppi et al.

Our current approach is certainly much simpler than the

above-mentioned methods.

Glucose-6P as potential signal metabolite in

triggering sugar repression

Our experimental data predict that glucose-6P is a media-

tor of sugar repression in H. polymorpha. This prediction

is supported by following facts: (1) phosphorylation of a

sugar by hexokinase or glucokinase is required for repres-

sion (Kramarenko et al., 2000; see also Fig. 2); (2) a glu-

cose analogue 2DG mimics the effect of glucose as a

repressor despite it not being metabolized beyond the

phosphorylation step (see further). According to our

hypothesis, Glc6P also signals for fructose repression,

because after phosphorylation of fructose in hexokinase

reaction it can be isomerized to Glc6P. According to our

regulation model, H. polymorpha is capable of sensing

intracellular Glc6P concentration and responds accord-

ingly by transcriptional down-regulation of target genes,

for example those of methanol and disaccharide catabo-

lism (see Fig. 5).

To find out whether Glc6P production rate may depend

on sugar concentration in the medium as well as on the

hexose kinase pattern of the strain, we measured glucose

phosphorylation in cell extracts. The wild-type H. poly-

morpha 201 and hxk1 strains were grown in liquid culture

on high (2%) and low (0.2%) glucose. Table 2 shows that

the glucose phosphorylating activity of cells depends on

glucose concentration in the medium being much lower in

the case of low glucose. Also, the glucose-phosphorylating

activity of the hxk1 strain was about twice as low as that of

the wild type. We consider that low glucose-phosphorylat-

ing activity means a reduced amount of Glc6P in the cell,

allowing promoter derepression. Hexokinase-negative

mutant does not phosphorylate fructose and is therefore

not capable of producing Glc6P. According to our hypoth-

esis, this feature explains the lack of fructose repression in

hkx1 H. polymorpha (Figs 2 and 5). The growth pattern of

strains on methanol + 2DG medium agrees with our

model. The wild-type, hxk1 and hxk1glk1 strains were

streaked onto YNB agar containing 1% methanol at

different concentrations (200, 400 and 1000 mg L�1) of

2DG. Only hxk1glk1 strain was able to grow.

2DG is the substrate for the H. polymorpha glucose

transport system (Karp & Alam€ae, 1998) and for hexoki-

nase and glucokinase as well (Laht et al., 2002; Karp

et al., 2003). 2DG does not provide growth, and it cannot

be isomerized and converted to Fru6P (Klein & Stitt,

1998). We interpret the repressive effect of 2DG on meth-

anol growth as follows. 2DG is transported to H. poly-

morpha cells and phosphorylated by hexokinase and

glucokinase producing 2DG-6P, which mimics the effect

of repression mediator Glc6P. Therefore methanol-specific

promoters will be repressed making growth on methanol

impossible. Double kinase-negative mutants do not phos-

phorylate 2DG, and they can therefore induce methanol-

specific functions and grow on methanol in the presence

of 2DG.

Disaccharides repress expression of methanol-

specific promoters only if transported into the

cell, hydrolyzed to monosaccharides and

phosphorylated

Disaccharides, maltose and sucrose, repress methanol-

specific promoters. Having in hand disruption mutants of

maltase and maltose permease genes, we asked two ques-

tions: (1) should a disaccharide be transported into the

cell to exert repression and (2) should a disaccharide be

hydrolyzed inside the cell to exert repression? To get the

answer, reporter analysis of MOX promoter was carried

out on strains cultivated on methanol + a disaccharide.

(a)

(d)

(b) (c)

Fig. 4. Glucose limitation is created at growth of yeast on glucose-

containing agar medium. Wild-type Hansenula polymorpha was

grown on small (diameter 4 cm) Petri plates with YNB agar

containing 2% (111 mM) glucose as shown in (a). On days 2, 4, 6

and 8, cells were scraped off the agar, the surface of the agar plate

was gently washed with distilled water, and discs were cut off from

the medium for determination of glucose concentration. Two discs

were excised from under the cells (the area encircled by broken line)

and two from the adjacent area as indicated in (c). The time course

of glucose consumption is shown in (d). On day 4, the presence of

glucose in the medium was developed in a chromogenic assay (b).



Figure 6 shows that the MOX promoter is repressed by

maltose and sucrose in the wild type. If either permease

or maltase gene is inactivated, the repressing ability of a

disaccharide is abolished and the MOX promoter can be

induced by methanol. In mutants grown on malt-

ose + methanol, the MOX promoter was more highly

induced than at sucrose + methanol growth. We do not

know the reason for that. However, measurement of alco-

hol oxidase activity of same cells disclosed the same trend

(data not shown).

Next, we inspected the MOX promoter repression at

sucrose growth of H. polymorpha strains. As hxk1

H. polymorpha cannot metabolize fructose moiety of

sucrose, this disaccharide was predicted to cause less

repression on MOX promoter in this strain. For reporter

assay, the strains were grown on (1) 1% sucrose and (2)

0.5% glucose + 0.5% fructose. At growth on both media,

Gus activity of the wild type was very low, below 9 Miller

units. As expected, the hxk1 strain had much higher Gus

activity (up to 78 Miller units) at growth on both media.

We conclude from these results that to repress a metha-

nol-specific promoter, maltose and sucrose should be

transported into the cell, split by maltase to monosaccha-

ridic products and then phosphorylated by hexokinase

and/or glucokinase.

Fig. 5. Proposed position of glucose-

6-phosphate (Glc6P) in sugar repression

signalling pathway in Hansenula polymorpha.

Table 2. Glucose phosphorylation in cell extracts of wild-type and

hexokinase-negative strains of Hansenula polymorpha grown at

different glucose concentrations

Strain

Carbon source in

the medium

Glucose

phosphorylation,

U mg�1

Wild type (WT) 2% glucose 1.60 � 0.12

Wild type (WT) 0.2% glucose 0.47 � 0.01

Hexokinase-negative (hxk1) 2% glucose 1.05 � 0.11

Hexokinase-negative (hxk1) 0.2% glucose 0.21 � 0.02

Cells for preparing the extracts were grown in liquid batch culture

and harvested for enzymatic assay in the mid-exponential growth

phase. Mean values � standard deviation are presented for two sepa-

rate cultures of the wild type and three different hxk1 strains.

Fig. 6. Gus expression from the MOX promoter in wild-type

Hansenula polymorpha (WT), maltose permease-negative mutant

(mal2) and maltase-negative mutant (mal1) harbouring the reporter

plasmid with the MOX promoter. Cells were grown on agar media

containing 2% sucrose + 1% methanol and 2% maltose + 1%

methanol for 3 days and Gus activity was measured in cells. Average

activities in Miller units and standard deviation are shown for three

transformants of each strain.


226 S. Suppi et al.

In double kinase-negative H. polymorpha,

glucose activates the promoters that are

considered glucose-repressible

We have earlier recorded a peculiar feature of double

kinase-negative mutants of H. polymorpha LR9 – the cells

grown on methanol + glucose had much higher alcohol

oxidase activity than those grown on methanol alone

(Kramarenko et al., 2000; Laht et al., 2002). Here, we

reinvestigated this matter in the hxk1glk1 strain of

H. polymorpha 201.

Figure 7 shows a typical glucose repression phenotype

for the wild type: 2% glucose in the medium prevents

induction of MOX, FMD and MPP1 promoters by metha-

nol, whereas 0.1% glucose allows induction. The MPP1

promoter was more sensitive to glucose than the MOX

and FMD promoters – it had less methanol induction in

the presence of glucose. Most intriguingly, in the hxk1glk1

strain, both tested concentrations (2% and 0.1%) of

glucose clearly enhanced expression from all three

promoters – it was always higher than in respective meth-

anol-grown cells. The highest expression level (almost

2900 Miller units) was recorded for the MOX promoter

at growth of hxk1glk1 strain on 1% methanol + 0.1%

glucose. This value is more than four times higher than

the respective activity in methanol-grown wild type. So,

glucose clearly activates methanol-specific promoters in

hxk1glk1 H. polymorpha.

We had shown earlier that the glycerol-grown double

kinase-negative mutant of H. polymorpha LR9 has a much

higher alcohol oxidase activity than the respective wild

type control (our unpublished data). Here, we inspected

hxk1glk1 H. polymorpha 201 from the same aspect. The

wild type and hxk1glk1 strains of H. polymorpha 201 car-

rying the MOX promoter reporter plasmid were grown in

liquid medium with 2% glycerol and assayed for reporter

activity in cell extracts. Gus activity of the wild type was

300 mU mg�1, whereas that of the hxk1glk1 strain was

over twice as high, 793 mU mg�1. Given that glucose

activated the MOX promoter in the hxk1glk1 strain

(Fig. 7), we suspected that glycerol-grown hxk1glk1

H. polymorpha may contain glucose. Determination of

intracellular glucose in wild-type, hxk1 and hxk1glk1

strains grown on agar plates with 2% glycerol revealed

that hxk1glk1 cells contained 13 mM glucose in cell water,

whereas no free glucose was detected in wild-type and

hxk1 cells. In accordance with our data, Miseta et al.

(2003) recorded a significant amount of intracellular

glucose in triple kinase-negative mutant of S. cerevisiae

grown on lactate.

At growth on non-sugar substrates such as glycerol,

methanol, lactate and ethanol, gluconeogenesis reactions

are obligatory to supply the cell with building blocks for

cell wall, glycolipids, storage sugars (glycogen) and stress

protectants (trehalose) (Arg€uelles, 2000; T€urkel, 2006). If

sugar-containing structures and molecules are recycled

and storage sugars are hydrolyzed, unphosphorylated

glucose should accumulate in the cell lacking glucose-

phosphorylating enzymes.

Regulation of MAL1 promoter by carbon

sources in H. polymorpha

First, we compared regulation of the MAL1 promoter

between the wild type and hxk1 strains of H. polymorpha.

Table 3 shows that high glycerol and glucose concentra-

tions repressed the MAL1 promoter in both strains. In

both strains, 2% glucose in the medium prevented induc-

tion from MAL1 promoter by disaccharides, whereas 2%

fructose did so only in the wild type. Our results also

indicate that hydrolysis products of maltose and sucrose

repress the MAL1 promoter and that their phosphoryla-

tion is obligatory for the repression. This conclusion is

based on following results: (1) lower disaccharide concen-

trations in the medium provide higher MAL1 promoter

expression level, and (2) at growth on disaccharides, the

MAL1 promoter was more highly activated in the hxk1

mutant.

As glucose activated methanol-specific promoters in

hxk1glk1 H. polymorpha, the MAL1 promoter was

addressed from the same aspect. Respective reporter

strains were grown on glycerol-containing agar plates

with addition of different sugars. Figure 8 clearly indi-

cates that glucose, fructose, maltose and sucrose have an

Fig. 7. Glucose acts as inducer of methanol-specific promoters in

double kinase-negative Hansenula polymorpha. Wild-type

H. polymorpha (WT) and double kinase-negative mutant (hxk1glk1)

harbouring Gus reporter plasmids for MOX, FMD and MPP1

promoters were grown on agar plates containing 1% methanol (met),

and on the mixture of methanol and either 2% or 0.1% of glucose

(glc) for 3 days and then assayed for b-glucuronidase activity.

Average Gus activities in Miller units of at least three transformants

and standard deviations are presented.



inducing effect on MAL1 promoter in hxk1glk1 strain.

In the case of all studied carbon source combinations, the

Gus activities in hxk1glk1 strain were higher than in malt-

ose- or sucrose-growing wild type. Mesurement of glucose

concentration in the medium indicated that although the

hxk1glk1 strain cannot grow on sucrose and maltose,

transport and hydrolysis of these sugars certainly took

place. At least part of the glucose produced from disac-

charide metabolism was excreted to the medium, most

probably using some glucose transporter. After 2 days of

growth of hxk1glk1 strain on 2% glycerol + 2% sucrose,

the glucose concentration in the agar medium below the

yeast growth area was 35 mM, whereas it was about twice

as high (68 mM) in the case of growth on 2% glyc-

erol + 2% maltose. Respective samples for wild-type cells

contained no detectable glucose. Jansen et al. (2002) have

reported for maltose-growing S. cerevisiae that if the cell

cannot manage the metabolism of glucose resulting from

maltose hydrolysis in the cell, intracellular glucose con-

centration rises and excess glucose will be exported by

hexose transporters. Our data therefore suggest that accu-

mulation of unphosphorylated hydrolysis products of

sucrose and maltose in hxk1glk1 strain may cause the

MAL promoter activation shown in Fig. 8.

We also carried out an experiment in which maltase

induction by glucose was assayed in liquid culture. Wild

type, hxk1 and hxk1glk1 strains were grown in liquid YNB

medium with 2% glycerol and maltase activity was mea-

sured in cell extracts. Table 4 shows that glycerol-grown

hxk1glk1 strain has over 20 times higher maltase activity

than that of the wild-type and hxk1 strains. Already very

high maltase activity was further increased if glucose was

added to glycerol-growing hxk1glk1 cells.

Growth of yeasts on disaccharides:

unanswered questions

It is yet not clear how yeasts detect the presence of disac-

charides in the medium to induce disaccharide-specific

(MAL) genes. Wang et al. (2002) showed for S. cerevisiae

that maltose permease is required for maltase induction

by maltose. In that paper, the authors conclude that

permease does not act as maltose sensor but rather pro-

vides the inducer for MAL genes inside the cell. Quite

recently, it was proposed that the transcriptional activator

of MAL genes (MAL activator) may function as intracel-

lular sensor for maltose in S. cerevisiae. Ran et al. (2008)

reported that the MAL63 activator forms a complex

Table 3. Regulation of MAL1 promoter by carbon sources in wild-

type (WT) and hexokinase-negative (hxk1) strains of Hansenula

polymorpha

Carbon sources

b-glucuronidase activity

(Miller units)

WT hxk1

2% glucose 9 � 2 13 � 4

2% glycerol 6 � 2 5 � 2

2% maltose 87 � 9 249 � 22

0.5% maltose 186 � 14 266 � 33

0.2% maltose 216 � 22 397 � 44

2% sucrose 151 � 18 266 � 30

0.5% sucrose 167 � 10 402 � 47

2% glucose + 2% maltose 29 � 7 43 � 8

2% glucose + 2% sucrose 40 � 6 35 � 7

2% fructose + 2% maltose 18 � 4 370 � 44

2% fructose + 2% sucrose 17 � 6 497 � 46

The strains harbouring Gus reporter plasmid with MAL1 promoter

were grown on agar media for 3 days. Mean b-glucuronidase val-

ues � standard deviation for at least three different transformants of

each strain are shown.

Fig. 8. Glucose acts as inducer of maltase gene promoter in double

kinase-negative Hansenula polymorpha. Wild-type H. polymorpha

(WT) and double kinase-negative mutant (hxk1 glk1) harbouring Gus

reporter plasmid with MAL1 promoter were grown on agar plates

containing 2% glycerol (gly) and 2% glycerol supplemented with 2%

of glucose (glc), fructose (fru), maltose (mal), or sucrose (suc) for

3 days and then assayed for b-glucuronidase activity. Average Gus

activities in Miller units of at least three transformants and standard

deviations are presented.

Table 4. Maltase activity (mU mg�1) in cell extracts of Hansenula

polymorpha strains grown in YNB liquid medium containing 2%

glycerol

Strain Carbon source Maltase activity

Wild type (WT) 2% glycerol 24 � 1

Hexokinase-negative

(hxk1)

2% glycerol 21 � 3

Double kinase-negative

(hxk1glk1)

2% glycerol 560 � 58

Double kinase-negative

(hxk1glk1)

2% glycerol + 0.1% glc* 2829 � 156

*Cells grown on 2% glycerol were washed, suspended in fresh medium

containing 2% glycerol + 0.1% glucose and further grown for 9 h.


228 S. Suppi et al.

inside the cell with several chaperon proteins, including

yeast homologues of Hsp70 and Hsp90. When maltose

was added to the growth medium, MAL63 was released

from the complex and maltase was induced. The authors

suggest that binding of maltose to either MAL63 or its

chaperon was responsible for the release. Our data on

H. polymorpha also show that a-glucoside permease is

required for the induction of maltase by sucrose and

maltose (Viigand & Alam€ae, 2007). A putative MAL

activator gene is present in genomic MAL locus of

H. polymorpha (Viigand & Alam€ae, 2007), but its func-

tion has yet to be proved. Results of current study show

that hydrolysis products of maltose and sucrose activate

maltase expression if they are not phosphorylated in the

cell, i.e. in hxk1glk1 H. polymorpha (Table 4, Fig. 8). We

consider that this is how initial derepression of MAL

genes may take place, and it will probably be followed by

up-regulation of these genes by yet unknown specific

inducers. We have shown that H. polymorpha grown on

non-sugar substrates is prepared to transport and split

maltose and sucrose (Viigand et al., 2005; Viigand &

Alam€ae, 2007). In good accordance with our data, van

Zutphen et al. (2010) showed that the maltase and the

permease genes were respectively 89- and and 181-fold

derepressed after the shift of H. polymorpha from glucose

to methanol medium. Maltase has very low affinity for

disaccharides (Liiv et al., 2001). Therefore, maltose and

sucrose should be concentrated first into the cell by

energy-dependent transport (Viigand & Alam€ae, 2007),

and only then will their efficient hydrolysis proceed. The

hydrolysis reaction should produce a significant amount

of glucose (and fructose) inside the cell, and because

glucose- and fructose- phosphorylating activity of

H. polymorpha growing on gluconeogenic carbon sources

is low (Parpinello et al., 1998; Kramarenko et al., 2000),

at least part of glucose and fructose should stay unphos-

phorylated and act as inducer for MAL genes.

Induction of glucose-repressed genes by low glucose

has been reported for Aspergillus fungi. So, acetate-

growing Aspergillus oryzae did not produce a-amylase,

whereas addition of small amount of glucose induced its

production (Carlsen & Nielsen, 2001). Murakoshi et al.

(2012) showed for Aspergillus nidulans that aside of

transglucosylation products of maltose (e.g. isomaltose

and kojibiose) also glucose acted as physiological inducer

of a-amylase. All these sugars promoted nuclear entry of

respective activator protein AmyR, whereas for maltose

as inducer, maltase activity was required. Although glu-

cose induces a-amylase production in A. nidulans, this

activity is masked to a certain extent by CreA-dependent

catabolite repression. Accordingly, glucose acted as

a much more potent inducer of a-amylase in a creA-

defective strain (Murakoshi et al., 2012). Here, a parallel

can be drawn with our results showing that glucose

reveals its inducing ability in glucose repression-deficient

(hxk1glk1) mutants.

Summing up, we consider that growth of H. polymor-

pha (and probably other yeasts) on disaccharides is

complicated, because hydrolysis products of disaccharides

that promote initial derepression of MAL genes will later

cause repression due to accumulation of their phosphory-

lated species. Therefore, disaccharide transport and

maltase expression should be finely accommodated with

glycolytic flux to provide an appropriate expression level

of disaccharide-specific genes.

Coregulation of MOX, FMD, MPP1 and MAL1

genes

This study suggests that H. polymorpha senses by an as

yet unknown mechanism the presence and amount of

glucose-6P in the cell to trigger repression of methanol-

specific and MAL genes. We have evidence that these two

sets of genes can be coregulated. Namely, in a regulatory

mutant L63 of H. polymorpha, a single recessive mutation

released maltase, alcohol oxidase and catalase from

glucose repression (Alam€ae & Liiv, 1998) suggesting a

defect or absence of a shared repressor protein. We spec-

ulate that the availability of glucose-6P in the cytosol may

modulate the activity of this hypothetical repressor, for

example, to promote its nuclear entry. So, if glucose-6P is

present and sensed in the cytosol, the glucose-repressed

promoters will remain down-regulated. In the absence of

free glucose-6P, the repressor will stay in the cytosol and

allow derepression of the promoters. According to our

data, unphosphorylated glucose and fructose also stimu-

late derepression of methanol-specific and MAL genes.

We speculate that they may participate in maintaining

cytosolic location of the hypothetical common repressor

protein.

In addition to derepression, specific induction is also

contributing to gene expression. It would make sense if

specific activator genes were induced first in response to

a new carbon source. After 2 h of transfer of glucose-

grown H. polymorpha to methanol, the MPP1 gene is

extremely highly (394 times) induced (van Zutphen

et al., 2010). So, the MPP1 protein may indeed act as

the main transcriptional activator of methanol-specific

genes. The mechanism of induction is yet not known,

but certain inducer metabolites may also have a role in

it. To draw a parallel, acetaldehyde was shown to be a

physiological inducer of ethanol-specific genes in

A. nidulans (Flipphi et al., 2002). Regarding maltase

expression, we have shown here that unphosphorylated

glucose and fructose activate MAL1 promoter expression

in H. polymorpha.



Concluding remarks

Analysis of reporter gene expression from MOX, FMD,

MPP1 and MAL1 promoters in wild-type, hexokinase-

negative (hxk1) and double kinase-negative (hxk1glk1)

strains of H. polymorpha suggested that most probably

the rate of sugar flux in the cell is sensed to initiate

repression of these promoters. We propose glucose-6P as

candidate for repression signalling metabolite. Intrigu-

ingly, our data on hxk1glk1 H. polymorpha show that

unphosphorylated sugars in the cells promote derepres-

sion of MOX, FMD, MPP1 and MAL1 promoters. We

consider that under certain conditions, unphosphorylated

sugars may accumulate also in wild-type cells, causing

initial derepression of methanol- and disaccharide-

specific promoters that will be followed by growth

substrate-specific induction. New data on regulation of

putative peroxisomal activator protein gene MPP1 were

presented. We showed that with regard to sugar repres-

sion, the MPP1 promoter was regulated similarly to

MOX and FMD promoters. Yet, its expression was

slightly more sensitive to glucose and its methanol-

induced strength was lower compared with MOX pro-

moter. We also show that cultivation of yeasts on agar

plates created nutrient-limitation allowing assessment of

promoter derepression. According to our results,

hexokinase-negative and double kinase-negative strains

of H. polymorpha are considered perspective hosts for

foreign protein production from sugar-repressed

promoters.

Acknowledgements

This work was supported by grants 7528 and 9072 from

the Estonian Science Foundation and by targeted financ-

ing grant SF0180088s08.

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Primers used in this work.


232 S. Suppi et al.

repression vs. activation of mox , fmd , mpp1 and mal1 promoters by sugars in hansenula polymorpha :...

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