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Applied and Environmental Microbiology, January 1999, p. 53-60, Vol. 65, No. 1
Department of Cell and Developmental Biology,
Pasteur Institute-Cenci Bolognetti Foundation, University of Rome "La
Sapienza," 00185 Rome, Italy,1 and
Biotechnology Department, Rhone-Poulenc Rorer, 94403 Vitry
cedex, France2
Received 13 July 1998/Accepted 8 October 1998
KlADH4 is a gene of Kluyveromyces lactis
encoding a mitochondrial alcohol dehydrogenase activity which is
specifically induced by ethanol. The promoter of this gene was used for
the expression of heterologous proteins in K. lactis, a
very promising organism which can be used as an alternative host to
Saccharomyces cerevisiae due to its good secretory
properties. In this paper we report the ethanol-driven expression in
K. lactis of the bacterial Kluyveromyces lactis is
an aerobic yeast which is able to grow on lactose as the sole carbon
source. Due to this property, which is rare among yeasts, K. lactis has been used for the purification of the enzyme lactase
( Regulated promoters have been obtained from the K. lactis
genes lactase (LAC4) (18, 26, 33, 46) and acid
phosphatase (KlPHO5) (14, 15). These genes are
regulated in a very similar way to the corresponding GAL and
PHO5 genes of S. cerevisiae, which are induced at
the transcriptional level in the presence of lactose or galactose and
of low phosphate concentrations, respectively (40, 45).
KlADH4 is a K. lactis gene induced by ethanol,
encoding a mitochondrial alcohol dehydrogenase (ADH) activity with
interesting regulatory properties (35). Such regulation can
be observed in Rag All these findings suggested to us the possibility of using the
promoter regions of KlADH4 for ethanol-dependent expression of heterologous genes in K. lactis Rag In this paper, we report the isolation of the KlADH4
promoter and its use for the regulated expression of bacterial and
human genes. We also identified a small region of the promoter which is
responsible for the induction of the gene in the presence of ethanol.
In K. lactis cells, an efficient production of heterologous
proteins with enzymatic activity, such as prochymosine (46) and To optimize the production of recombinant HSA (rHSA) under the control
of the KlADH4 promoter, we focused on the medium composition and growth conditions; in preliminary attempts, we obtained good yields
of rHSA in fed-batch fermentation.
Strains.
The following K. lactis strains were
used in this work. MW98-8C ( Media and culture conditions.
YPD consists of (grams/liter)
glucose, 20; yeast extract (Difco), 10; and Bacto Peptone (Difco), 20;
supplemented, when required, with 100 mg of Geneticin G418 (Sigma) per
liter. When indicated, ethanol (YPE), glycerol (YPG), or glucose plus
ethanol (YPDE) at 20, 30, and 20 + 10 g/liter, respectively, were
added instead of glucose.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of the KlADH4 Promoter for
Ethanol-Dependent Production of Recombinant Human Serum Albumin in
Kluyveromyces lactis
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-glucuronidase and of the
human serum albumin (HSA) genes under the control of the
KlADH4 promoter. In particular, we studied the
extracellular production of recombinant HSA (rHSA) with integrative and
replicative vectors and obtained a significant increase in the amount
of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By
deletion analysis of the promoter, we identified an element
(UASE) which is sufficient for the induction of
KlADH4 by ethanol and, when inserted in the respective
promoters, allows ethanol-dependent activation of other yeast genes,
such as PGK and LAC4. We also analyzed the
effect of medium composition on cell growth and protein secretion. A
clear improvement in the production of the recombinant protein was
achieved by shifting from batch cultures (0.3 g/liter) to fed-batch
cultures (1 g/liter) with ethanol as the preferred carbon source.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-galactosidase) and for the production of low-lactose milk in the
dairy industry (22), and the conditions used for cultivation
of this organism on the large scale have been well established. In the
last few years, K. lactis has been successfully used as an
alternative host to Saccharomyces cerevisiae for
heterologous gene expression, and good transformation systems and
stable multicopy vectors are now available for the genetic manipulation
of this yeast (for reviews, see references 16, 32,
and 44). Expression of heterologous genes in
K. lactis can be achieved by the use of constitutive
promoters which have also been isolated in S. cerevisiae;
such promoters are interchangeable between the two yeasts. However,
S. cerevisiae inducible promoters are not always tightly
regulated in K. lactis cells, suggesting the existence of
different regulatory circuits on gene expression in the two organisms.
(resistance to antibiotics on glucose)
strains of K. lactis, which are unable to grow on glucose in
the presence of mitochondrial inhibitors (19). In these
strains, which are impaired in sugar fermentation and are poor
producers of ethanol, the addition of ethanol specifically induced
KlADH4 while the addition of glycerol and other
nonfermentable carbon sources did not induce the expression of this
gene. Moreover, differently from the ADH2 gene of S. cerevisiae (8-10), the induction of KlADH4
is not sensitive to glucose repression, since the gene is expressed
when both glucose and ethanol are present in the culture medium
(29, 34).
strains. Such strains can be either naturally isolated or constructed in the laboratory by disrupting genes which affect the glycolytic flux,
such as KlPGI1 (29), the gene encoding
phosphoglucoisomerase, or genes directly involved in ethanol
production, such as KlPDC1, the gene encoding pyruvate
decarboxylase of K. lactis (3). By contrast, in
Rag+ strains, the KlADH4 promoter is also active
in cells growing in glucose and other fermentable carbon sources
because of the ethanol produced by cells through fermentation, but its
transcriptional activity can be still increased (two- to threefold) by
the addition of exogenous ethanol (data not shown).
-galactosidase (1), and proteins of therapeutic
interest, such as interleukin-1 (4, 17), hepatitis B
surface antigen (28), granulocyte colony-stimulating factor
(21), and human serum albumin (HSA) (5, 18), have
been efficiently produced (for a review, see reference
44).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References
uraA lys arg rag1 pgi1 adh3)
is the parental strain (2). MS1 is a derivative of MW98-8C
carrying an integrated copy of plasmid P4GUS into the KlADH4
chromosomal locus (12) CMK5 (a thr lys pgi1 adh3
adh1::URA3 adh2::URA3), which expresses only
KlADH4, was obtained by crossing strains that lacked single ADH activities (29). CBS 293.91 is a prototrophic natural
K. lactis isolate (12). Y 721 is an isogenic
derivative of CBS 293.91 carrying a disrupted copy of the
phosphoglycerate kinase (PGK) gene (12). This
strain, used for many generations in batch and fed-batch fermentations,
showed a Rag+ phenotype for the presence of the
PGK gene on plasmid pYG156. Since the gene is essential for
the growth on fermentable carbon sources, such a host-vector
combination resulted in the stabilization of the expression system.
Shake flask cultures. Flasks (300 ml) containing 50 ml of medium were seeded with 0.5 ml of an inoculum grown to the stationary phase on the same medium. Cultures were grown for 3 days at 28°C on a rotary shaker (220 rpm) for rHSA production.
Fed-batch cultures. Fermentations were carried out in a 2-liter fermentor (SETRIC France) containing 0.8 liter of medium. The fermentor was inoculated with 40 ml of a fresh preculture grown to the stationary phase on the same medium. During fermentation, the pH was automatically controlled and adjusted between 6.5 and 7.0 with 10% (wt/vol) ammonia and the temperature was maintained at 28°C. Aeration was maintained at 75 liters/h under 0.2 × 105 Pa, and the dissolved oxygen was controlled at ~30% air saturation by the impeller speed controller and, when necessary, by enrichment of the air stream with pure O2. All the process variables (pH, temperature, agitation, dissolved oxygen, and O2 and CO2 contents in the exhaust gases) were transduced on-line and interfaced by a PH3852 unit to a Hewlett-Packard 9000 computer. The compositions of the feed media were carbon source, 340 g/liter; yeast extract, 100 g/liter; ammonium acetate, 50 g/liter; or carbon source, 340 g/liter; sodium glutamate, 40 g/liter; ammonium acetate, 70 g/liter; and vitamins for complex and defined media, respectively.
General methods. Restriction enzyme digestions, plasmid engineering, and standard techniques were performed as specified by Sambrook et al. (37). Escherichia coli and yeast transformation was performed by electroporation with a Bio-Rad Gene-Pulser apparatus as specified by the manufacturer.
PCR amplification and cloning of KlADH4 promoter
fragments.
The oligonucleotides used in PCR amplifications are
listed in Table 1. PCRs were performed
with the GeneAmp kit (Perkin-Elmer Cetus), using as template a 4.0-kbp
genomic DNA region of K. lactis, which contained the
KlADH4 gene, cloned on a plasmid. The reaction mixture (100 µl) contained 0.2 mM each primer, 0.2 mM each deoxynucleoside triphosphate, 1× PCR buffer, 25 ng of DNA, 2 mM MgCl2, and
2.5 U of Ampli-Taq DNA polymerase. PCR was carried out for 30 cycles of
94°C for 1 min, 52°C for 2 min, and 72°C for 3 min. After
amplification, the DNA fragments were purified on agarose gels,
digested with the appropriate enzyme, cloned into the pKSII plasmid
(Stratagene cloning system), and transferred to K. lactis
replicating plasmids by standard procedures.
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Vector constructions.
E. coli -glucuronidase (GUS)
and HSA expression vectors pP4-GUS, pYG132, and pYG156 (Fig.
1) were engineered as derivatives of
pKD1, a natural plasmid originally isolated from Kluyveromyces drosophilarum (7, 13). pKD1 is structurally related to
the S. cerevisiae plasmid 2µm and can stably replicate in
K. lactis (2).
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Construction of KlADH4 promoter-HSA fusion vectors. In contrast to pP4-GUS, plasmid pYG132 contains the entire sequence of pKD1, linearized at the unique EcoRI site, as well as an HSA expression cassette consisting of the KlADH4 SalI-HindIII portable promoter, the prepro-HSA cDNA gene, and the S. cerevisiae phosphoglycerate kinase (PGK) terminator. pYG156 contains the entire pKD1 and the K. lactis PGK gene that can be used as a selectable marker in pgk mutant host strains (12). The common features of all three vectors are the presence of the E. coli origin of replication, allowing the plasmids to work as shuttle vectors, and the presence of the bla and aph genes conferring ampicillin and geneticin resistance to E. coli and yeast, respectively. In addition, pP4-GUS carries the S. cerevisiae URA3 gene as an auxotrophic marker.
The vectors pYG107 and pYG108 contain the entire pKD1 sequence and the HSA cDNA fused to LAC4 and PGK promoters, respectively (18). To construct pYG534 and pYG397, the region from
536 to 13 was
obtained by PCR amplification with oligonucleotides 5 and 8, and the
region from 399 to 13 was obtained with oligonucleotides 7S and 8. After SalI-HindIII digestion, these fragments
were inserted into plasmid pYG108 to give pYG534 and pYG397, respectively.
To construct pYG108/700M and pYG108
UAS, the region from 1199 to
519 was obtained by PCR amplification with oligonucleotides 1 and 6. This SalI-HindIII fragment was cloned into
the polylinker of pKSII plasmid. The HindIII site was
eliminated by filling in with Klenow polymerase to give plasmid
pKSII/700M. The 700-bp fragment was cut by a
SalI-NotI digestion and cloned into plasmid pYG108 to give plasmid pYG108/700M. In plasmid pYG108/700M, the region
of the PGK promoter from 1499 to 402 containing the
upstream activation site (UAS) was replaced by the 700-bp fragment of
the KlADH4 promoter. As a control, the pYG108 plasmid was
digested with SalI-NotI, filled in with Klenow
DNA polymerase, and religated with DNA T4 ligase. The resulting plasmid
was called pYG108UAS.
Plasmid pYG108/2-4 contains the promoter regions from 953 to 741
(amplified with oligonucleotides 2 and 4) cloned into the SalI-EcoRI sites of plasmid pYG108/700M.
Plasmid pCM13 carries the region of the KlADH4 promoter
spanning from 399 to 13 (amplified with oligonucleotides 7E and 8).
The amplified fragment was inserted into the
EcoRI-HindIII sites of plasmid pYG108/700M.
In this way, the PGK promoter TATA box was replaced with the
KlADH4 TATA box.
Plasmid pCM15 was obtained by cloning the promoter fragment contained
in plasmids pYG108/2-4 into the SalI-EcoRI sites
of pCM13. The promoter regions from 758 to 519 (amplified with oligonucleotides 3 and 6) were cloned in the
SalI-HindIII sites of the pKSII polylinker to
yield plasmid pKS/3-6. After SalI-EcoRI digestion, the promoter fragment was cloned into pCM13 plasmid to yield
plasmid pCM16.
In pCM17, two of the three UAS elements contained in the
LAC4 promoter were replaced with the 200-bp fragment
containing UASE.
Protein assays. Cultures (10 ml) of K. lactis were grown to the early stationary phase in YP medium containing different carbon sources as specified in the text. The cells were broken with glass beads in Eppendorf tubes and centrifuged, and the supernatants were analyzed by electrophoresis on 5% nondenaturing acrylamide minigels. Gel preparation and buffers were essentially as described by Williamson et al., (47). Samples corresponding to 20 to 40 µg of proteins were run at 4°C for 60 min at 20 mA. For the detection of ADH activity, the gels were stained as described by Lutstorf and Megnet (27). GUS activity (39) was detected by soaking the gels in a staining solution containing 50 mM Na2HPO4 (pH 7.0) and 50 µg of 5-bromo-4-chloro-3-indolyl glucuronide (X-Glu) per ml dissolved in dimethylformamide (5 mg/ml). The gels were then incubated at 37°C until bands corresponding to the enzymatic activities became visibly stained.
Albumin detection. To detect albumin, 5 × 105 cells/ml were inoculated into YPD or YPDE medium and supernatants were tested after 3 days of fermentation. Aliquots of the supernatants were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and the protein was revealed by Coomassie brilliant blue staining or by Western blotting. When the latter method was used, aliquots of supernatants were separated on sodium dodecyl sulfate-10% polyacrylamide gels and proteins were electroblotted onto Immobilon P membranes (Millipore) with a Bio-Rad wet blotting system. Filters were saturated by incubation for 30 min at room temperature in phosphate-buffered saline (PBS) with 5% nonfat dry milk. The blots were washed twice in PBST (PBS, 0.05% Tween 20) and incubated for 2 h with the anti-HSA monoclonal antibody (Sigma Co.A-0433) diluted 1:16,000 in PBS. After two washes with PBST, the filters were incubated for 1 h with the secondary antibody linked to the alkaline phosphatase. After two final washes, the blots were developed with the 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazolium system (20).
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Cloning of the K. lactis KlADH4 promoter. The KlADH4 gene was isolated as an 8.4-kb BamHI fragment from a K. lactis genomic bank by using an S. cerevisiae ADH2-derived probe (36). Based on the nucleotide sequence of the KlADH4 coding region (35), we located the ATG initiation codon close to a HindIII site. Accordingly, we subcloned a 2.2-kb HindIII fragment containing the KlADH4 upstream region and the first 14 codons of the coding region into plasmid pTZ19 (Pharmacia). This construct was used to introduce a BamHI site 10 nucleotides upstream from the ATG codon by site-direct mutagenesis by using the double-primer procedures (37). The resulting plasmid allowed the isolation of a 1.2-kb BglII-BamHI portable promoter used in the construction of the pP4-GUS expression vector (Fig. 1). A promoter variant in which the BglII and BamHI sites were replaced by SalI and HindIII sites, respectively, was obtained by the PCR procedure (pBluescript, mung bean nuclease). This variant was used in the engineering of albumin expression vectors pYG132 and pYG156 (Fig. 1).
Ethanol-regulated expression of bacterial GUS. We then wondered whether the KlADH4 promoter could be successfully used for the regulated expression of heterologous genes. First we used the E. coli GUS gene as a reporter gene under control of the KlADH4 upstream region (see Materials and Methods). The resulting plasmid, named pP4-GUS (Fig. 1), was used to transform K. lactis MW98-8C to uracil auxotrophy. This autonomously replicating sequence-containing vector, which is unstable in K. lactis, was used to select for chromosomal integration of the expression cassette. One of the isolated Ura+ transformants, named MS1, expressed GUS activity over many generations. Southern analysis revealed that integration of the expression vector occurred at the chromosomal KlADH4 locus by homologous recombination, which resulted in the duplication of the promoter region without interfering with the integrity of the KlADH4 structural gene (data not shown). In fact, as demonstrated in Fig. 2A, both genes are functionally expressed and they are coregulated in an ethanol-dependent manner. Furthermore, these results show that all elements relevant for regulation are present on the 1.2-kb portable KlADH4 promoter. Moreover, as shown by Northern analysis, GUS- and KlADH4-specific transcripts can be detected as soon as 30 min after induction by ethanol (Fig. 2B). For the GUS gene, besides the mRNA of the expected length (about 2,300 nucleotides), another, larger transcript which migrated at the level of the large rRNA was detected. This transcript, regulated in the same way as the mature mRNA, could represent a precursor molecule.
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Production of HSA under control of the KlADH4 promoter on multicopy vectors. Recently, increasing interest has been focused on the production of HSA in K. lactis. The HSA cDNA gene has been cloned into pKD1-derived vectors under the control of the constitutive PGK promoter of S. cerevisiae and the regulated LAC4 promoter of K. lactis, and good production of the protein has been obtained under different growth conditions (batch, fed batch, and chemostat) (5, 18).
To test whether the KlADH4 promoter could be efficiently used for the biotechnological production of a protein of industrial interest, we engineered vectors carrying the HSA cDNA gene under the control of this promoter. In the first experiment, we integrated the PKlADH4-prepro-HSA expression cassette into the genome of K. lactis. Since the resulting integrants showed very low levels of secreted HSA which could be detected only by immunological methods (data not shown), we examined the production of this protein in K. lactis strains transformed with the pKD1-derived multicopy vector pYG132 (Fig. 1A), which carries 1.2 kbp of the KlADH4 promoter fused to the HSA cDNA. This plasmid was introduced into the nonfermenting strain CMK5, and after 3 days of growth in YP medium containing different carbon sources, aliquots of supernatants were analyzed for the presence of HSA by gel electrophoresis under denaturing conditions. The protein was revealed either by Coomassie blue staining or by Western blot analysis with an anti-HSA monoclonal antibody (Fig. 3A and B). In CMK5, as in other nonfermenting strains, the HSA was present only in the supernatant of cells grown in ethanol (lane 3) and not in the supernatant of glucose- or glycerol-grown cells (lanes 2 and 4).
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Identification of an ethanol-responsive element.
To identify
possible cis-acting elements, we introduced deletions into
the KlADH4 promoter and parts of it were also cloned upstream of the TATA box of the heterologous PGK promoter of
S. cerevisiae. All constructions were introduced into CMK5
cells, and the level of HSA produced by transformant clones was
analyzed by gel electrophoresis. The results of our analysis are
reported in Fig. 4. HSA production was
not observed on ethanol with KlADH4 promoter deletions
upstream of position 536 (plasmids pYG534 and pYG397), suggesting
that the putative activating sequences were localized within the
deleted regions. On the other hand, we could observe ethanol-driven
expression of the HSA cDNA when the region from bp 1197 to
536 was fused to the TATA box of both the KlADH4 (pCM13)
and the PGK (pYG108/700M) genes. For this reason, we
performed a more detailed analysis of this region.
|
,
KlADH4 promoter; 
, PGK promoter;
, LAC4
promoter.
953 to 741 fused to the TATA box of the KlADH4 and
PGK genes, respectively. Both plasmids allowed
ethanol-dependent expression of the HSA cDNA. In contrast,
when this 200-bp region was missing (plasmid pCM16), the regulatory
property of the promoter was completely lost. No HSA production could
be detected with the control vector pYG108UAS, which contained only
the TATA element of PGK.
To further confirm that the region from bp 953 to 741 contained the
ethanol-responsive element(s), we substituted this fragment to the
upstream activation site (UAS) region of the promoter of LAC4. This is the -galactosidase-encoding gene of
K. lactis, which is induced by lactose and is repressed in
the presence of glucose (1, 26, 33, 38). The resulting
hybrid LAC4-KlADH4 promoter was fused to the HSA
cDNA (plasmid pCM17), and the production of HSA on different carbon
sources was monitored. As shown in Fig. 4, this promoter could drive
HSA production only in ethanol-grown cells and, as expected, was no
longer inducible by lactose (data not shown). In control experiments
(plasmid pYG107), the LAC4 promoter fused to the
HSA cDNA failed to drive HSA production on glucose, glucose
plus ethanol, and glycerol, whereas it allowed HSA production in
lactose-grown cells (not shown).
All the above results indicate that the element(s) necessary and
sufficient for the induction of KlADH4 by ethanol is located within the 200-bp fragment spanning bp 953 to 741. This element, which also proved functional when inserted in heterologous promoters, was named UASE.
pKlADH4-driven rHSA secretion in fed-batch fermentations. In shaken-flask cultures, the production of rHSA under inducing conditions reached 200 to 250 mg/liter in defined medium and 300 mg/liter in complex medium, which corresponded to yields of 20 to 30 mg of rHSA/g of dry biomass. These values represent 5 to 7% of the yeast total proteins and 80 to 90% of the total soluble proteins (data not shown).
To improve the rHSA yields, we performed fed-batch fermentations (0.8 liter) on different media. In these experiments, the vector used was pYG156, which carries the PGK gene for selection (see Materials and Methods) and does not confer resistance to the aminoglycoside antibiotic G418. After initial batchwise growth of strain Y 721 in defined medium (DM1) and consumption of the carbon substrate, the feed medium was added to the fermentor by using a peristaltic pump coupled to a preprogrammed time-based feeding profile, deduced from previous experiments where the feeding rate was coupled to the carbon dioxide evolution rate. The carbon substrate used was a mixture of lactose and ethanol (final concentration, 20 g/liter) in different ratios: 50/50, 12/88, and 0/100. As shown in Fig. 5A, when 100 g of carbon sources was added, 45% of the carbon was recovered in the biomass when 100% ethanol was used, and this value increased to 53% when lactose plus ethanol (12/88) was used. On the other side, the best results in terms of rHSA production were obtained with ethanol as the only carbon substrate (Fig. 5B). In fact, under these conditions, about 800 mg of the protein per liter was produced, compared to 200 and 350 mg/liter obtained when different mixtures of ethanol and lactose were used. In conclusion, at the end of the fermentation time (72 h) with ethanol as the only carbon source, we obtained 71 g (dry-cell weight) of biomass per liter with a yield (Yx/s) of 45%. At the same time, 620 mg of rHSA per liter was produced, with a yield (Yp/x) of 8.7 mg of rHSA/g of biomass and a productivity of 8.7 mg of rHSA/liter · h.
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, lactose-ethanol (50/50); 
, lactose-ethanol
(12/88); 
, lactose-ethanol (0/100).
Kinetics of rHSA secretion in fed-batch fermentations. The growth and production kinetics of rHSA in complex medium (ethanol, yeast extract, and ammonium acetate) and a defined medium (ethanol, sodium glutamate, ammonium acetate, vitamins) have been compared. As shown in Fig. 6A and B, the kinetics of growth and the production levels of rHSA were very similar in the two media during the first 3 days, and we could observe an increase in the specific productivity of rHSA from 5 to 30 mg/liter · h between 24 and 72 h of fermentation. After 4 days of culture, a high biomass production occurred in both complex and defined media (70 and 80 g [dry-cell weight]/liter, respectively), with a yield of about 45% as a result of the added ethanol. In this period, the amount of rHSA produced was 1.05 g/liter in complex medium and 0.85 g/liter in defined medium, which corresponded to a productivity of 15 and 10 mg of rHSA/g of biomass, respectively. In both complex and defined media, biomass and rHSA yields were well correlated with the ethanol added during the fed-batch culture (Fig. 6C).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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HSA is the most abundant protein in human plasma involved in the maintenance of a normal osmolarity and also in the transport of hydrophobic molecules. This protein has a big market requirement in that it can be used as replacement fluid during septic or traumatic shock, to compensate for blood loss, and to treat burn victims. At present, HSA is largely produced (300 tons/year) by conventional techniques involving fractionation of plasma obtained from blood donors. It would be a great advantage to be able to use genetic engineering to obtain rHSA in good yield and at lower cost, with no danger of contamination by human pathogens. For this reason, great efforts have been dedicated to the production of this protein on a large scale by genetically engineered microorganisms. For this purpose, rHSA production has been studied in E. coli (24, 25) Bacillus subtilis (38), S. cerevisiae (11, 23, 31, 42, 43), Pichia pastoris (30), and plants (41).
K. lactis is a very promising nonpathogenic organism because
of its good secretory capabilities (17, 18, 44, 46), and new
promoters with specific properties can be derived from this yeast for
the regulated expression of heterologous proteins. In fact, we isolated
a new promoter from the KlADH4 gene of K. lactis.
This promoter, also present in multicopy vectors, allows regulated or
constitutive expression of heterologous genes depending on the host
strain used. In nonfermenting strains (Rag), which are
unable to produce intracellular ethanol from fermentable carbon
sources, the transcriptional activation of the KlADH4
promoter is dependent on the addition of ethanol to the culture medium. Nonfermenting K. lactis mutants can be easily generated from
fermenting strains with particular industrially relevant properties
(i.e., biomass yield and secretion efficiency) by disrupting genes
which affect ethanol production.
Another interesting aspect of the regulation of this promoter is its insensitivity to glucose repression. This property, combined with the specificity of ethanol as the activator, allows the expression of cloned genes after the addition of the inducer, independently of the carbon source present in the medium.
We identified an ethanol-responsive element (UASE) which is
located within a 200-bp region spanning from 953 to 741 of the promoter. Deletions of UASE render the KlADH4
promoter no longer inducible in the presence of ethanol. On the other
hand, UASE constitutes an interesting "transportable"
cis-acting element for the construction of a regulated
chimeric promoter. In fact, UASE can confer
ethanol-dependent promoter activation, such as of the S. cerevisiae PGK and K. lactis LAC4 promoters, which are usually not induced by this substrate.
Finally, we studied the production of rHSA under the control of the KlADH4 promoter in K. lactis cells grown in complex and defined media under different cultivation conditions. Considering the results obtained in terms of production per liter of culture, high biomass production (70 to 80 g [dry-cell weight]/liter) was obtained either in both complex and defined media, and a significant amount of rHSA production was observed in fed-batch cultures (1 g/liter) as compared to batch cultures (0.2 to 0.3 g/liter). These values are similar to those obtained under the same conditions with the HSA gene under the control of the PGK and LAC4 promoters (18).
On the basis of other studies, it should certainly be possible to increase the level of production of rHSA to at least 2 g/liter by working on the fermentation parameters. As an example, under the culture conditions used, the oxygen uptake rate never exceeded 2 mM/min.
In conclusion, we believe that the yields of rHSA obtained can be improved and that the process can be scaled up.
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ACKNOWLEDGMENTS |
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Michele Saliola and Cristina Mazzoni contributed equally to this work.
Cristina Mazzoni was supported by a fellowship from the Istituto Pasteur-Fondazione Cenci Bolognetti. This work was supported by EC contract BIO4-CT96-0003 and partially supported by MURST (Ministero della Ricerca Scientifica e Tecnologica).
We would like to thank F. Castelli for technical assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Cell and Developmental Biology, Pasteur Institute-Cenci Bolognetti Foundation, University of Rome "La Sapienza," Piazzale A. Moro, 00185 Rome, Italy. Phone: (39) 0649912278. Fax: (39) 0649912256. E-mail: FALCONEC{at}AXCASP.CASPUR.IT.
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REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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