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Applied and Environmental Microbiology, February 2007, p. 922-929, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts{triangledown}

Andrea Camattari,1 Michele M. Bianchi,2 Paola Branduardi,1 Danilo Porro,1 and Luca Brambilla1*

Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy,1 Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma "La Sapienza," P.le A. Moro 5, 00185 Rome, Italy2

Received 26 July 2006/ Accepted 24 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1ß was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get ß-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heterologous protein production is a strategic market for different biotechnological industries. Recent business analyses estimated the worth of the recombinant DNA-derived products market at 32 billion dollars in 2003 in the United States alone, considering only the human-related drugs (20). Yeast cells have been frequently proposed as hosts for heterologous gene expression due to some advantageous characteristics (23, 26, 30). Yeasts are in fact microorganisms able to carry out several posttranslational modifications that are not performed by bacteria. Moreover, the presence of a secretory apparatus facilitates industrial processing, making yeasts suitable hosts for large-scale manufacturing. For the production of heterologous proteins, a large number of promoters, either constitutive or inducible, are available. The regulation of the expression of proteins of interest allows the separation of the growth phase from the production phase. This helps in avoiding the selection of fast-growing nonproducing cells and also permits the synthesis of proteins that could be detrimental to the host cells (23). Although the two-stage strategy is theoretically advantageous, very often constitutive promoters and one-phase processes are chosen at the industrial level, since inducible promoters usually require the addition of expensive inducers and/or compel rigorous formulation on growth medium composition. Ideally, a good induction system should be economical and easy to manage; activation of gene transcription should be fast and strong enough to ensure a good induction ratio over the uninduced conditions.

Kluyveromyces lactis is a nonconventional yeast with a "generally regarded as safe" status; it is able to perform posttranslational processing and modification; and it can grow with good yields on cheap substrates, like whey. In addition, genetic manipulations are facilitated by the availability of integrative and episomal vectors and by the publication of its entire genome (7). Considering its good secretion ability, K. lactis constitutes a valid alternative to the classical baker's yeast, Saccharomyces cerevisiae, for the expression of heterologous products, often being the best producer when a direct comparison has been attempted between these two yeasts (25). This good biotechnological potential is confirmed by the expression of several proteins for industrial and medical use (reviewed in reference 29). KlPDC1 is the unique gene coding for pyruvate decarboxylase (PDC) activity in K. lactis (2). Transcription of KlPDC1 is regulated by the carbon source, induced by glucose and repressed by ethanol, and repressed by autoregulation (5). Moreover, PDC activity depends on oxygen availability. In fact, both enzymatic activities and KlPDC1 transcription are induced by hypoxic growth conditions (5, 13).

The possibility of increasing production of heterologous products by hypoxia is very interesting from an applicative point of view, because hypoxic conditions are easily and cheaply obtained in a bioreactor by reducing the air supply and neither modulation of medium composition nor addition of inducers is required.

In the present work, the possibility of a practical application of the hypoxic regulation of the KlPDC1 promoter is investigated by cloning the whole promoter sequence upstream to several heterologous genes coding for proteins widely heterogeneous in their characteristics. A careful optimization of the induction protocol, as well as the rearrangement of the promoter sequence, obtained by genetic manipulation, allowed a further increase in the performance of this inducible system. Finally, the possibility of transferring this promoter system to other related yeasts has been explored.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Yeast strains and plasmids.
A list of yeast strains and plasmids utilized in this work is given in Table 1. The detailed description of the construction of Z2{Delta}L, a leu2 deletion mutant strain of Zygosaccharomyces bailii, is the subject of a separate study (P. Branduardi et al., unpublished data). Construction of the plasmids developed for this work is described below.


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  		TABLE 1. Yeast strains and plasmids used in this work

 
Plasmids pLC12 and pLC12RR were obtained as follows. The bacterial lacZ gene was excised from plasmids pMD12 (5) and pMD12RR (5) by HindIII digestion and replaced with a HindIII fragment containing the cDNA of the laccase gene lcc1 from the fungus Trametes trogii (4). The plasmid harboring the lcc1 cDNA was a kind gift of M. Ruzzi. The resulting vectors were named pLC12 and pLC12RR, respectively.

Plasmid pS13-PIL was constructed as follows. The interleukin 1 beta (IL-1ß) gene and the S. cerevisiae PHO5 terminator were amplified by PCR from plasmid pYG81 (8). XbaI recognition sites were generated at the ends of the amplified DNA fragment, which was subsequently cloned into the XbaI site of plasmid pKSMD8/7 (21) downstream of the KlPDC1 promoter's whole sequence. The new plasmid was called pKSPPI/3. The expression cassette, composed of the KlPDC1 promoter, the IL-1ß gene, and the ScPHO5 terminator, was controlled by sequencing, excised from pKSPPI/3 as a SalI fragment, and cloned into the single SalI site of the multicopy pKD1-based plasmid pS13 (1). The final construct was called pS13-PIL.

Plasmid pC-3312 was obtained by the insertion of a DNA fragment containing the C33 coding sequence downstream of the KlPDC1 wild-type promoter in pMD12, previously deprived of the lacZ gene. To this purpose, pYX-C33 plasmid (18) was cut with PvuII and XbaI restriction enzymes, blunt ended with DNA polymerase, and ligated to the receiving vector pMD12, previously cut with HindIII, blunt ended, and dephosphorylated.

Plasmids pAC12 and pAC12RR were obtained as follows. Plasmids pMD12 and pMD12RR (5) were cut with NheI, blunt ended, and cut again with HindIII. The resulting fragments, containing the lacZ gene along with the whole KlPDC1 promoter or its modified form, were cloned in the centromeric vector YCplac111, previously cut with the HindIII and SmaI enzymes. The final constructs were called pAC12 and pAC12RR, respectively.

Growth media and fermentation conditions.
If not differently specified, a general scheme was followed for all fermentations. Cells were pregrown overnight in minimal medium, with the following basic formulation: yeast nitrogen base (0.67 g/liter; Difco) and glucose (20 g/liter; Merck), supplemented with 100 mg/liter of leucine, lysine, methionine (Merck), or 100 mg/liter of Geneticin (G418 sulfate; Gibco) when required. Fermentations were carried out at 30°C in a 2-liter bioreactor (Biostat B; B-Braun) containing yeast-peptone-dextrose (YPD) medium: yeast extract (10 g/liter; Biolife), peptone (20 g/liter; Biolife), and glucose (20 g/liter; Merck), pH 5. Temperature and pH were controlled at 30°C and 5.0, respectively. During cell growth, the dissolved oxygen tension (DOT) value was maintained at a level higher than 40% of saturation by stirring, with a 2-vvm aeration rate. Promoter activation was trigged by complete interruption of aeration and by the concomitant reduction of agitation. The promoter was induced at a cellular concentration between optical densities at 660 nm (OD660s) of 1 and 10, corresponding to a dry weight of 25 to 250 mg/liter, respectively. Cell growth was monitored by measuring the OD660.

Determination of plasmid stability.
At various stages of growth, samples were withdrawn from the bioreactor, diluted with sterile water, and plated in triplicate onto selective or nonselective medium. We estimated the percentage of the population still maintaining the plasmid by dividing the average number of colonies growing on selective plates against the average number of colonies growing without any pressure.

Glucose and ethanol quantification.
Glucose and ethanol were measured by high-performance liquid chromatography (Jasco) at 35°C with an HPX-87 H Aminex column (Bio-Rad) and a refractive index detector. The mobile phase was 4 mM H2(SO4)2 at 0.6 ml/min.

Enzymatic assays.
ß-Galactosidase activity was detected after cellular permeabilization with chloroform and sodium dodecyl sulfate (SDS), using o-nitrophenyl-ß-galactopyranoside (Sigma) as the substrate, essentially as described previously (15). One unit of ß-galactosidase activity is the amount of enzyme able to catalyze the oxidation of 1 nmol of O-nitrophenyl-ß-galactopyranoside per minute.

Laccase activity was assessed by measuring the enzymatic oxidation of 0.02 M 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) in McIlvine buffer (pH 3.4) at 30°C (9). The buffer solution was saturated with air by bubbling prior to the assay. One nkat of activity was defined as the amount of enzyme that oxidizes 1 nmol of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid per second.

Glucoamylase (GAM) activity was determined by measuring spectrophotometrically the decrease of the iodine-starch complex in the presence of culture supernatants, as described previously (27). One unit of GAM activity per milliliter is defined as the amount of enzyme that is able to decrease the A580 value by 1 U in 1 min.

Immunoassays.
Cellular extracts from cells expressing interleukin-1ß and c33 have been obtained by trichloroacetic acid (TCA) extraction (3). Briefly, after washing with water, pellets corresponding to 108 cells were washed twice with TCA (20% [vol/vol]) and once with TCA (5% [vol/vol]) and finally resuspended in 150 µl of SDS sample buffer.

Supernatants from interleukin-1ß-producing cells were directly diluted 1:2 with SDS sample buffer.

Identical volumes of samples were loaded on a 10% SDS-polyacrylamide gel, electrophoresed, and transferred onto a nitrocellulose membrane. Immunodecoration of filters was performed essentially as described previously (3, 18) for interleukin-1ß and c-33, respectively. Protein concentrations were estimated by densitometric analysis, using commercial standards of known concentrations.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
KlPDC1 hypoxic expression.
We explored the possibility of using induction by hypoxia of the KlPDC1 promoter as an alternative to other inducible systems, like induction by lactose/galactose of the lac genes or induction of phosphatases by low phosphate concentrations, currently used with K. lactis (29). In order to measure the level of hypoxic induction, we used the bacterial reporter gene lacZ fused to the KlPDC1 promoter on a centromeric plasmid, plasmid pMD12 (Table 1), which has been previously exploited to determine glucose induction, ethanol repression, and autoregulation of the KlPDC1 gene (5). A mutant strain without ß-galactosidase activity (lac4-8 mutation) was transformed with pMD12 and grown in a bioreactor on YPD medium. In the first part of the experiment, biomass was produced and the aerobic metabolism was ensured by vigorous aeration (2 vvm), which, coupled with controlled stirring, maintained the DOT above 40% of the saturation value. In spite of the lack of selective pressure, the stability of pMD12 remained higher than 95% over the course of the experiment. Similarly high stability values were obtained with all the centromeric vectors used in this study independently of the heterologous gene or the promoter portion present in the vector (data not shown).

Production of the heterologous enzyme was induced during exponential growth by simply closing the air inlet of the bioreactor. To verify the time course of promoter activation, biomass and ß-galactosidase activity were followed for at least 24 h. Experiments were run at least in duplicate, yielding reproducible data. Results of a typical experiment are shown in Fig. 1A. Interruption of the air supply resulted in a very fast (10 min) drop of DOT to zero, suggesting a strong respiratory metabolism of the exponentially growing cells. This DOT value was maintained for the rest of the fermentation. Under these conditions the growth rate became linear, probably due to oxygen limitation. Glucose metabolism was mainly fermentative, since ethanol was produced. The scarcity of oxygen determined a low biomass yield; in fact, the cell density reached a final value of an OD660 of 13 in 24 h, while the fully aerated control cultures reached a cellular density of an OD660 of 35 (data not shown). The value of specific ß-galactosidase activity, expressed as units/OD660, showed a sharp increase after the induction, reaching a value of 32 U/OD660 (about 20-fold the basal level). This behavior was related to hypoxic induction, since cells growing under the same conditions, but without induction, displayed a basal level of about 1.7 ± 0.3 U/OD660 for the entire duration of the fermentation process (data not shown).


Figure 1
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  		FIG. 1. A: Hypoxic induction of MW341-5(pMD12) cells in YPD medium. Cells were grown aerobically in YPD medium. Induction of the system was achieved at time zero by stopping air inlet when the optical density ({blacksquare}) was 1. In a few minutes, the value of dissolved oxygen ({circ}) decreased to 0% and ethanol ({blacklozenge}) was produced. The value of specific (units/OD) ß-galactosidase activity ({square}) increased from a basal level to the induced maximum level within a few hours. B: Hypoxic induction of MW341-5(pMD12) cells in YP medium. Cells were grown in YP medium and induced with the same procedure used for Fig. 1A. Values of optical density ({blacksquare}), dissolved oxygen ({circ}), ethanol concentration ({blacklozenge}), and specific ß-galactosidase activity ({square}) are reported.

 
The KlPDC1 promoter is regulated by carbon sources (5). In order to exclude any contribution to induction by specific carbon sources, cells were inoculated in a bioreactor containing YP medium (yeast extract and peptone, without any added specific carbon compound), where the carbon source is composite and consists basically of a mixture of amino acids. Figure 1B shows a typical experiment performed under those conditions. First, cell growth stopped immediately after hypoxic conditions were induced. Then, in a difference from the YPD process, ethanol was not produced, consistent with the absence of fermentable carbon sources in the growing medium. The level of expression found in YP medium was similar to that measured in YPD medium, except that we registered a lower basal level (1.1 ± 0.3 versus 1.7 ± 0.2 U/OD660) in YP. The specific ß-galactosidase activity reported in Fig. 1A decreased with time, from 32 to 27 U/OD660 in 21 h, while the volumetric activity increased over the course of the fermentation, reaching a value of about 330 U/ml. On the contrary, the absence of growth after the hypoxic induction shown in Fig. 1B resulted in a stable level of ß-galactosidase, at least for several hours. The comparison of the two processes suggested that the KlPDC1 promoter is scarcely induced by glucose during aerobic growth.

Another interesting mechanism of KlPDC1 regulation that might have a potential biotechnological application is autoregulation (5, 27). In order to assess whether the presence of KlPdc1p might influence hypoxic induction, K. lactis strain MM1-12D, carrying the Klpdc1{Delta} deletion, was transformed with pMD12. The resulting clones were grown in a bioreactor in YP medium and induced by hypoxia, following the same protocol. We found that without functional KlPdc1p, the phenomenon of oxygen regulation of the KlPDC1 promoter was almost completely masked by the strong effect of the absence of autoregulation. In fact, we measured a very high basal activity (70 ± 8 U/OD660) during aerobic growth, which was increased only threefold, reaching about 200 ± 36 U/OD660 after 24 h of induction by hypoxia (data not shown).

KlPDC1 promoter robustness.
Having assessed the extent of KlPDC1 promoter hypoxic induction and the technical feasibility of the procedure, we decided to further test the biotechnological potential of the system and to evaluate its efficiency in greater detail. Since the performance of an expression system is heavily affected by the characteristics of the heterologous product, we cloned under the control of the KlPDC1 promoter some other heterologous proteins of biotechnological interest, chosen to encompass a panel of different characteristics, as shown in Table 2. The proteins differed in their origin and dimension, in the presence of posttranslational modifications, and in their localization.


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  		TABLE 2. Heterologous proteins expressed under hypoxic induction of KlPDC1 promoter

 
Each construct was assayed at least in duplicate under the same experimental conditions described above, i.e., batch growth on YPD medium, hypoxic induction at a cellular density between OD660s of 1 and 10, and determination of overall protein production in the following 24 h. The ratio of induction was calculated, considering the highest production obtained with respect to the basal level, determined just before induction. Depending on the protein, we could estimate the total amount of protein produced by means of densitometric analysis of Western blots, or when specific antibodies against the heterologous product were not available, we estimated the production of correctly folded and bioactive molecules by using enzymatic assays. Average data for all production analyses are summarized in Table 3. Strain CBS2359/152F was used as a host for heterologous production. Individual processes are described briefly below.


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  		TABLE 3. Levels of heterologous protein production under KlPDC1 promotera

 
Human interleukin-1ß production.
IL-1ß is a monokine with a broad spectrum of biological activity. Besides pure immunological activities, many neurological, metabolic, and endocrinological effects have been described (6). The gene of human recombinant IL-1ß, in which a glycosylation site has been removed to avoid yeast's posttranslational modifications (8), was inserted downstream to the KlPDC1 promoter on a multicopy vector (pS13-PIL) that was used to transform K. lactis strain CBS2359/152F. Transformed cells were grown in a bioreactor (YPD medium), while biomass supernatants were processed at different times after the induction. Western blot analyses using specific antibodies were performed (Fig. 2A). A progressive increase in the signal detected in the supernatants (upper panel) was observed after 2 h from induction and accumulation in the medium continued for 4 to 6 h. The analysis of cellular extracts (lower panel) revealed a transient accumulation of the heterologous product immediately after the induction and before product release in the medium. The intracellular product of higher mobility was probably a truncated form that did not complete the secretory pathway, since it was not detectable in the supernatants. Densitometric analysis revealed very good production, with an estimated average concentration of 112 mg/liter of heterologous secreted product and an induction ratio of 8.4 (see Table 3). Figure 2B shows the effects of hypoxic induction on the specific production, expressed as mg of recombinant IL-1ß per gram of biomass, for cells induced and for the noninduced control. Transformed cells accumulated much more heterologous product in the absence of aeration.


Figure 2
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  		FIG. 2. A: Recombinant IL-1ß production after hypoxic induction. Identical volumes (10 µl) of supernatant (upper panel) and amounts of cell extract corresponding to 7 x 106 cells (lower panel) were loaded on gels together with a sample of commercial IL-1ß (c) and analyzed by the Western procedure. Numbers represent hours after induction. The arrow indicates a product with higher mobility, probably due to a truncated form. B: Specific interleukin-1ß production after hypoxic induction. Levels of secreted interleukin-1ß weighted by cellular biomass (dry weight, g/liter) for induced ({blacksquare}) and noninduced ({square}) transformed cells. C: Expression of heterologous laccase after hypoxic induction. Volumetric laccase production for induced ({blacksquare}) and noninduced ({square}) cells (nkat/ml).

 
T. trogii laccase production.
Laccases are typical fungal enzymes, involved in lignin synthesis, lignin degradation, and morphogenesis (9). They are multicopper enzymes, highly glycosylated, employed by fungal organisms to colonize the environment, especially roots or, in general, the tree's surface. These enzymes are very attractive for biotechnological applications, mainly in the paper and textile industries, and the interest in their production is strong (9). Strain CBS2359/152F of K. lactis was transformed with the centromeric vector pLC12, carrying lcc1, a laccase gene from T. trogii, and subjected to the usual induction procedure. Progression of laccase accumulation was monitored by means of a specific laccase enzymatic assay on supernatants against a noninduced control (Fig. 2C). Also in this case, we calculated an average induction ratio of about 10 times, starting from a basal level of 0.01 nkat/ml of bioactive molecule (Table 3).

Glucoamylase production by Arxula adeninivorans.
Glucoamylases are secreted enzymes typical of various fungus species, such as Aspergillus or Rhizopus. They can hydrolyze {alpha}1-4 bonds and, to a lesser extent, {alpha}1-6 bonds present on starch, yielding the almost complete conversion of starch to glucose. For this reason, these enzymes are commonly utilized in the preparation of glucose syrups from starch. The episomal pDC-GAM plasmid carries the GAM gene from A. adeninivorans, including its natural leader sequence, under the control of KlPDC1 promoter (27). Strain CBS2359/152F was transformed with pDC-GAM, and glucoamylase accumulation in the medium was followed upon hypoxic induction. Since this protein was unstable at pH 5.0, we introduced a variation in the usual protocol, maintaining the pH value at 6.5 during the whole fermentation process. Although we could detect a satisfactory activity (about 0.10 U/ml), the value of the basal aerated level measured was quite high (about 0.025 U/ml), yielding an induction ratio of four times (Table 3).

Production of recombinant c-33 protein from hepatitis C virus (HCV).
Among the genes of hepatitis C virus, the sequence named c-33, coding for NTPase/helicase activities, could represent a valid target for the development of an immunological diagnostic assay (18). When K. lactis CBS2359/152F cells transformed with the centromeric pC-3312 plasmid were induced by following the standard procedure, we could detect by Western analysis an intracellular accumulation of c-33 HCV protein that resembled the dynamic of induction already observed for the other heterologous products (not shown). Starting from the basal level, the protein concentration increases in a few hours, reaching a value of about 15 mg/liter, with an induction ratio of 3.5 times (Table 3).

KlPDC1 promoter sequence optimization.
All of the data presented above showed an increase in protein production when cells were subjected to oxygen limitation. This consideration led us conclude that the KlPDC1 promoter can be exploited as an effective inducible promoter, characterized by versatility and a very easy and cheap induction procedure. We decided to further investigate the promoter potential in order to increase the level of production.

The well-documented influence of several positive effectors upon KlPDC1 expression (glucose metabolism [2, 17, 24], hypoxia [5], and the presence of several putative consensus sequences for transcription factors [5]) suggests that the promoter activity could be the result of different stimuli which might not necessarily be synergic. In order to get rid of interfering regulations and to identify a promoter sequence specifically responding to hypoxia, we decided to assay a panel of promoter sequences, generated by enzymatic digestions and/or PCR amplification of the KlPDC1 promoter sequence, as hypoxic mediators of lacZ gene expression. This panel, already used to study the regulation by carbon sources and autoregulation (5), has been implemented with plasmids pMD3 and pMD3.2, which contained the proximal region of the promoter with the TATA box (from –1 to –238) and the same region (–1 to –238) plus the fragment –444 to –626, where the Rap1p/Gcr1p boxes and ERA-like sequences were present. Promoter sequences present in the vectors are schematically reported in Fig. 3. Each deletion construct was tested in a bioreactor to analyze its capability to allow hypoxic induction. In order to exclude any interference with glucose regulation, cells were grown in YP medium and induced with the usual procedure. In Fig. 3 are also reported specific ß-galactosidase activities of the aerated basal level, the maximum value reached after induction, and the induction ratio. As is described in the legend to Fig. 1B, the interruption of aeration resulted in a block of growth and in a stable level of specific heterologous activity for at least 12 h (data not shown). Some promoter arrangements resulted in higher activities than were seen with the whole promoter of plasmid pMD12. In particular, plasmid pMD12RR showed higher activity than pMD12 and the highest induction rate (more than 120-fold), thanks to a low basal activity, making it an attractive candidate for process improvement. For this reason, we decided to use the promoter sequence harbored by plasmid pMD12RR as a "second-generation" hypoxia-inducible promoter. Expression of the lcc1 gene under the control of this promoter, obtained in cells transformed with pLC12RR, confirmed the above findings. In fact, after the typical induction procedure already described, we could measure a final average activity of 0.18 ± 0.020 nkat/ml (versus 0.11 ± 0.001 nkat/ml for the wild-type promoter), starting from a basal level under the detection limits of our assay.


Figure 3
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  		FIG. 3. Effects of KlPDC1 promoter rearrangements on hypoxic induction. Specific ß-galactosidase activities (U/OD) obtained with a MW341-5 strain transformed with a plasmid carrying different configurations of the KlPDC1 promoter are reported. Activities were measured in aerobic (basal) or hypoxic (induced) cells. Induction ratios (induced/basal) are also reported. Each value represents an average of at least three different experiments (SD ± 8%). A schematic representation of the promoter portions present in each plasmid is shown in the left part of the figure. Letters indicate the positions of Gcr1 (G) and Rap1 (R) recognition sequences, the ERA-like (E) sequences, and the TATA box (T).

 
Optimization of induction procedure.
We observed that the above-described shift towards hypoxic conditions had a strong impact on cellular growth. In fact, as shown in Fig. 1, the growth rate slowed down after the air inlet was closed, and cell density reached only about an OD660 of 13, which was one-third of the density reached without hypoxic induction (not shown). In order to guarantee hypoxic induction without reducing cell growth, we found that a complete depletion of inlet air for 2 h, followed by the restoration of a minimum air flux (0.05 vvm), allowed the full induction of the system, and at the end of the growth, much higher cell densities were reached (an OD660 of about 30; data not shown). Under these conditions, DOT was below 2%, a value sufficient to pursue a better biomass yield but still low enough to derepress the KlPDC1 promoter. In fact, due to the triplication of the biomass, the trend of specific productivity that we obtained was identical to that described in the legend to Fig. 1A but with a dramatic increase of volumetric productivity. Table 4 shows data obtained with this two-stage induction procedure for the production of some heterologous protein using the wild-type and/or the rearranged promoter sequence from pMD12RR. In all cases, we were able to increase the final protein production levels, as well as the induction ratios. In particular, laccase activity seemed to benefit from this new procedure of induction.


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  		TABLE 4. Heterologous protein production with two-stage induction strategya

 
Transferability of the expression system.
Having assessed the efficacy of the expression system based upon hypoxic induction of KlPDC1 in K. lactis, we tested the possibility of transferring this system to other yeasts. To this purpose, we constructed vectors pAC12 and pAC12RR, harboring the wild-type and modified KlPDC1 promoter sequences (pMD12 and pMD12RR; see Fig. 3), respectively, upstream of the lacZ reporter gene on the S. cerevisiae centromeric plasmid YCplac111. S. cerevisiae and the nonconventional acidophilic yeast Z. bailii (3) were transformed with these plasmids. The transformed clones were grown in a bioreactor until early exponential phase, induced by closing the air inlet, and monitored for 24 h. Figure 4 reports the basal and maximum levels of ß-galactosidase activity (U/OD660) detected in S. cerevisiae and Z. bailii, together with the values obtained with K. lactis under identical conditions. In S. cerevisiae transformants, a significant difference between results under aerated and hypoxic conditions could not be detected, but interestingly, the modified promoter yielded fivefold-higher activity than the wild type, independently of the presence of oxygen. For Z. bailii, on the contrary, and similarly to K. lactis, an evident induction was found, especially with the modified promoter. Since genetic tools for exploiting protein expression in Z. bailii are still scarce and no homologous inducible promoters are available (3), the utilization with this yeast of the KlPDC1 promoter, especially in the rearranged form, could be very useful.


Figure 4
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  		FIG. 4. Hypoxic induction of heterologous ß-galactosidase activity from the KlPDC1 promoter in different yeast species. Basal (black blocks) or induced (white blocks) level of specific (U/OD) ß-galactosidase activity obtained with the whole KlPDC1 promoter (plasmids pMD12 and pAC12) or with the rearranged form (plasmids pMD12RR and pAC12RR) with K. lactis, S. cerevisiae, or Z. bailii. K. lactis values are those reported in Fig. 3. Experiments were run in duplicate, with a standard deviation of ±8%.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
K. lactis displays some advantageous properties with respect to S. cerevisiae for the production of heterologous proteins, such as a notable secretory capacity, good growth performances in a bioreactor, and the ability to use a large number of substrates. As a consequence, many heterologous products have been successfully obtained with this yeast (28, 29). Although many promoters are available for K. lactis, few of them are inducible, and they are not friendly usable in large-scale productions, because of the cost of the inductor or the procedures required to obtain the induction (10). Since the level of dissolved oxygen is a parameter that drops during industrial fermentations, usually efforts are made to keep the DOT value high. Consequently, a system able to trigger heterologous expression at low DOT values should be easy to implement. Moreover, the procedure for establishing hypoxic conditions is very simple and cheap, achieved by closing the air inlet to the bioreactor. A similar system has already been proposed for the production of heterologous proteins with another nonconventional yeast, Pichia stipitis, using the oxygen-regulated ADH2 homologous promoter (19). As with our two-stage induction strategy, in this case the levels of heterologous production benefit from a small amount of air inlet (11). In this paper we demonstrate the possibility of a practical application of hypoxic induction of the KlPDC1 promoter (5) for the production of five different heterologous proteins in K. lactis. After hypoxic induction, an increase of protein production, varying between 4- and 100-fold, was obtained. Our choice to use a unique protocol of induction most probably did not result in the optimization of the process for each heterologous product. In spite of the above considerations, absolute levels of production obtained are generally in agreement with those published for the same proteins expressed in K. lactis or in related yeasts. For example, it is reported that the expression of glucoamylase under the control of the strong glycolytic glyceraldehyde-3-phosphate dehydrogenase promoter yielded 0.053 to 0.143 U/ml (27), while c-33 production obtained with S. cerevisiae yielded, depending on the strain, between 27 and 51 mg/liter (18). Levels of recombinant human interleukin obtained with K. lactis under the control of the inducible PHO5 promoter (100 mg/liter) were lower than ours and were obtained with an elaborate induction procedure, i.e., the transfer of the culture to a low-phosphate medium, or were increased by gene dosage amplification (16).

The energetic shortage due to the shift to fermentative metabolism could impair either the cytoplasmic heterologous protein synthesis or the protein secretion process, a multistep metabolic process that requires ATP. In this respect, a significant improvement of the whole process can be obtained by inflating small amounts of air 2 h after oxygen starvation. Cells exposed to these conditions are probably able to gain larger amounts of energy, which can be utilized for their biosynthetic needs, including the synthesis and the secretion of a heterologous protein. In fact, applying the two-stage strategy, cells reach a final biomass level similar to that of fully aerated cultures, and as a consequence, volumetric production results consistently increased for all the proteins assayed. We also could observe the accumulation in the growing medium of satisfactory amounts of laccase, a protein that is usually poorly expressed in yeasts compared to the expression levels obtained with fungi (9, 12). Moreover, levels of laccase much higher than those reported in the present paper have been obtained with plasmid pLC12 with K. lactis strains different from the one described here (M. M. Bianchi, personal communication), suggesting the possibility of further process improvement. The induction and expression under hypoxia seem not to be affected by gene dosage, as indicated by GAM and IL-1ß induction ratios from multicopy pKD1-based episomal plasmid (see Table 1).

The sequence of the KlPDC1 promoter contains putative consensus sequences for transcriptional regulators, such as Gcr1p and Rap1p, and sequences responsive to ethanol repression and autoregulation (ERA-like sequences [5]). Although a detailed and single-nucleotide-level analysis of the KlPDC1 promoter is beyond the scope of this paper, the presented molecular dissection of the promoter and the hypoxic induction data of ß-galactosidase activity indicate the contribution of different regions of the promoter to the transcriptional oxygen response of KlPDC1. It is evident that a region between the ERA-like sequences and the TATA box, which was not previously characterized for regulation of expression, is essential for hypoxic induction or derepression (pMD4 versus pMD3), while the ERA-like sequences themselves and the Rap1 binding site are not essential (pMD3.2). However, in the distal region of the promoter, contained only in plasmids pMD12, pMD12R, and pMD12RR, a locus required for maximal hypoxic expression might be present. A sequence necessary for complete repression under aerobic conditions might reside in the distal region of pMD10, since constructions having this sequence showed lower aerobic activity values. Careful researching of the KlPDC1 promoter of consensus sequences for known hypoxic factors of other organisms (yeasts and humans) did not reveal the presence of significantly conserved elements (data not shown).

The particular promoter rearrangement of plasmid pMD12RR has proved to be the best combination for our purposes, since it guarantees high values of expression coupled with the highest induction ratio allowed by the lowest basal level. Both of these characteristics are valuable points in the choice of an inducible system. Actually, the highest activity level can be obtained with mutant strains deleted of the KlPDC1 gene. In this background, the absence of repression by autoregulation is added to the hypoxic induction/derepression of transcription. However, because the factors involved in autoregulation appear to be unaffected by aerobic conditions, the basal level is likewise high and makes this host-vector system less versatile and more similar to the constitutive expression systems.

Finally, interesting observations can be made on the different behaviors of our promoter/induction system with S. cerevisiae and Z. bailii. Given that a transcriptional hypoxic response does exist in S. cerevisiae, the absence of hypoxic induction of the KlPDC1 promoter in this yeast could be ascribed either to the species specificity of the regulatory sequences of the promoter or to differences in the transcription factors activated by low-oxygen sensing and signaling in the two yeasts. In this respect, an interesting annotation is that in S. cerevisiae the PDC genes are not induced by hypoxia (14). Although the PDC promoters of Z. bailii have not yet been isolated and sequenced and we do not know if the PDC gene(s) of this yeast is induced by hypoxia, it is clear that hypoxic regulatory sequences of the KlPDC1 promoter are recognized by the final effectors of the hypoxic regulatory cascade of Z. bailii. Isolation and characterization of the PDC gene(s) of Z. bailii and analysis of pyruvate metabolism will contribute to understanding the behavior of this host. Since to date, inducible promoter systems have not been described for Z. bailii, we propose using the KlPDC1 promoter-based expression vectors to achieve heterologous production in this yeast with a simple and cheap procedure.


   ACKNOWLEDGMENTS
 
We thank M. Ruzzi for the kind gift of the lcc1 gene, G. Rebuzzini for his skillful technical assistance, and C. Smeraldi for her accurate review of the manuscript.

This work was partially supported by FAR 2003 and FAR 2006 to D.P.

We thank Instituto Pasteur Fondazione Cenci-Bolognetti, Centro di Eccellenza di Biologia e Medicina Molecolari de La Sapienza.


   FOOTNOTES
 
* Corresponding author. Mailing address: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy. Phone: 390264483451. Fax: 390264483565. E-mail: luca.brambilla{at}unimib.it. Back

{triangledown} Published ahead of print on 1 December 2006. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, February 2007, p. 922-929, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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