The ALEU2 gene--a new component for an Arxula adeninivorans-based expression platform

The ALEU2 gene, encoding beta-isopropylmalate dehydrogenase, was isolated from the non-conventional yeast Arxula adeninivorans. The isolated gene harbours an open reading frame of 1086 bp, encoding a putative protein of 362 amino acids. The derived protein sequence shares a high degree of homology with other fungal beta-isopropylmalate dehydrogenases thus confirming the identity of the gene. The isolated ALEU2 gene was tested for its suitability to complement the auxotrophy of an A. adeninivorans aleu2 host. For this purpose the plasmid pAL-ALEU2m which contains the ALEU2 gene as a selection marker and the 25S rDNA for targeting was employed in transformation experiments. Transformants harboured a single copy of the heterologous DNA and were found to be mitotically stable. For assessment of heterologous gene expression, two model genes were incorporated into the vector: the GFP gene, encoding intracellular green fluorescent protein, and the HSA gene, encoding the secreted human serum albumin. For expression control, both gene sequences were fused to the constitutive A. adeninivorans-derived TEF1 promoter and the Saccharomyces cerevisiae-derived PHO5 terminator. In the respective recombinant strains the GFP was localised in the cytoplasm, whereas more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials using a 200-ml shake flask, maximal HSA product levels were observed after 96 h of cultivation.     

High-level production and secretion of recombinant proteins by the dimorphic yeast Arxula adeninivorans

The non-conventional dimorphic thermo- and salt-resistant yeast Arxula adeninivorans has been developed as a host for heterologous gene expression. For assessment of the system two model genes have been selected: the GFP gene encoding the intracellular green fluorescent protein, and the HSA gene encoding the secreted human serum albumin. The expression system includes two host strains, namely A. adeninivorans LS3, which forms budding cells at 30 degrees C and mycelia at >42 degrees C, and the strain A. adeninivorans 135, which forms mycelia at temperatures as low as 30 degrees C. For expression control the constitutive A. adeninivorans-derived TEF1-promoter and S. cerevisiae-derived PHO5-terminator were selected. The basic A. adeninivorans transformation/expression vector pAL-HPH1 is further equipped with the Escherichia coli-derived hph gene conferring hygromycin B resistance and the 25S rDNA from A. adeninivorans for rDNA targeting. Transformants were obtained for both budding cells and mycelia. In both cell types similar expression levels were achieved and the GFP was localised in the cytoplasm while more than 95% of the HSA accumulated in the culture medium. In initial fermentation trials on a 200-ml shake flask scale maximal HSA product levels were observed after 96 h of cultivation.     

Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts

The high-level production of rhamnolipid biosurfactants is a unique feature of Pseudomonas aeruginosa and is strictly regulated in response to environmental conditions. The final step in rhamnolipid biosynthesis is catalyzed by the rhlAB genes encoding a rhamnosyltransferase. The expression of the cloned rhlAB genes was studied in heterologous hosts, either under the control of the rhlR and rhlI rhamnolipid regulatory elements or under the control of the tac promoter. A recombinant P. fluorescens strain harboring multiple plasmid-encoded copies of the rhamnolipid gene cluster produced rhamnolipids (0.25 g liter(sup-1)) when grown under nitrogen-limiting conditions. The highest yields (0.6 g liter(sup-1)) and productivities (24 mg liter(sup-1) h(sup-1)) were obtained in a recombinant Pseudomonas putida strain, KT2442, harboring promoterless rhlAB genes fused to the tac promoter on a plasmid. Active rhamnosyltransferase was synthesized, but no rhamnolipids were produced, by recombinant Escherichia coli upon induction of rhlAB gene expression.     

The <Emphasis Type="Italic">Arxula adeninivorans</Emphasis><Emphasis Type="Italic">ATAL</Emphasis> gene encoding transaldolase-gene characterization and biotechnological exploitation

The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20°C. The preferred substrates for the enzyme include d-erythrose-4-phosphate and d-fructose-6-phosphate, whereas d-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.     

Secretory expression of the human serum albumin gene in the yeast, Saccharomyces cerevisiae

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.     

Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein

To construct a recombinant strain of Listeria monocytogenes for the expression of heterologous genes, homologous recombination was utilized for insertional mutation, targeting its listeriolysin O gene (hly). The gene encoding green fluorescent protein (GFP) was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream from its promoter and signal sequence by an overlapping extension polymerase chain reaction, and was then cloned into the shuttle plasmid pKSV7 for allelic exchange with the L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at a non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenes hly-gfp lost its hemolytic activity as visualized on the blood agar or when analyzed with the culture supernatant samples. Such insertional mutation resulted in a reduced virulence of about 2 logs less than its parent strain L. monocytogenes 10403s as shown by the 50%-lethal-dose assays in the mouse and embryonated chicken egg models. These results thus demonstrate that mutated L. monocytogeues could be a potential carrier for the expression of heterologous passenger genes or could act as an indicator organism in the food industry.     

Transformation vector based on promoter and intron sequences of a replacement histone H3 gene. A tool for high,constitutive gene expression in plants

This study explored the possibility of using non-viral, plant-based gene sequences to create strong and constitutive expression vectors. Replacement histone H3 genes are highly and constitutively expressed in all plants. Sequences of the cloned alfalfa histone H3.2 gene MsH3g1 were tested. Constructs of the -glucuronidase (GUS) reporter gene were produced with H3.2 gene promoter and intron sequences. Their efficiency was compared with that of the commonly used strong 35S cauliflower mosaic virus promoter in transgenic tobacco plants. Combination of the H3.2 promoter and intron produced significantly higher GUS expression than the strong viral 35S promoter. Histochemical GUS analysis revealed a constitutive pattern of expression. Thus, alfalfa replacement H3 gene sequences can be used instead of viral promoters to drive heterologous gene expression in plants, avoiding perceived risks of viral sequences.     

Strain and process development for the production of human cytokines in Hansenula polymorpha

The early status of strain development for the production of interleukin (IL)-6, IL-8, IL-10, and interferon (IFN) gamma is described. The general approach to generating such strains was to amplify gene sequences encoding the mature forms of the various cytokines by PCR from commercially available cDNA sources. The design of the amplificates allowed an in-frame fusion to an MFalpha1 leader segment contained in two basic expression vectors, pFPMT121-MFalpha1 and pTPSMT-MFalpha1. The two vectors differ in that one harbors the methanol-inducible FMD promoter and the other the constitutive TPS1 promoter as control elements for heterologous gene expression. The most advanced process development example is that of IFNalpha-2a. Here, the MOX promoter derived from another key gene of methanol metabolism is used for expression control. The successful development of a production process for Hansenula polymorpha-derived IFNalpha-2a is summarized. This was achieved by combining genetic engineering of suitable production strains with improved processing capabilities for the secreted cytokine, and by purification procedures from cultures grown in yeast extract-peptone-glycerol-based media.     

Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene

The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP). Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha.     

Production of Man5GlcNAc2-type sugar chain by the methylotrophic yeast Ogataea minuta

The methylotrophic yeast Ogataea minuta IFO 10746 was selected as a suitable strain for producing human-compatible glycoproteins by means of analyses of its cell-wall mannoproteins. First, the OmURA3 gene encoding an orotidine-5'-phosphate decarboxylase was cloned and disrupted to generate a host strain with a uracil auxotrophic marker. Second, both the promoters and the terminators from the OmAOX1 gene encoding an alcohol oxidase for an inducible promoter, or those from the OmTDH1 gene encoding a glyceraldehyde-3-phosphate dehydrogenase for a constitutive promoter, were isolated to construct an expression vector system for heterologous genes. Next, the OmOCH1 gene encoding a starting enzyme with alpha-1,6-mannosyltransferase activity to form a backbone of the N-linked outer sugar chain peculiar to yeast was disrupted, and an alpha-1,2-mannosidase gene from Aspergillus saitoi with an endoplasmic reticulum retention signal (HDEL) under the control of the OmAOX1 promoter was introduced to convert the sugar chain to Man5GlcNAc2 in O. minuta. As a result, we succeeded in breeding a new methylotrophic yeast, O. minuta, producing a Man5GlcNAc2-high-mannose-type sugar chain as a prototype of a human-compatible sugar chain. We also elucidate here the usefulness of the strategy for producing human-compatible sugar chains in yeast.     

Stress-response sigma factor σΗ is essential for morphological differentiation of Streptomyces coelicolor A3(2)

We previously cloned the sigH gene encoding a stress-response sigma factor sigma(H) in Streptomyces coelicolor A3(2), located in an operon with the gene encoding proposed anti-sigma factor UshX. To clarify the in vivo function of sigma(H), a stable null mutant of sigH was prepared by homologous recombination. This mutation appeared to have no obvious effect on vegetative growth, but dramatically affected morphological differentiation. Microscopy showed that the sigH mutant produced undifferentiated hyphae with rare spore chains, giving the colony a pale gray color compared to the dark gray wild-type spores. The sigH mutation partially affected growth under conditions of high osmolarity. Expression of the sigH operon was investigated in the S. coelicolor sigH mutant. Out of four promoters directing expression of the sigH operon, the sigH-P2 promoter--the only promoter preferentially induced by salt-stress conditions--was inactive in the sigH mutant. The results indicated that the sigH-P2 promoter is dependent (directly or indirectly) upon sigma(H) and that the operon is autocatalytically activated. We propose that in S. coelicolor sigma(H) has a dual role, regulating the osmotic response and morphological differentiation.     

Use of the KlADH4 Promoter for Ethanol-Dependent Production of Recombinant Human Serum Albumin in Kluyveromyces lactis

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 β-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 (UAS_E) 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.     

Construction of expression vectors for protein production in Gluconobacter oxydans

The characteristic ability of Gluconobacter oxydans to incompletely oxidize numerous sugars, sugar acids, polyols, and alcohols has been exploited in several biotechnological processes, for example vitamin C production. The genome sequence of G. oxydans 621H is known but molecular tools are needed for the characterization of putative proteins and for the improvement of industrial strains by heterologous and homologous gene expression. To this end, promoter regions for the genes encoding G. oxydans ribosomal proteins L35 and L13 were introduced into the broad-host-range plasmid pBBR1MCS-2 to construct two new expression vectors for gene expression in Gluconobacter spp. These vectors were named pBBR1p264 and pBBR1p452, respectively, and have many advantages over current vectors for Gluconobacter spp. The uidA gene encoding β-D-glucuronidase was inserted downstream of the promoter regions and these promoter-reporter fusions were used to assess relative promoter strength. The constructs displayed distinct promoter strengths and strong (pBBR1p264), moderate (pBBR1p452) and weak (pBBR1MCS-2 carrying the intrinsic lac promoter) promoters were identified.