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.     

The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform

An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved. This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.     

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.     

Construction of an Arxula adeninivorans host-vector system based on trp1 complementation

A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium.     

Assessment of Hansenula polymorpha and Arxula adeninivorans-derived rDNA-targeting elements for the design of Arxula adeninivorans expression vectors

Different targeting sequences derived from the Arxula adeninivorans and Hansenula polymorpha rDNA clusters were tested in A. adeninivorans integration/expression vectors. For element identification, the rDNA unit of A. adeninivorans (accession number ) was first isolated and characterized in addition to the known H. polymorpha unit. The rDNA is a cluster of some forty 7653-bp units without the 5S rDNA gene. The selected elements were integrated into a set of A. adeninivorans expression/integration vectors harbouring a TEF1 promoter - amyA ORF - PHO5 terminator sequence as reporter gene. No differences in mitotic stability, copy number and transformation frequency were observed. All transformants harboured a single copy integrated into the rDNA by a homologous recombination. In contrast, the choice of the rDNA targeting sequence was found to be of impact on productivity. Use of ETS-18S-5.8S fragments from both organisms resulted in a more than 50% increase in comparison to the use of other elements, independent of the orientation within the vector.     

Characterization of the AINV gene and the encoded invertase from the dimorphic yeast Arxula adeninivorans

The invertase-encoding of AINV gene Arxula adeninivorans was isolated and characterized. The gene includes a coding sequence of 2700 bp encoding a putative 899 amino acid protein of 101.7 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of alpha-glucosidases from different sources. The gene activity is regulated by carbon source. In media supplemented with sucrose induction of the AINV gene and accumulation of the encoded invertase in the medium was observed. In addition the extracellular enzyme level is influenced by the morphological status of the organism, with mycelia secreting the enzyme in titres higher than those observed in budding yeasts. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AINV gene under control of the strong A. adeninivorans -derived TEF1 promoter. For both proteins a molecular mass of 600 kDa was determined, a pH optimum at pH 4.5 and a temperature optimum at 55 degrees C. The preferred substrates for the enzyme included the ss-D-fructofuranosides sucrose, inulin and raffinose. Only a weak enzyme activity was observed for the alpha-D-glucopyranosides maltotriose, maltose and isomaltose. Thus the invertase primarily is a ss-fructosidase and not an alpha-glucosidase as suggested by the homology to such enzymes.     

Time-lapsed confocal microscopy reveals temporal and spatial expression of the lysine epsilon-aminotransferase gene in Streptomyces clavuligerus

To investigate the temporal and spatial expression patterns of the gene (lat ) encoding lysine epsilon-aminotransferase (LAT) for cephamycin C biosynthesis, a mutant form of green fluorescent protein (mut1GFP) was integrated into the Streptomyces clavuligerus chromosome (strain LH369), resulting in a translational fusion with lat. LAT activity and fluorescence profiles of the recombinant protein paralleled the native LAT enzyme activity profile in wild-type S. clavuligerus, which peaked during exponential growth phase and decreased slowly towards stationary phase. These results indicate that the LAT-Mut1GFP fusion protein retains both LAT and GFP functionality in S. clavuligerus LH369. LH369 produced wild-type levels of cephamycin C in minimal medium culture conditions supplemented with lysine. Time-lapsed confocal microscopy of the S. clavuligerus LH369 strain revealed the temporal and spatial characteristics of lat gene expression and demonstrated that physiological development of S. clavuligerus colonies leading to cephamycin C biosynthesis is limited to the substrate mycelia.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

SCS1, a multicopy suppressor of hsp60-ts mutant alleles, does not encode a mitochondrially targeted protein.

We identified and isolated a Saccharomyces cerevisiae gene which, when overexpressed, suppressed the temperature-sensitive phenotype of cells expressing a mutant allele of the gene encoding the mitochondrial chaperonin, Hsp60. This gene, SCS1 (suppressor of chaperonin sixty-1), encodes a 757-amino-acid protein of as yet unknown function which, nonetheless, has human, rice, and Caenorhabditis elegans homologs with high degrees (ca. 60%) of amino acid sequence identity. SCS1 is not an essential gene, but SCS1-null strains do not grow above 37 degrees C and show some growth-related defects at 30 degrees C as well. This gene is expressed at both 30 and 38 degrees C, producing little or no differences in mRNA levels at these two temperatures. Overexpression of SCS1 could not complement an HSP60-null allele, indicating that suppression was not due to the bypassing of Hsp60 activity. Of 10 other hsp60-ts alleles tested, five could also be suppressed by SCS1 overexpression. There were no common mutant phenotypes of the strains expressing these alleles that give any clue as to why they were suppressible while others were not. An epitope (influenza virus hemagglutinin)-tagged form of SCS1 in single copy complemented an SCS1-null allele. The Scs1-hemagglutinin protein was found to be at comparable levels and in similar multiply modified forms in cells growing at both 30 and 38 degrees C. Surprisingly, when localized either by cell fractionation procedures or by immunocytochemistry, these proteins were found not in mitochondria but in the cytosol. The overexpression of SCS1 had significant effects on the cellular levels of mRNAs encoding the proteins Cpn10 and Mgel, two other mitochondrial protein cochaperones, but not on mRNAs encoding a number of other mitochondrial or cytosolic proteins analyzed. The implications of these findings are discussed.     

Simultaneous analysis of spatio-temporal gene expression for cephamycin biosynthesis in Streptomyces clavuligerus.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.     

Enhanced fluorescent properties of an OmpT site deleted mutant of Green Fluorescent Protein

Background  

The green fluorescent protein has revolutionized many areas of cell biology and biotechnology since it is widely used in determining gene expression and for localization of protein expression. Expression of recombinant GFP in E. coli K12 host from pBAD24M-GFP construct upon arabinose induction was significantly lower than that seen in E. coli B cells with higher expression at 30°C as compared to 37°C in E. coli K12 hosts. Since OmpT levels are higher at 37°C than at 30°C, it prompted us to modify the OmpT proteolytic sites of GFP and examine such an effect on GFP expression and fluorescence. Upon modification of one of the two putative OmpT cleavage sites of GFP, we observed several folds enhanced fluorescence of GFP as compared to unmodified GFPuv (Wild Type-WT). The western blot studies of the WT and the SDM II GFP mutant using anti-GFP antibody showed prominent degradation of GFP with negligible degradation in case of SDM II GFP mutant while no such degradation of GFP was seen for both the clones when expressed in BL21 cells. The SDM II GFP mutant also showed enhanced GFP fluorescence in other E. coli K12 OmpT hosts like E. coli JM109 and LE 392 in comparison to WT GFPuv. Inclusion of an OmpT inhibitor, like zinc with WT GFP lysate expressed from an E. coli K12 host was found to reduce degradation of GFP fluorescence by two fold.     

Engineering of a Sagiyama alphavirus RNA-based transient expression vector

Sagiyama virus (SAGV), a strain of Getah virus in the genus Alphavirus in the family Togaviridae, has a broad host range in vertebrates and invertebrates but is not pathogenic for humans. We engineered the SAGV genome as an efficient transient expression vector using the full-length infectious cDNA clone pSAG2 as the background. A green fluorescent protein (GFP) gene was used as a reporter gene and expressed from a subgenomic mRNA. When the GFP gene was placed downstream of the intact capsid protein gene or an internally deleted capsid protein gene encoding the N-terminal 9 amino acids and C-terminal 149 amino acids, autoproteolysis occurred efficiently at the boundary site to release GFP from the N-terminally-fused capsid-protease domain. GFP was also expressed efficiently without the 5'-terminal region of the capsid protein gene, suggesting that SAGV capsid protein gene does not have a translation enhancer sequence. To provide structural proteins for pseudovirion formation, a nonviable mutant construct, pSAG2.3L, which contains a Gly-to-Leu substitution at the - 2 position of the nsP3/4 cleavage site, was used as a helper. GFP was expressed up to 50 pg from 1 X 10(6) BHK21 cells after inoculation of pseudovirions. The C6/36 mosquito cell was also a suitable host for a large scale expression of GFP using pseudovirions. In addition to high-level transient expression, safeness of SAGV should give an advantage over other alphavirus expression vectors.     

Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506

The green fluorescent protein (GFP) gene, gfp, of the jellyfish Aequorea victoria is being used as a reporter system for gene expression and as a marker for tracking prokaryotes and eukaryotes. Cells that have been genetically altered with the gfp gene produce a protein that fluoresces when it is excited by UV light. This unique phenotype allows gfp-tagged cells to be specifically monitored by nondestructive means. In this study we determined whether a gfp-tagged strain of Pseudomonas fluorescens continued to fluoresce under conditions under which the cells were starved, viable but nonculturable (VBNC), or dead. Epifluorescent microscopy, flow cytometry, and spectrofluorometry were used to measure fluorescence intensity in starved, VBNC, and dead or dying cells. Results obtained by using flow cytometry indicated that microcosms containing VBNC cells, which were obtained by incubation under stress conditions (starvation at 37.5 degrees C), fluoresced at an intensity that was at least 80% of the intensity of nonstressed cultures. Similarly, microcosms containing starved cells incubated at 5 and 30 degrees C had fluorescence intensities that were 90 to 110% of the intensity of nonstressed cells. VBNC cells remained fluorescent during the entire 6-month incubation period. In addition, cells starved at 5 or 30 degrees C remained fluorescent for at least 11 months. Treatment of the cells with UV light or incubation at 39 or 50 degrees C resulted in a loss of GFP from the cells. There was a strong correlation between cell death and leakage of GFP from the cells, although the extent of leakage varied depending on the treatment. Most dead cells were not GFP fluorescent, but a small proportion of the dead cells retained some GFP at a lower concentration than the concentration in live cells. Our results suggest that gfp-tagged cells remain fluorescent following starvation and entry into the VBNC state but that fluorescence is lost when the cells die, presumably because membrane integrity is lost.     

Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa).

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.     

Vesicular monoamine transporters heterologously expressed in the yeast Saccharomyces cerevisiae display high-affinity tetrabenazine binding

A mammalian vesicular neurotransmitter transporter has been expressed in the yeast Saccharomyces cerevisiae. The gene encoding the rat vesicular monoamine transporter (rVMAT(1)) was cloned in several expression plasmids. The transporter was expressed at detectable levels only when short sequences using codons favored by S. cerevisiae were fused preceding the start of translation of rVMAT(1). The scarce expression of the wild-type protein was, most likely, due to the fact that part of the N-terminus of the protein is encoded by codons not preferred in S. cerevisiae. Furthermore, low growth temperatures increased rVMAT(1) expression and altered its processing. Whereas at 30 degrees C the protein is not glycosylated, at lower temperatures ( approximately 16 degrees C) half of the expressed transporters undergo core glycosylation. In addition, under these conditions the levels of protein expression significantly increase. Using a functional chimeric protein composed by VMAT and the green fluorescent protein (GFP), it is shown that the punctate pattern of intracellular distribution remains invariable at the different temperatures. Using a similar fusion sequence, the bovine VMAT isoform 2 (bVMAT(2)) was also expressed in yeast. The yeast-expressed bVMAT(2) binds [(3)H]dihydrotetrabenazine ([(3)H]TBZOH) with the same characteristics found in the native protein from bovine chromaffin granules. Dodecyl maltoside-solubilized bVMAT(2) retains the conformation required for [(3)H]TBZOH binding. We exploited the robust binding to follow the transporter during purification assays on a Ni(2+)-chelating column. In this report we describe for the first time the heterologous expression of a neurotransmitter transporter in the yeast S. cerevisiae.