Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production in Ashbya gossypii

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg⁻¹ was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.     

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.     

Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii

Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1α. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UAS_rpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UAS_rpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UAS_rpg homologous sequences.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii,maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Sequence and promoter analysis of the highly expressed TEF gene of the filamentous fungus Ashbya gossypii

Ashbya gossypii carries only a single gene (TEF) coding for the abundant translation elongation factor 1. Cloning and sequencing of this gene and deletion analysis of the promoter region revealed an extremely high degree of similarity with the well studied TEF genes of the yeast Saccharomyces cerevisiae including promoter upstream activation sequence (UAS) elements. The open reading frames in both species are 458 codons long and show 88.6% identity at the DNA level and 93.7% identity at the protein level. A short DNA segment in the promoter, between nucleotides -268 and -213 upstream of the ATG start codon, is essential for high-level expression of the A. gossypii TEF gene. It carries two sequences, GCCCATACAT and ATCCATACAT, with high homology to the UAS_rpg sequence of S. cerevisiae, which is an essential promoter element in genes coding for highly expressed components of the translational apparatus. UAS_rpg sequences are binding sites for the S. cerevisiae protein TUF, also called RAP1 or GRF1. In gel retardation with A. gossypii protein extracts we demonstrated specific protein binding to the short TEF promoter segment carrying the UAS_rpg homologous sequences.     

Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence

Summary This study presents the first evidence that the 5 promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus — thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5 flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.     

The Ashbya gossypii EF-1α promoter of the ubiquitously used MX cassettes is toxic to Saccharomyces cerevisiae

Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF-1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone.     

Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis

Background

Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins.

Results

After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG’s annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii’s metabolism in comparison to the one of S. cerevisiae (post-WGD – whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism.

Conclusions

This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii’s metabolism.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-810) contains supplementary material, which is available to authorized users.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Expression of alanine:glyoxylate aminotransferase gene from Saccharomyces cerevisiae in Ashbya gossypii

Two plasmids containing an autonomously replicating sequence from Saccharomyces cerevisiae were constructed. Using these vectors, the AGX1 gene encoding alanine:glyoxylate aminotransferase (AGT) from S. cerevisiae, which converts glyoxylate into glycine but is not present in Ashbya gossypii, was expressed in A. gossypii. Geneticin-resistant transformants with the plasmid having the kanamycin resistance gene under the control of the translation elongation factor 1 α (TEF) promoter and terminator from A. gossypii were obtained with a transformation efficiency of approximately 10–20 transformants per microgram of plasmid DNA. The specific AGT activities of A. gossypii pYPKTPAT carrying the AGX1 gene in glucose- and rapeseed-oil-containing media were 40 and 160 mU mg⁻¹ of wet mycelial weight, respectively. The riboflavin concentrations of A. gossypii pYPKTPAT carrying AGX1 gene in glucose- and rapeseed-oil-containing media were 20 and 150 mg l⁻¹, respectively. In the presence of 50 mM glyoxylate, the riboflavin concentration and the specific riboflavin concentration of A. gossypii pYPKTPAT were 2- and 1.3-fold those of A. gossypii pYPKT without the AGX1 gene.     

Expression of bacterial xylose isomerase in Saccharomyces cerevisiae under galactose supplemented condition

We constructed recombinant Saccharomyces cerevisiae harboring the xylose isomerase (XI) gene isolated from Clostridium phytofermentans to metabolize xylose and use it as a carbon and energy source. In this study, the effect of supplementation using co-substrate such as glucose or galactose on xylose utilization was studied in recombinant S. cerevisiae. Glucose, which is transported with high affinity by the same transport system as is xylose, was not affected by the heterologous expression of XI, thus xylose utilization was not observed in recombinant S. cerevisiae. However, supplemental galactose added to the recombinant S. cerevisiae stimulated xylose utilization as well as the expression of XI protein. Recombinant S. cerevisiae consumed up to 23.48 g/L of xylose when grown in media containing 40 g/L of xylose and supplemented with 20 g/L of galactose. These cells also produced 15.89 g/L of ethanol. Therefore, expression of the bacterial XI in recombinant S. cerevisiae was highly induced by the addition of supplemental galactose as a co-substrate with xylose, and supplemented galactose enabled the yeast strain to grow on xylose and ferment xylose to ethanol.     

Construction of xylose-assimilating Saccharomyces cerevisiae

The xylose reductase gene originating from Pichia stipitis was subcloned on an expression vector with the enolase promoter and terminator from Saccharomyces cerevisiae. The transformants of S. cerevisiae harboring the resultant plasmids produced xylose reductase constitutively at a rate about 3 times higher than P. stipitis, but could not assimilate xylose due to the deficient conversion of xylitol to xylulose. The xylitol dehydrogenase gene was also isolated from the gene library of P. stipitis by plaque hybridization using a probe specific for its N-terminal amino acid sequence. The gene transferred into S. cerevisiae was well expressed. Furthermore, high expressions of the xylose reductase and xylitol dehydrogenase genes in S. cerevisiae were achieved by introducing both genes on the same or coexisting plasmids. The transformants could grow on a medium containing xylose as the sole carbon source, but ethanol production from xylose was less than that by P. stipitis and a significant amount of xylitol was excreted into the culture broth.     

Display of heterologous proteins on the Saccharomyces cerevisiae surface display system using a single constitutive expression vector

In this study, we constructed a novel and simple yeast surface display system with a single expression vector. The newly established system uses a bidirectional expression vector carrying the AGA1 gene driven by the PGK1 promoter in one direction and the AGA2‐expression cassette driven by the TEF1 promoter in the reverse direction, and uses the geneticin, a G418‐resistant gene, as the selection marker for transformants. Because all the display elements are put into one expression vector, the new system is much simpler to use, and there is no need for any genetic modification of the host strains; therefore, the new system can be used in wild type as well as laboratory strains of Saccharomyces cerevisiae. The display efficiency of heterologous proteins using the new system has been confirmed by displaying enhanced green fluorescent protein and Eimeria tenella (a chicken protozoan parasite) microneme protein2 (EtMic2) on several S. cerevisiae strains. We also tested the new system with an aga2 mutant strain of S. cerevisiae. The results indicate that the native expressed Aga2 protein has no effect on the display efficiency of heterologous proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:443–450, 2014     

Use of the glyceraldehyde-3-phosphate dehydrogenase promoter from a thermotolerant yeast,Pichia thermomethanolica,for heterologous gene expression,especially at elevated temperature

The glyceraldehyde-3-phosphate dehydrogenase (GAP) gene from the thermotolerant yeast strain Pichia thermomethanolica BCC16875 was characterized. To investigate the efficiency of the GAP promoter for heterologous expression, especially at high temperature in various carbon sources, the promoter was employed for constitutive expression of a phytase reporter gene. The results showed that this promoter was able to drive efficient expression of phytase at 30 °C; the native promoter was highly robust compared with the heterologous GAP promoter from Pichia pastoris. More importantly, the GAP promoter was shown to be able to function at higher temperatures up to 42 °C, which could be useful for large-scale protein production to help reduce cooling costs in the fermenter. Expression in different carbon sources revealed that the GAP promoter was functional in glucose-, glycerol-, and methanol-containing media, with the highest level of expression in YPD medium. This strong promoter will help promote high expression of heterologous protein expression in P. thermomethanolica, especially in large-scale fermentation. In addition, a new tool for heterologous expression in yeast has been gained.     

Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter

The methylotrophic yeast, Candida boidinii, was investigated as a new efficient host for heterologous gene expression. The Saccharomyces cerevisiae adenylate kinase gene (ADK1) was used as the first example for heterologous enzyme production in C. boidinii. C. boidinii cells were transformed with plasmids harboring the S. cerevisiae ADK1 gene under the alcohol oxidase (C. boidinii AOD1) promoter. The chromosome-integrant strains produced adenylate kinase protein corresponding to 22%–28% of the total soluble proteins in an enzymatically active form. When the three-copy integrative transformant was grown for 60 h on methanol-glycerol medium in a 1.5-l jar fermentor, adenylate kinase was produced intracellularly with a yield of up to 2 g/l culture medium. As the expression of the S. cerevisiae ADK1 in C. boidinii was under similar regulation to that of the C. boidinii AOD1, the previously cloned 1.7-kb AOD1 promoter fragment was proved to harbor sufficient cis elements for AOD1 regulation and found to be an efficient promoter for heterologous gene expression.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.