Auxotrophic Markers Research Articles

Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens.

Golden Gate-based metabolic engineering strategy for wild-type strains of Yarrowia lipolytica.

The yeast Yarrowia lipolytica represents a future microbial cell factory for numerous applications in a bio-based economy. Outstanding feature of this yeast is the metabolic flexibility in utilising various substrates (sugars, fatty acids, glycerol, etc.). The potential of wild-type isolates of Y. lipolytica to convert glycerol into various value-added compounds is attracting attention of academia and industry. However, the already established tools for efficient engineering of the metabolism of Y. lipolytica are often dependent on genetic features like auxotrophic markers. With the present work we want to introduce a new set of vectors for metabolic engineering strategies, including CRISPR/Cas9 technology. The system is based on GoldenMOCS, a recently established rapid Golden Gate cloning strategy applicable in multiple organisms. We could show that our new GoldenMOCS plasmids are suitable for the extrachromosomal overexpression of the gene glycerol kinase (GUT1) in wild-type isolates of Y. lipolytica resulting in enhanced conversion of glycerol to erythritol and citric acid. Moreover, a GoldenMOCS plasmid for CRISPR/Cas9 mediated genome editing has been designed, which facilitates single gene knock-outs with efficiencies between 6% and 25% in strains with genetic wild-type background.

Combining 26s rDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica.

Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multicopy metabolic pathways in Y. lipolytica. We demonstrated the efficient genome integration of a plant-derived flavonoid pathway at random sites with multiple copies. Transient expression of Cre recombinase enabled efficient marker removal and allowed for the next round of genome integration. Investigating the recombination events demonstrated that the iterative integration is happening at sufficiently high rates (more than 80%) without disrupting the previous integration. Both the flavonoid precursor pathway and the plant-derived cytochrome c P450 enzymes were functionally integrated to improve flavonoid and hydroxylated flavonoid production. The engineered strains produced 71.2 mg/L naringenin, 54.2 mg/L eriodyctiol, and 48.1 mg/L taxifolin. The reported work provides a versatile platform to iteratively integrate functional gene clusters at high copy numbers. This work may streamline and expand our capability to build efficient microbial cell factories for high-value natural products and commodity chemical production in Y. lipolytica.

Methods for studying the regulation of membrane traffic by ubiquitin and the ESCRT pathway.

Covalent modification of proteins with ubiquitin dynamically regulates their function and fate. The ubiquitination of most plasma membrane proteins initiates endocytosis and ESCRT-mediated sorting to the lysosomal lumen for degradation. Powerful genetic approaches in the budding yeast Saccharomyces cerevisiae have been particularly instrumental in the discovery and elucidation of these molecular mechanisms, which are conserved in all eukaryotes. Here we provide two detailed protocols and tools for studying ubiquitination-dependent membrane trafficking mechanisms in yeast. The first utilizes fusions between a protein of interest and an auxotrophic marker to screen for mutants that affect ubiquitin-mediated endocytosis. The second method artificially ubiquitinates a protein of interest, allowing downstream trafficking steps to be studied independently from the regulatory signals that initiate endocytosis.

Challenging SNP impact on caspofungin resistance by full-length FKS1 allele replacement in Candida lusitaniae.

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically engineered for full-length replacement of the FKS1 gene encoding the target of echinocandin antifungals in order to assess the impact of FKS mutations on echinocandin resistance and reduced echinocandin susceptibility (RES). FKS1 allelic exchange was achieved by transforming C. lusitaniae with two DNA fragments covering the entire FKS1 ORF. Both fragments overlap a 40 bp region where SNPs or small indels of interest were inserted. To target integration at the FKS1 locus, each DNA fragment was fused with split auxotrophic markers of which complementary truncated parts were previously inserted into the chromosomal regions flanking FKS1, allowing selection on minimal medium. Three SNPs described in the FKS1 hotspot (HS) regions HS1 or HS2 of clinical isolates of Candida albicans were expressed at an equivalent position in C. lusitaniae and were confirmed to confer either reduced susceptibility (F641V) or full resistance (S645P and R1361G) to caspofungin. The F659 deletion reported in an FKS2 allele of Candida glabrata and the naturally occurring P660A substitution in FKS1 of Candida parapsilosis were shown to confer a 256-fold and 6-fold increase in caspofungin MIC, respectively, when introduced into an FKS1 allele of C. lusitaniae. We have successfully developed a C. lusitaniae strain for the expression of full-length FKS1 alleles harbouring known mutations contributing to reduced susceptibility or resistance to caspofungin, thus opening the way for the screening of other FKS1/FKS2 mutations potentially involved in RES.

Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES

SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. Here we describe a reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. Furthermore, by analyzing the tolerant strains, we are able to identify ACE2, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, this work not only establishes a generic platform to rapidly identify strains of interest by SCRaMbLE, but also provides methods to dissect the underlying mechanisms of resistance.

Engineered CRISPR/Cas9 system for multiplex genome engineering of polyploid industrial yeast strains

The CRISPR/Cas9 system has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. However, its application in manipulating industrial yeast strains is less successful, probably due to the genome complexity and low copy numbers of gRNA expression plasmids. Here we developed an efficient CRISPR/Cas9 system for industrial yeast strain engineering by using our previously engineered plasmids with increased copy numbers. Four genes in both a diploid strain (Ethanol Red, 8 alleles in total) and a triploid strain (ATCC 4124, 12 alleles in total) were knocked out in a single step with 100% efficiency. This system was used to construct xylose-fermenting, lactate-producing industrial yeast strains, in which ALD6, PHO13, LEU2, and URA3 were disrupted in a single step followed by the introduction of a xylose utilization pathway and a lactate biosynthetic pathway on auxotrophic marker plasmids. The optimized CRISPR/Cas9 system provides a powerful tool for the development of industrial yeast based microbial cell factories.

Construction of Designer Selectable Marker Deletions with a CRISPR-Cas9 Toolbox in Schizosaccharomyces pombe and New Design of Common Entry Vectors.

Vectors encoding selectable markers have been widely used in yeast to maintain or express exogenous DNA fragments. In the fission yeast Schizosaccharomyces pombe, several engineered markers have been reported and widely used, such as ura4+ and ScLEU2 from Saccharomyces cerevisiae, which complement ura4 and leu1 mutations, respectively. These two auxotrophic markers share no homology with the S. pombe genome; however, most others can recombine with the genome due to sequence homology shared between the genomic and plasmid-borne copies of the markers. Here, we describe a CRISPR-Cas9 toolbox that can be used to quickly introduce “designer” auxotrophic marker deletions into host strains, including leu1-Δ0, his3-Δ0, and lys9-Δ0. Together with ura4-D18, this brings the total number of available designer deletion auxotrophic markers to four. The toolbox consists of a Cas9-gRNA expression vector and a donor DNA plasmid pair for each designer deletion. Using this toolbox, a set of auxotrophic S. pombe strains was constructed. Further, we reorganized essential components in the commonly used pREP series of plasmids and assembled the corresponding auxotrophic marker gene onto these plasmids. This toolbox for producing designer deletions, together with the newly developed strains and plasmids, will benefit the whole yeast community.

Application of the Cre/lox System to Construct Auxotrophic Markers for Quantitative Genetic Analyses in Fusarium graminearum.

The bacteriophage P1 Cre/lox system has been utilized in diverse fungi for marker recycling and exchange, generation of targeted chromosome translocations, and targeted deletion of interstitial chromosome segments. Here we show the application of this tool in the wheat and maize pathogen, Fusarium graminearum. We explored three different ways to introduce Cre into strains with floxed genes, namely transformation with an episomal or integrative plasmid (pLC28), fusion of protoplasts of strains carrying floxed genes with strains expressing Cre by forcing heterokaryons, and crosses between strains with floxed genes and strains expressing Cre to isolate progeny in which the target genes had been deleted during the cross. We used this system for the construction of strains bearing auxotrophic markers that were generated by gene replacement with positively selectable markers followed by Cre-mediated marker excision. In addition, updated protocols for transformation and crosses for F. graminearum are provided. In combination, strains and tools developed here add to the arsenal of methods that can be used to carry out molecular genetics with F. graminearum.

Construction of prototrophic glutathione-high-producing yeast strain mediated by CRISPR/Cas9

Glutathione (GSH), a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9-associated-system (CRISPR-Cas), in which gRNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1b/FGP. As a result, the prototrophic strain W303-1b/FGPPT showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1b/FGPPT that might have great interests in industrial fermentation.

A CTG Clade Candida Yeast Genetically Engineered for the Genotype-Phenotype Characterization of Azole Antifungal Resistance in Human-Pathogenic Yeasts.

A strain of the opportunistic pathogenic yeast Candida lusitaniae was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase ERG11 mutations on azole resistance. Candida lusitaniae was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the Candida species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the ERG11 locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of ERG11 Homologous and heterologous ERG11 alleles are expressed from the resident ERG11 promoter of C. lusitaniae, allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in C. lusitaniae different ERG11 alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, C. albicans and Kluyveromyces marxianusCandida lusitaniae constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the ERG11 donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in Candida species, and the equivalent position K151 in K. marxianus, plays a critical role in fluconazole resistance.

Testing of Auxotrophic Selection Markers for Use in the Moss Physcomitrella Provides New Insights into the Mechanisms of Targeted Recombination.

The moss Physcomitrella patens is unique among plants in that homologous recombination can be used to knock out genes, just like in yeast. Furthermore, transformed plasmids can be rescued from Physcomitrella back into Escherichia coli, similar to yeast. In the present study, we have tested if a third important tool from yeast molecular genetics, auxotrophic selection markers, can be used in Physcomitrella. Two auxotrophic moss strains were made by knocking out the PpHIS3 gene encoding imidazoleglycerol-phosphate dehydratase, and the PpTRP1 gene encoding phosphoribosylanthranilate isomerase, disrupting the biosynthesis of histidine and tryptophan, respectively. The resulting PpHIS3Δ and PpTRP1Δ knockout strains were unable to grow on medium lacking histidine or tryptophan. The PpHIS3Δ strain was used to test selection of transformants by complementation of an auxotrophic marker. We found that the PpHIS3Δ strain could be complemented by transformation with a plasmid expressing the PpHIS3 gene from the CaMV 35S promoter, allowing the strain to grow on medium lacking histidine. Both linearized plasmids and circular supercoiled plasmids could complement the auxotrophic marker, and plasmids from both types of transformants could be rescued back into E. coli. Plasmids rescued from circular transformants were identical to the original plasmid, whereas plasmids rescued from linearized transformants had deletions generated by recombination between micro-homologies in the plasmids. Our results show that cloning by complementation of an auxotrophic marker works in Physcomitrella, which opens the door for using auxotrophic selection markers in moss molecular genetics. This will facilitate the adaptation of shuttle plasmid dependent methods from yeast molecular genetics for use in Physcomitrella.

Construction of cell factories for high production of nerolidol in Saccharomyces cerevisiae

Nerolidol is an important constituent of terpenoid essential oil and has excellent anti-tumor, anti-bacterial, and anti-oxidative properties. For realizing heterogenous production of nerolidol, our research firstly integrated the codon-optimized Actinidia sinensis nerolidol synthase gene (NES) into the terpenoid chassis strain FPP-001, and obtained NES-001 that could produce 2.71 mg•L⁻¹ nerolidol. Then, the N-terminal of the NES was fused with FPS by linker peptide GGGS. With this strategy, nerolidol production improved by 59.80-fold, reaching 162.07 mg•L⁻¹. Finally, by introduction of auxotrophic marker TRP1 in NES-002, the resulting strain NES-003 could produce 1 711.53 mg•L⁻¹ by high cell density fermentation method. This study provides the basis for the fermentative production of nerolidol and other sesquiterpenoids.

Selecting Schizosaccharomyces pombe Diploids.

Here we describe procedures for the selection of diploid Schizosaccharomyces pombeade6-M210/ade6-M216 heteroallelic complementation is widely used to select for Ade+ diploids. Such diploids will readily sporulate when starved of nitrogen. For some investigations, stable diploids are preferable (e.g., for genetic complementation tests), and in these cases mating an h- strain with an h90 mat2-Pi-102 strain can be used to prevent sporulation. When ade6-M210/ade6-M216 mutations impact on, or show synthetic interactions with, the gene of interest, two different auxotrophic markers can be used to select complementation.

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

BackgroundA commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.ResultsA K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.ConclusionsIn this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.

Heterologous Expression of theCarrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.), Hsp17.7, was inserted into the yeast genome via homologous recombination to increase tolerance to stress conditions that can occur during industrial culture. A DNA construct, Translational elongation factor gene promoter-carrot Hsp17.7 gene-Phosphoribosyl-anthranilate isomerase gene (an auxotrophic marker), was generated by a series of PCRs and introduced into the chromosome IV of the yeast genome. Immunoblot analysis showed that carrot Hsp17.7 accumulated in the transformed yeast cell lines. Growth rates and cell viability of these cell lines were higher than control cell lines under heat, cold, acid, and hyperosmotic stress conditions. Soluble protein levels were higher in the transgenic cell lines than control cell lines under heat and cold conditions, suggesting the molecular chaperone function of the recombinant Hsp17.7. This study showed that a recombinant DNA construct containing a HSP gene from carrot was successfully expressed in yeast by homologous recombination and increased tolerances to abiotic stress conditions.

EYK1 encoding erythrulose kinase as a catabolic selectable marker for genome editing in the non-conventional yeast Yarrowia lipolytica

We report here on EYK1, encoding erythrulose kinase, as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers, EYK1 increases the growth rate of transformants and allows improved efficiency of transformation. The utility of the marker EYK1 in a replicative vector was also demonstrated.

HisB as novel selection marker for gene targeting approaches in Aspergillus niger

BackgroundFor Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger.ResultsA histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB-, pyrG- strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus.ConclusionTaken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.

Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox

A robust and versatile tool for multigene pathway assembly is a key to the biosynthesis of high-value chemicals. Here we report the rapid construction of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative toolbox (pUMRI) developed in our research group, which has features of ready-to-use, convenient marker recycling, arbitrary element replacement, shuttle plasmid, auxotrophic marker independence, GAL regulation, and decentralized assembly. Functional isoprenoid biosynthesis pathways containing 4–11 genes with lengths ranging from ~10 to ~22 kb were assembled using this toolbox within 1–5 rounds of reiterative recombination. In combination with GAL-regulated metabolic engineering, high production of isoprenoids (e.g., 16.3 mg∙g–1 dcw carotenoids) was achieved. These results demonstrate the wide range of application and the efficiency of the pUMRI toolbox in multigene pathway construction of S. cerevisiae. Open image in new window

Performing Yeast One-Hybrid Library Screens.

Yeast one-hybrid (Y1H) assays are used to identify which transcription factor (TF) "prey" molecules can bind a DNA fragment of interest that is used as "bait". Y1H assays involve introducing plasmids that encode TFs into a yeast "bait strain" in which the DNA fragment of interest is integrated upstream of one or more reporters, and activation of these reporters indicates that a TF-DNA interaction has occurred. These plasmids express each TF as a hybrid protein (hence the "one-hybrid" name) fused to the activation domain (AD) of the yeast TF Gal4. The AD moiety activates reporter expression even if the TF to which it is fused typically functions as a repressor. Here, we describe how to perform a Y1H screen of a library of cDNA fragments cloned into a pPC86 plasmid expressing the protein encoded by the cDNA as an AD fusion. The method assumes availability of either commercially available libraries or libraries generated in house using mRNA extracted from a tissue of interest. We also assume that users have access to a yeast bait strain that possesses the DNA fragment of interest integrated upstream of two different reporters-HIS3, an auxotrophic marker, and LacZ, a colorimetric marker that changes colorless X-gal into a blue compound. Briefly, the screen involves transforming the AD-cDNA library into the yeast bait strain, identifying colonies that show activation of both reporters, retesting the interaction in a freshly grown bait strain, and sequencing the cDNA insert to identify the interacting TF.