Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Efficient production of bacterial antibiotics aminoriboflavin and roseoflavin in eukaryotic microorganisms, yeasts.

BACKGROUND: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known. RESULTS: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L- 1 (C. famata), and that of roseoflavin was 5 mg L- 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L- 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all. CONCLUSIONS: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated.

Insights into the glycerol transport of Yarrowia lipolytica.

Cellular membranes separate cells from the environment and hence, from molecules essential for their survival. To overcome this hurdle, cells developed specialized transport proteins for the transfer of metabolites across these membranes. Crucial metabolites that need to cross the membrane of each living organism, are the carbon sources. While many organisms prefer glucose as a carbon source, the yeast Yarrowia lipolytica seems to favor glycerol over glucose. The fast growth of Y. lipolytica on glycerol and its flexible metabolism renders this yeast a fascinating organism to study the glycerol metabolism. Based on sequence similarities to the known fungal glycerol transporter ScStl1p and glycerol channel ScFps1p, ten proteins of Y. lipolytica were found that are potentially involved in glycerol uptake. To evaluate, which of these proteins is able to transport glycerol in vivo, a complementation assay with a glycerol transport-deficient strain of Saccharomyces cerevisiae was performed. Six of the ten putative transporters enabled the growth of S. cerevisiae stl1Δ on glycerol and thus, were confirmed as glycerol transporting proteins. Disruption of the transporters in Y. lipolytica abolished its growth on 25 g/L glycerol, but the individual expression of five of the identified glycerol transporters restored growth. Surprisingly, the transporter-disrupted Y. lipolytica strain retained its ability to grow on high glycerol concentrations. This study provides insight into the glycerol uptake of Y. lipolytica at low glycerol concentrations through the characterization of six glycerol transporters and indicates the existence of further mechanisms active at high glycerol concentrations.

Identification of the citrate exporter Cex1 of Yarrowia lipolytica.

Yarrowia lipolytica is a yeast with many talents, one of them being the production of citric acid. Although the citrate biosynthesis is well studied, little is known about the transport mechanism by which citrate is exported. To gain better insight into this mechanism, we set out to identify a transporter involved in citrate export of Y. lipolytica. A total of five proteins were selected for analysis based on their similarity to a known citrate exporter, but neither a citrate transport activity nor any other phenotypic function could be attributed to them. Differential gene expression analysis of two strains with a distinct citrate productivity revealed another three putative transporters, one of which is YALI0D20196p. Disrupting YALI0D20196g in Y. lipolytica abolished citrate production, while extrachromosomal expression enhanced citrate production 5.2-fold in a low producing wildtype. Furthermore, heterologous expression of YALI0D20196p in the non-citrate secreting yeast Saccharomyces cerevisiae facilitated citrate export. Likewise, expression of YALI0D20196p complemented the ability to secrete citrate in an export-deficient strain of Aspergillus niger, confirming a citrate export function of YALI0D20196p. This report on the identification of the first citrate exporter in Y. lipolytica, termed Cex1, represents a valuable starting point for further investigations of the complex transport processes in yeasts.

Complete genome sequence and transcriptome regulation of the pentose utilizing yeast Sugiyamaella lignohabitans.

Efficient conversion of hexoses and pentoses into value-added chemicals represents one core step for establishing economically feasible biorefineries from lignocellulosic material. While extensive research efforts have recently provided advances in the overall process performance, the quest for new microbial cell factories and novel enzymes sources is still open. As demonstrated recently the yeast Sugiyamaella lignohabitans (formerly Candida lignohabitans) represents a promising microbial cell factory for the production of organic acids from lignocellulosic hydrolysates. We report here the de novo genome assembly of S. lignohabitans using the Single Molecule Real-Time platform, with gene prediction refined by using RNA-seq. The sequencing revealed a 15.98 Mb genome, subdivided into four chromosomes. By phylogenetic analysis, Blastobotrys (Arxula) adeninivorans and Yarrowia lipolytica were found to be close relatives of S. lignohabitans Differential gene expression was evaluated in typical growth conditions on glucose and xylose and allowed a first insight into the transcriptional response of S. lignohabitans to different carbon sources and different oxygenation conditions. Novel sequences for enzymes and transporters involved in the central carbon metabolism, and therefore of potential biotechnological interest, were identified. These data open the way for a better understanding of the metabolism of S. lignohabitans and provide resources for further metabolic engineering.

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters.

Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

Organic acids from lignocellulose: Candida lignohabitans as a new microbial cell factory.

Biorefinery applications require microbial cell factories for the conversion of various sugars derived from lignocellulosic material into value-added chemicals. Here, the capabilities of the yeast Candida lignohabitans to utilize a range of such sugars is characterized. Substrates efficiently converted by this yeast include the pentoses xylose and arabinose. Genetic engineering of C. lignohabitans with the isolated endogenous GAP promoter and GAP terminator was successful. GFP expression was used as a proof of functionality for the isolated transcription elements. Expression of lactate dehydrogenase and cis-aconitate decarboxylase resulted in stable and reproducible production of lactic acid and itaconic acid, respectively. The desired organic acids were accumulated converting pure sugars as well as lignocellulosic hydrolysates. C. lignohabitans proved therefore to be a promising reliable microbial host for production of organic acids from lignocellulosic material.

Six novel constitutive promoters for metabolic engineering of Aspergillus niger.

Genetic tools for the fine-tuning of gene expression levels are a prerequisite for rational strain optimization through metabolic engineering. While Aspergillus niger is an industrially important fungus, widely used for production of organic acids and heterologous proteins, the available genetic tool box for this organism is still rather limited. Here, we characterize six novel constitutive promoters of A. niger providing different expression levels. The selection of the promoters was based on published transcription data of A. niger. The promoter strength was determined with the ß-glucuronidase (gusA) reporter gene of Escherichia coli. The six promoters covered a GUS activity range of two to three orders of magnitude depending on the strain background. In order to demonstrate the power of the newly characterized promoters for metabolic engineering, they were used for heterologous expression of the cis-aconitate decarboxylase (cad1) gene of Aspergillus terreus, allowing the production of the building block chemical itaconic acid with A. niger. The CAD activity, dependent on the choice of promoter, showed a positive correlation with the specific productivity of itaconic acid. Product titers from the detection limit to up to 570 mg/L proved that the set of constitutive promoters is a powerful tool for the fine-tuning of metabolic pathways for the improvement of industrial production processes.

Process engineering towards an oxidative cellular state improves 3-hydroxypropionic acid production with Lentilactobacillus diolivorans.

3-hydroxypropionic acid (3-HP) is among the top platform chemicals proposed for bio based production by microbial fermentation from renewable resources. A promising renewable substrate for 3-HP production is crude glycerol. Only a few microorganisms can efficiently convert glycerol to 3-HP. Among the most promising organisms is Lentilactobacillus diolivorans. In this study, an already established fed-batch process, accumulating 28 g/L 3-HP, was used as a starting point for process engineering. The engineering approaches focused on modulating the cellular redox household towards a more oxidized state, as these conditions favour 3-HP production. Variations of oxygen and glucose availability (controlled by the glucose/glycerol ratio in the feed medium) individually already improved 3-HP production. However, the combination of both optimal parameters (30% O2, 0.025 mol/mol glu/gly) led to the production of 67.7 g/L 3-HP after 180 h of cultivation, which is so far the highest titer reported for 3-HP production using Lactobacillus spp.

From rumen to industry.

The rumen is one of the most complicated and most fascinating microbial ecosystems in nature. A wide variety of microbial species, including bacteria, fungi and protozoa act together to bioconvert (ligno)cellulosic plant material into compounds, which can be taken up and metabolized by the ruminant. Thus, the rumen perfectly resembles a solution to a current industrial problem: the biorefinery, which aims at the bioconversion of lignocellulosic material into fuels and chemicals. We suggest to intensify the studies of the ruminal microbial ecosystem from an industrial microbiologists point of view in order to make use of this rich source of organisms and enzymes.

Genome sequence of the ruminal bacterium Megasphaera elsdenii.

Megasphaera elsdenii is a Gram-negative ruminal bacterium. It is being investigated as a probiotic supplement for ruminants as it may provide benefits for energy balance and animal productivity. Furthermore, it is of biotechnological interest due to its capability of producing various volatile fatty acids. Here we report the complete genome sequence of M. elsdenii DSM 20460, the type strain for the species.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

Slow Growth and Increased Spontaneous Mutation Frequency in Respiratory Deficient afo1- Yeast Suppressed by a Dominant Mutation in ATP3.

A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1- yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1- strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the respiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.

Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus.

The yeast Pichia pastoris is a widely used host organism for heterologous protein production. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number integrants of P. pastoris are achievable only by screening of random events or by cloning of gene concatemers. Methods for rapid and reliable multicopy integration of the expression cassette are therefore desirable. Here we present such a method based on vector integration into the rDNA locus and post-transformational vector amplification by repeated selection on increased antibiotic concentrations. Data are presented for two exemplary products: human serum albumin, which is secreted into the supernatant, and human superoxide dismutase, which is accumulated in the cytoplasm of the cells. The striking picture evolving is that intracellular protein production is tightly correlated with gene copy number, while use of the secretory pathway introduces a high clonal variability and the correlation with gene copy number is valid only for low gene copy numbers.

Microbial 2-butanol production with Lactobacillus diolivorans.

BACKGROUND: Biobutanol has great potential as biofuel of the future. However, only a few organisms have the natural ability to produce butanol. Amongst them, Clostridium spp. are the most efficient producers. The high toxicity of biobutanol constitutes one of the bottlenecks within the biobutanol production process which often suffers from low final butanol concentrations and yields. Butanol tolerance is a key driver for process optimisation and, therefore, in the search for alternative butanol production hosts. Many Lactobacillus species show a remarkable tolerance to solvents and some Lactobacillus spp. are known to naturally produce 2-butanol from meso-2,3-butanediol (meso-2,3-BTD) during anaerobic sugar fermentations. Lactobacillus diolivorans showed already to be highly efficient in the production of other bulk chemicals using a simple two-step metabolic pathway. Exactly, the same pathway enables this cell factory for 2-butanol production. RESULTS: Due to the inability of L. diolivorans to produce meso-2,3-BTD, a two-step cultivation processes with Serratia marcescens has been developed. S. marcescens is a very efficient producer of meso-2,3-BTD from glucose. The process yielded a butanol concentration of 10 g/L relying on wild-type bacterial strains. A further improvement of the maximum butanol titer was achieved using an engineered L. diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on the engineered strain led to a maximum 2-butanol titer of 13.4 g/L, which is an increase of 34%. CONCLUSION: In this study, L. diolivorans is for the first time described as a good natural producer for 2-butanol from meso-2,3-butanediol. Through the application of a two-step cultivation process with S. marcescens, 2-butanol can be produced from glucose in a one-vessel, two-step microbial process.

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.

Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris.

BACKGROUND: High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. RESULTS: Overexpression of the first gene of the riboflavin biosynthetic pathway (RIB1) is already sufficient to obtain yellow colonies and the accumulation of riboflavin in the supernatant of shake flask cultures growing on glucose. Sequential deregulation of all the genes, by exchange of their native promoter with the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) increases the riboflavin accumulation significantly. CONCLUSION: The regulation of the pathway is distributed over more than one gene. High cell density cultivations of a P. pastoris strain overexpressing all six RIB genes allow the accumulation of 175 mg/L riboflavin in the supernatant. The basis for rational engineering of riboflavin production in P. pastoris has thus been established.

3-Hydroxypropionaldehyde production from crude glycerol by Lactobacillus diolivorans with enhanced glycerol uptake.

BACKGROUND: In their quest for sustainable development and effective management of greenhouse gas emissions, our societies pursue a shift away from fossil-based resources towards renewable resources. With 95% of our current transportation energy being petroleum based, the application of alternative, carbon-neutral products-among them biodiesel-is inevitable. In order to enhance the cost structure of biodiesel biorefineries, the valorization of the crude glycerol waste stream into high-value platform chemicals is of major importance. RESULTS: The purpose of this study is the production of 3-hydroxypropionaldehyde (3-HPA) from biodiesel-derived crude glycerol by Lactobacillus diolivorans. Particular focus is given on overcoming potential limitations of glycerol transport into the cell, in order to use the cells' total glycerol dehydratase capability towards the formation of 3-HPA as the main product. Recombinant overexpression of the endogenous glycerol uptake facilitating protein PduF results in a significant increase of glycerol conversion by a factor of 1.3. Concomitantly, glycerol dehydratase activity increased from initially 1.70 ± 0.03 U/mg protein to 2.23 ± 0.11 U/mg protein. With this approach, an average productivity of 4.8 g3-HPA/(gCDM h) yielding up to 35.9 g/L 3-HPA and 0.91 mol3-HPA/molGlycerol have been obtained. CONCLUSION: Lactobacillus diolivorans proves to be a valuable cell factory for the utilization of crude glycerol delivering high-value C3 chemicals like 3-HPA, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP). Enhancing the glycerol influx into the cell by genetic engineering was successful paving the way towards the commercial production of 3-HPA.

Effect of carbon pulsing on the redox household of Lactobacillus diolivorans in order to enhance 1,3-propanediol production.

This study investigates potential limitations of 1,3-propanediol formation by Lactobacillus diolivorans. Particular focus is given to enhanced glycerol utilization as well as the elimination of by-product formation. The key aspect is a modulation of the redox household by process engineering through the application of carbon pulses. A shift in the product pattern of C3 products was achieved, improving the ratio of 1,3-propanediol versus 3-hydroxypropionic acid up to a level of 20:1. Moreover, in combination with alternative feeding strategies this ratio was enhanced up to 45:1 and the maximum observed productivity of 1,3-propanediol could be significantly increased to 1.7g/Lh.

Metabolic Flexibility of Yarrowia lipolytica Growing on Glycerol.

The yeast Yarrowia lipolytica is a fascinating microorganism with an amazing metabolic flexibility. This yeast grows very well on a wide variety of carbon sources from alkanes over lipids, to sugars and glycerol. Y. lipolytica accumulates a wide array of industrially relevant metabolites. It is very tolerant to many environmental factors, above all the pH value. It grows perfectly well over a wide pH range, but it has been described, that the pH has a decisive influence on the metabolite pattern accumulated by this yeast. Here, we set out to characterize the metabolism of different Y. lipolytica strains, isolated from various environments, growing on glycerol at different pH values. The conditions applied for strain characterization are of utmost importance. Shake flask cultures lead to very different results, when compared to controlled conditions in bioreactors regarding pH and aeration. Only one of the tested strains was able to accumulate high amounts of citric acid in shake flask experiments, whereas a group of six strains turned out to accumulate citric acid efficiently under controlled conditions. The present study shows that strains isolated from dairy products predominantly accumulate sugar alcohols at any given pH, when grown on glycerol under nitrogen-limitation.