Systematic development of a two-stage fed-batch process for lipid accumulation in Rhodotorula glutinis.

The application of oleaginous yeast cells as feed supplement, for instance in aqua culture, can be a meaningful alternative for fish meal and oil additives. Therefore, a two-stage fed-batch process split into growth and lipogenesis phase was systematically developed to enrich the oleaginous yeast Rhodotorula glutinis Rh-00301 with high amounts of lipids at industrial relevant biomasses. Thereby, the different carbon sources glucose, sucrose and glycerol were investigated concerning their abilities to serve as a suited raw material for growth and/or lipid accumulation. With the background of economic efficiency C/N ratios of 40, 50 and 70 were investigated as well. It became apparent that glycerol is an improper carbon source most likely because of the passive diffusion of this compound caused by absence of active transporters. The opposite was observed for sucrose, which is the main carbon source in molasses. Finally, an industrially applicable process was successfully established that ensures biomasses of 106±2gL-1 combined with an attractive lipid content of 63±6% and a high lipid-substrate yield (YL/S) of 0.18±0.02gg-1 in a short period of time (84h). Furthermore, during these studies a non-negligible formation of the by-product glycerol was detected. This characteristic of R. glutinis is discussed related to other oleaginous yeasts, where glycerol formation is absent. Nevertheless, due to modifications in the feeding procedure, the formation of glycerol could have been reduced but not avoided.

Medium optimization based on yeast's elemental composition for glutathione production in Saccharomyces cerevisiae.

The production of glutathione (GSH) or GSH enriched yeast is still in the focus of research driven by a high industrial interest. In this study, an optimal growth rate for GSH production via Saccharomyces cerevisiae Sa-07346 was investigated. To further improve the fermentation process in a way that it is independent of lots, the influence of different WMIX medium compositions on biomass and GSH production was studied. Thereby, the fermentation medium was adjusted based on yeast's elemental composition. The resulting chemically defined fermentation medium led to high cell densities in fed-batches. Therefore, it has the potential to be applied successfully for other high cell density yeast fermentation processes. As cysteine is the key component for GSH production, different cysteine addition strategies were studied and finally, a continuous cysteine feeding was applied in the late stage of fermentation. Thereby, a GSH concentration of 1459 ± 57 mg/l was reached by continuously feeding cysteine, which meant an increase to 253% compared to the control without cysteine addition (577 mg/l GSH).

Enhanced incorporation yield of cysteine for glutathione overproduction by fed-batch fermentation of Saccharomyces cerevisiae.

In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).

Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs.

Single bacteria movement tracking by online microscopy--a proof of concept study.

In this technical report we demonstrate a low-cost online unit allowing movement tracking of flagellated bacteria on a single-cell level during fermentation processes. The system's ability to distinguish different metabolic states (viability) of bacteria by movement velocity was investigated. A flow-through cuvette with automatically adjustable layer thickness was developed. The cuvette can be used with most commercially available laboratory microscopes equipped with 40× amplification and a digital camera. In addition, an automated sample preparation unit and a software module was developed measuring size, moved distance, and speed of bacteria. In a proof of principle study the movement velocities of Bacillus amyloliquefaciens FZB42 during three batch fermentation processes were investigated. In this process the bacteria went through different metabolic states, vegetative growth, diauxic shift, vegetative growth after diauxic shift, and sporulation. It was shown that the movement velocities during the different metabolic states significantly differ from each other. Therefore, the described setup has the potential to be used as a bacteria viability monitoring tool. In contrast to some other techniques, such as electro-optical techniques, this method can even be used in turbid production media.

Control of cell morphology of probiotic Lactobacillus acidophilus for enhanced cell stability during industrial processing.

The viability of bacteria during industrial processing is an essential quality criterion for bacterial preparations, such as probiotics and starter cultures. Therefore, producing stable microbial cultures during proliferation is of great interest. A strong correlation between the culture medium and cellular morphology was observed for the lactic acid bacterium Lactobacillus acidophilus NCFM, which is commonly used in the dairy industry as a probiotic supplement and as a starter culture. The cell shapes ranged from single short rods to long filamentous rods. The culture medium composition could control this phenomenon of pleomorphism, especially the use of peptone in combination with an adequate heating of the medium during preparation. Furthermore, we observed a correlation between the cell size and stability of the microorganisms during industrial processing steps, such as freeze-drying, extrusion encapsulation and storage following dried preparations. The results revealed that short cells are more stable than long cells during each of the industrially relevant processing steps. As demonstrated for L. acidophilus NCFM, the adaptation of the medium composition and optimized medium preparation offer the possibility to increase the concentration of viable cells during up- and survival rate during down-stream processing.

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (ß-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from ß-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

Stability of creatine derivatives during simulated digestion in an in vitro model.

Newly developed forms of creatine are often claimed to exhibit improved bioavailability and efficacy. They are of great interest for sports nutrition and therapeutic uses. However, for most newer creatine forms stability after ingestion under physiological conditions is insufficiently documented, relevant data are inconsistent or even missing. Therefore, we developed a controlled simulated digestion system for testing different creatine derivatives in specific simulated parts of the human digestive system. All derivatives showed high stability with negligible formation of creatinine.

Recovery of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Ralstonia eutropha cultures with non-halogenated solvents.

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA-based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non-halogenated solvents. Several non-halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17-30 mol%) remained completely in solution, while polymer with a lower HHx content (11-16 mol%) formed a gel-like phase. All PHA in solution could be precipitated by addition of threefold volumes of n-hexane or n-heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non-halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA.

Overexpression of genes of the fatty acid biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with deregulated sterol and fatty acid biosynthesis pathways were analysed for sterol and fatty acid content and mRNA profiles, with the aim of identifying interactions between lipid biosynthesis pathways. Acetyl CoA carboxylase ACC1 and fatty acid synthases FAS1/FAS2 were overexpressed in wild-type and squalene-overproducing strains. ACC1 overexpression led to decreased fatty acid content in the squalene-overproducing strain (factor of 0.7), while sterols and squalene were increased (factor of 1.5). In the wild-type strain, ACC1 overexpression led to increased levels of both fatty acids and squalene/sterols (factors of 4.0 and 1.7, respectively). This parallel activation of the two pathways seems to be due to transcriptional co-regulation of ACC1 and HMG1. While FAS1 and FAS2 overexpression had no effect in the wild-type strain, FAS2 overexpression induced significant increase of sterols and squalene (factors of 7.2 and 1.3, respectively) and a concomitant decrease of both saturated and unsaturated fatty acids in the squalene-overproducing strain (factor of 0.6). The microarray expression profiles showed that genes upregulated in ACC1-overexpressing strains are FAS1, ERG11, ERG28, ERG5, ERG2 and ERG20, supporting the observed increase of zymosterol and saturated fatty acids. The high ACC1 expression level due to overexpression correlated with increased transcript levels of sphingolipid and sterol biosynthesis genes. The relationship between was shown using the Pathway Studio program.

Rapid determination of the attenuation limit of beer using middle-infrared (MIR) spectroscopy and a multivariate model.

A new approach for the determination of the attenuation limit of beer samples using the specific fingerprint region of middle-infrared (MIR) spectroscopy in combination with multiple regression by partial least-squares (PLS) was developed using an attenuated total reflectance (ATR) module. A specific spectral region between 1200 and 800 cm(-1) was identified as highly informative for the quantification of the limit of attenuation. The absorptions in this region are induced by vibrational bands of ethanol (1080, 1040, and 880 cm(-1)) and dissolved extract, in majority maltotriose (1160-1140 and 1040-980 cm(-1)). The multivariate calibration results in a root mean squared error of calibration (RMSEC) of 0.40% and a validation procedure with independent samples results in a root mean squared error of validation (RMSEV) of 0.50%. A repeatability test, concerning the precision of the developed MIR method as well as the reference method, was analyzed using Student's t test. The test has shown no significant difference between the two random samples.

Transcriptome sequencing and comparative transcriptome analysis of the scleroglucan producer Sclerotium rolfsii.

BACKGROUND: The plant pathogenic basidiomycete Sclerotium rolfsii produces the industrially exploited exopolysaccharide scleroglucan, a polymer that consists of (1 --> 3)-beta-linked glucose with a (1 --> 6)-beta-glycosyl branch on every third unit. Although the physicochemical properties of scleroglucan are well understood, almost nothing is known about the genetics of scleroglucan biosynthesis. Similarly, the biosynthetic pathway of oxalate, the main by-product during scleroglucan production, has not been elucidated yet. In order to provide a basis for genetic and metabolic engineering approaches, we studied scleroglucan and oxalate biosynthesis in S. rolfsii using different transcriptomic approaches. RESULTS: Two S. rolfsii transcriptomes obtained from scleroglucan-producing and scleroglucan-nonproducing conditions were pooled and sequenced using the 454 pyrosequencing technique yielding approximately 350,000 reads. These could be assembled into 21,937 contigs and 171,833 singletons, for which 6,951 had significant matches in public protein data bases. Sequence data were used to obtain first insights into the genomics of scleroglucan and oxalate production and to predict putative proteins involved in the synthesis of both metabolites. Using comparative transcriptomics, namely Agilent microarray hybridization and suppression subtractive hybridization, we identified approximately 800 unigenes which are differently expressed under scleroglucan-producing and non-producing conditions. From these, candidate genes were identified which could represent potential leads for targeted modification of the S. rolfsii metabolism for increased scleroglucan yields. CONCLUSIONS: The results presented in this paper provide for the first time genomic and transcriptomic data about S. rolfsii and demonstrate the power and usefulness of combined transcriptome sequencing and comparative microarray analysis. The data obtained allowed us to predict the biosynthetic pathways of scleroglucan and oxalate synthesis and to identify important genes putatively involved in determining scleroglucan yields. Moreover, our data establish the first sequence database for S. rolfsii, which allows research into other biological processes of S. rolfsii, such as host-pathogen interaction.

The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley.

Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.

Endocytic uptake of fluorescence labelled DNA in yeast.

After dispiriting results using viral vectors in gene therapy, by which a number of patients acquired cancer as a result of the use of retroviral vector constructs, the percentage of non-viral approaches has increased over recent years. To elucidate potential bottlenecks in the non-viral transfection process we here introduce a novel method to directly visualize endocytic non-viral DNA uptake in a transfection approach. This novel method allows for the first time to monitor the location of DNA which is taken up by endocytosis in yeast (Saccharomyces cerevisiae) wild type and mutant strains. More specifically it enables drawing conclusions about conditions favouring non-viral gene transfection.

The human c-fos and TNFalpha AU-rich elements show different effects on mRNA abundance and protein expression depending on the reporter in the yeast Pichia pastoris.

AU-rich elements (AREs) are located in the 3' untranslated region (3' UTR) of their host genes and tightly regulate mRNA degradation and expression. Examples for this kind of regulation are the human proto-oncogene c-fos and the cytokine TNFalpha. Despite large effort in this field, the exact mechanism of ARE-mediated mRNA turnover remains unclear. In this work we analysed the effects of c-fos- and TNFalpha AREs on mRNA abundance and protein expression of selected human cDNAs in the yeast Pichia pastoris. This yeast is exceedingly well known for its excellent protein production capacity; however, ARE-like mechanisms have not been studied in this yeast to date. Interestingly, we observed both stabilizing and destabilizing effects of the c-fos ARE, whereas the TNFalpha ARE has a destabilizing or expression-reducing function in all tested cDNAs. Based on this observation, we introduced a number of single-point mutations upstream of the introduced c-fos ARE into the 3' UTR of a single cDNA in order to demonstrate the importance of ARE-flanking sequences for their own regulation. In conclusion, we illustrate that the analysis of ARE-mediated effects on mRNA abundance and protein expression of a reporter depends on the sequence of the reporter itself as well as the ARE-surrounding sequences within the 3' UTR. For this reason, we question whether already established reporter constructs in other cellular systems display the true type of regulation of the tested AREs for its original host gene. Finally, we propose that AREs should be analysed in their native sequence context.

Tudor staphylococcal nuclease is an evolutionarily conserved component of the programmed cell death degradome.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

A 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus enhances acyl exchange between acyl-CoA and phosphatidylcholine.

The gene encoding a 10-kDa acyl-CoA-binding protein (ACBP) from Brassica napus was over-expressed in developing seeds of Arabidopsis thaliana. Biochemical analysis of T(2) and T(3) A. thaliana seeds revealed a significant increase in polyunsaturated fatty acids (FAs) (18:2(cisDelta9,12) and 18:3(cisDelta9,12,15)) at the expense of very long monounsaturated FA (20:1(cisDelta11)) and saturated FAs. In vitro assays demonstrated that recombinant B. napus ACBP (rBnACBP) strongly increases the formation of phosphatidylcholine (PC) in the absence of added lysophosphatidylcholine in microsomes from DeltaYOR175c yeast expressing A. thaliana lysophosphatidylcholine acyltransferase (AthLPCAT) cDNA or in microsomes from microspore-derived cell suspension cultures of B. napus L. cv. Jet Neuf. rBnACBP or bovine serum albumin (BSA) were also shown to be crucial for AthLPCAT to catalyse the transfer of acyl group from PC into acyl-CoA in vitro. These data suggest that the cytosolic 10-kDa ACBP has an effect on the equilibrium between metabolically active acyl pools (acyl-CoA and phospholipid pools) involved in FA modifications and triacylglycerol bioassembly in plants. Over-expression of ACBP during seed development may represent a useful biotechnological approach for altering the FA composition of seed oil.

Crystal structure of a class XIB phospholipase A2 (PLA2): rice (oryza sativa) isoform-2 pla2 and an octanoate complex.

Phospholipase A(2) catalyzes the specific hydrolysis of the sn-2 acyl bond of various glycerophospholipids, producing fatty acids and lysophospholipids. Phospholipase A(2)s (PLA(2)s) constitute a large superfamily of enzymes whose products are important for a multitude of signal transduction processes, lipid mediator release, lipid metabolism, development, plant stress responses, and host defense. The crystal structure of rice (Oryza sativa) isoform 2 phospholipase A(2) has been determined to 2.0 A resolution using sulfur SAD phasing, and shows that the class XIb phospholipases have a unique structure compared with other secreted PLA(2)s. The N-terminal half of the chain contains mainly loop structure, including the conserved Ca(2+)-binding loop, but starts with a short 3(10)-helix and also includes two short anti-parallel beta-strands. The C-terminal half is folded into three anti-parallel alpha-helices, of which the two first are also present in other secreted PLA(2)s and contain the conserved catalytic histidine and calcium liganding aspartate residues. The structure is stabilized by six disulfide bonds. The water structure around the calcium ion binding site suggests the involvement of a second water molecule in the mechanism for hydrolysis, the water-assisted calcium-coordinate oxyanion mechanism. The octanoate molecule in the complex structure is bound in a hydrophobic pocket, which extends to the likely membrane interface and is proposed to model the binding of the product fatty acid. Due to the differences in structure, the suggested surface for binding to the membrane has a different morphology in the rice PLA(2) compared with other phospholipases.

Characterization of two Arabidopsis thaliana acyltransferases with preference for lysophosphatidylethanolamine.

BACKGROUND: Two previously uncharacterized Arabidopsis genes that encode proteins with acyltransferase PlsC regions were selected for study based on their sequence similarity to a recently identified lung lysophosphatidylcholine acyltransferase (LPCAT). To identify their substrate specificity and biochemical properties, the two Arabidopsis acyltransferases, designated AtLPEAT1, (At1g80950), and AtLPEAT2 (At2g45670) were expressed in yeast knockout lines ale1 and slc1 that are deficient in microsomal lysophosphatidyl acyltransferase activities. RESULTS: Expression of AtLPEAT1 in the yeast knockout ale1 background exhibited strong acylation activity of lysophosphatidylethanolamine (LPE) and lysophosphatidate (LPA) with lower activity on lysophosphatidylcholine (LPC) and lysophosphatidylserine (LPS). AtLPEAT2 had specificities in the order of LPE > LPC > LPS and had no or very low activity with LPA. Both acyltransferases preferred 18:1-LPE over 16:0-LPE as acceptor and preferred palmitoyl-CoA as acyl donor in combination with 18:1-LPE. Both acyltransferases showed no or minor responses to Ca2+, despite the presence of a calcium binding EF-hand region in AtLPEAT2. AtLPEAT1 was more active at basic pH while AtLPEAT2 was equally active between pH 6.0 - 9.0. CONCLUSION: This study represents the first description of plant acyltransferases with a preference for LPE. In conclusion it is suggested that the two AtLPEATs, with their different biochemical and expression properties, have different roles in membrane metabolism/homoeostasis.

Defects in intracellular trafficking and endocytic/vacuolar acidification determine the efficiency of endocytotic DNA uptake in yeast.

The yeast Saccharomyces cerevisiae is a standard model system to study endocytosis. Here we describe the examination of a representative subset of deletion mutants to identify and locate steps in endocytic transport, endosomal/lysosomal acidification and in intracellular transport of hydrolases in non-viral transfection processes. When transport in late endocytosis is inhibited, transfection efficiency is significantly enhanced. Similarly, transfection efficiency is enhanced when the pH-value of the endosomal/vacuolar system is modified. Transfection efficiency is furthermore elevated when the N+/K+ transport in the endosomal system is disturbed. Finally, we observe enhanced transfection efficiency in mutants disturbed in the CVT/autophagy pathway and in hydrolase transport to the vacuole. In summary, non-viral transfection efficiency can be significantly increased by either (i) inhibiting the transport of endocytosed material before it enters the vacuole, or (ii) inducing a non-natural pH-value of the endosomal/vacuolar system, or (iii) slowing down degradative processes by inhibiting vacuolar hydrolases or the transport between Golgi and late endosome/vacuole.