Yarrowia lipolytica SRP54 Homolog and Translocation of Kar2p

To investigate the role of Srp54p in protein translocation, the Yarrowia lipolytica SRP54 homolog was cloned. Sequencing revealed an open reading frame of 536 amino acids coding for a 57·2 kilodalton polypeptide with 55 to 57% sequence identity to Srp54ps of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and mouse. Like these Srp54ps, Y. lipolytica Srp54p has an N-terminal domain with a highly conserved GTP-binding site and a methionine-rich C-terminal domain. Differing results regarding the essentiality of SRP subunits were obtained. SRP54 is important but not essential for growth, but it was reconfirmed that at least one SRP RNA gene is essential. Cells with SRP54 deleted grow about six times more slowly than wild type; faster-growing colonies, still growing much slower than wild type, appeared quite frequently. In srp54Δ cells, no untranslocated alkaline extracellular protease precursor was detected. Therefore, to develop another reporter molecule the Y. lipolytica KAR2 homolog was cloned and Kar2p antibodies were produced. For Kar2p an untranslocated precursor was detected in srp54Δ but not in wild-type cells, suggesting that its translocation was defective in the srp54Δ cells. These results confirm an in vivo role for SRP in protein translocation in Y. lipolytica, suggest that SRP RNA or an SRP core-particle has functions not shared by Srp54p, and show that, as in S. cerevisiae and Sz. pombe, reporter molecules differ in their dependency on SRP for translocation. The SRP54 and KAR2 sequences have been deposited in GenBank under Accession Numbers U42418 and U63136.     

Secretion and pH-dependent self-processing of the pro-form of the Yarrowia lipolytica acid extracellular protease

The secretion and maturation of the acid extracellular protease (AXP) of the yeast Yarrowia lipolytica have been characterized using antiserum raised against this enzyme. A 42 kDa pro-enzyme form of AXP was identified from lysates of radiolabelled Y. lipolytica cells and found to contain no N-linked carbohydrate moieties. Using pulse-chase immune precipitation it was demonstrated that the AXP precursor was secreted into the extracellular medium where, under conditions of low pH, it underwent autocatalytic activation forming the mature enzyme. Conversion of the AXP pro-form in the presence of the protease inhibitor pepstatin indicated that an intramolecularly-catalysed reaction mechanism was involved in AXP maturation. Further evidence supporting the role of autocatalytic processing came from the side-chain specificity of mature AXP towards the oxidized B-chain of insulin. © 1998 John Wiley & Sons, Ltd.     

Screening various Yarrowia lipolytica strains for citric acid production

Citric acid (CA) productivity by Yarrowia lipolytica dependents on strain type, carbon source, carbon to nitrogen (C/N) molar ratio as well as physicochemical conditions (pH, temperature, oxygen transfer rate, etc.). In the current study, 10 different Y. lipolytica strains were first challenged in shake-flask culture for CA production in a glucose-based media under nitrogen-limited conditions. For the most promising one, strain K57, CA productivity was monitored during culture in batch bioreactor for three initial C/N molar ratio (167, 367, and 567 Cmol/Nmol). The highest CA yield (0.77 g/g glucose), titre (72.3 g/L CA), and productivity (0.04 g/g.h) were found for C/N ratio of 367. However, the highest growth rate was obtained for an initial C/N ratio of 167. From these results, Y. lipolytica strain K57 could be considered to produce CA at higher titre on glucose-based medium in batch bioreactor than others Y. lipolytica strain reported in the literature.     

The impact of oxygen availability on stress survival and radical formation of Bacillus cereus

Both the growth and stress survival of two model Bacillus cereus strains, ATCC 14579 and ATCC 10987, were tested in three different conditions varying in oxygen availability, i.e., aerobic, microaerobic and anaerobic conditions. Both B. cereus strains displayed highest growth rates and yields under aerobic conditions, whereas the microaerobic and anaerobic cultures showed similar reduced growth performances. The cells grown and exposed microaerobically and anaerobically were more resistant to heat and acid than cells that were cultured and exposed aerobically. On the other hand, the anaerobically grown cells were more sensitive to hydrogen peroxide compared to the (micro)aerobically grown cells. The increased heat- and acid-induced inactivation in aerobic conditions appeared to be associated with intracellular accumulation of excess hydroxyl and/or peroxynitrite radicals, as determined by flow cytometry in combination with the fluorescent reporter dye 3′-(p-hydroxyphenyl) fluorescein. This suggests that radical formation may contribute to inactivation of bacteria in the presence of oxygen, such as in aerobic and microaerobic conditions. No evidence was found for radical formation upon exposure to salt and hydrogen peroxide. The increased resistance to heat and acid in microaerobic and anaerobic conditions shows that oxygen availability should be taken into account when behavior of bacteria, such as B. cereus, in food industry related conditions is investigated, because oxygen availability may affect the efficiency of food preservation conditions.     

Sterol synthesis and cell size distribution under oscillatory growth conditions in Saccharomyces cerevisiae scale‐down cultivations

Physiological responses of yeast to oscillatory environments as they appear in the liquid phase in large‐scale bioreactors have been the subject of past studies. So far, however, the impact on the sterol content and intracellular regulation remains to be investigated. Since oxygen is a cofactor in several reaction steps within sterol metabolism, changes in oxygen availability, as occurs in production‐scale aerated bioreactors, might have an influence on the regulation and incorporation of free sterols into the cell lipid layer. Therefore, sterol and fatty acid synthesis in two‐ and three‐compartment scale‐down Saccharomyces cerevisiae cultivation were studied and compared with typical values obtained in homogeneous lab‐scale cultivations. While cells were exposed to oscillating substrate and oxygen availability in the scale‐down cultivations, growth was reduced and accumulation of carboxylic acids was increased. Sterol synthesis was elevated to ergosterol at the same time. The higher fluxes led to increased concentrations of esterified sterols. The cells thus seem to utilize the increased availability of precursors to fill their sterol reservoirs; however, this seems to be limited in the three‐compartment reactor cultivation due to a prolonged exposure to oxygen limitation. Besides, a larger heterogeneity within the single‐cell size distribution was observed under oscillatory growth conditions with three‐dimensional holographic microscopy. Hence the impact of gradients is also observable at the morphological level. The consideration of such a single‐cell‐based analysis provides useful information about the homogeneity of responses among the population.     

An efficient method for production of kynurenic acid by Yarrowia lipolytica

Kynurenic acid (KYNA) is a compound derived from the tryptophan catabolic pathway. Antioxidant and neuroprotective properties have been confirmed for KYNA, which makes it an interesting and important metabolite of biomedical significance. In the present study, the yeast Yarrowia lipolytica was tested for KYNA biosynthesis. The results showed that Y. lipolytica strain S12 is able to produce KYNA in high concentrations (up to 21.38 μg/ml in culture broth and 494.16 μg/g cell dry weight in biomass) in optimized conditions in a medium supplemented with tryptophan. Different conditions of culture growth, including the source of carbon, its concentration and pH value of the medium, as well as the influence of an inhibitor or precursor of KYNA synthesis, were analysed. The obtained data confirmed the presence of KYNA metabolic pathway in the investigated yeast. To our best knowledge, this is the first study that reports KYNA production in the yeast Y. lipolytica in submerged fermentation.     

Comparison of expression systems in the yeasts Saccharomyces cerevisiae,Hansenula polymorpha,Klyveromyces lactis,Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4·5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1α and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters, Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF gene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509. © 1998 John Wiley & Sons, Ltd.     

Feeding strategy impacts heterologous protein production in Yarrowia lipolytica fed-batch cultures—Insight into the role of osmolarity

Fed-batch cultivation is the preferred bioprocessing strategy applied in microbial production of proteins. Feeding strategy is crucial parameters to be optimized upon development of a fed-batch process. In this study, we investigated impact of different feeding strategies on production of recombinant enzymatic protein in Yarrowia lipolytica cultures. From amongst tested strategies, comprising intermittent and continuous feedings, also in cascade with respiratory factors, intermittent feeding executed after complete exhaustion of glycerol from the medium, with moderate amplitude of osmolarity, was the most beneficial in terms of the secretory enzyme amount, its volumetric productivity and specific activity. Because adopted feeding strategies strongly modulated osmolarity of the cultures, the effect of osmotic pressure on production of the target heterologous protein was investigated in a series of batch cultivations with addition of osmoactive compounds (NaCl, sorbitol, sucrose, and glycerol) at different concentrations. Although obvious promoting effect of the osmoactive substances on the enzyme production was clear, no straightforward correlation between the medium osmolarity and the target enzyme's specific activity could be observed. These results suggest that not only the level of osmolarity but also chemical character of the osmoactive compound have both important impact on the production of secretory proteins in Y. lipolytica cultures.     

Change in transport activities of vacuoles of the yeast Yarrowia lipolytica during its growth on glucose

Vacuoles were isolated from Yarrowia lipolytica yeast cells taken at various growth phases under carbon or nitrogen limitation. Vacuoles from the cells at the logarithmic growth phase showed a high activity of vacuolar H⁺-ATPase (0·9–1·1 U/mg protein) and efficiently generated chemical proton gradient and membrane potential across the tonoplast. Ca²⁺- and citrate transport were found to be maximal at this growth phase. At growth retardation and then in the stationary phase all the parameters studied decreased irrespective of the method of growth limitation. The citrate-transporting activity of vacuoles completely disappeared at growth retardation, also irrespective of the limitation method and irrespective of whether yeast cells overproduced citrate in the culture medium. The citrate-transporting system of Y. lipolytica vacuolar membrane is concluded not to be involved in citrate efflux and this efflux is probably performed by the plasmalemma transport system.     

The technological characteristics of Debaryomyces hansenii and Yarrowia lipolytica and their potential as starter cultures for production of Danablu

Six strains of Debaryomyces hansenii var. hansenii and Yarrowia lipolytica, respectively, originating from blue mould cheeses were examined for their potential use as starter cultures for the production of Danablu in laboratory studies. D. hansenii showed strong growth and assimilation of lactose, galactose, lactate and five out of six strains assimilated citric acid under the environmental conditions prevailing in Danablu during maturation at 10°C. Y. lipolytica was more sensitive to NaCl and did not assimilate lactose and galactose. Both yeasts hydrolysed tributyrin with the highest activity observed for Y. lipolytica. D. hansenii showed little if any release of free fatty acids from butterfat at 10°C. Y. lipolytica was strongly lipolytic. The strains of D. hansenii were not able to hydrolyse casein at 10°C whereas 4 of the 6 strains of Y. lipolytica degraded all components of the casein. Strain-specific interactions, in cheese agar resulting in inhibition of mycelial growth and sporulation of P. roqueforti was observed, especially for Y. lipolytica.     

Hepatoprotective effect of chicory (Chicorium intybus) root extract against orotic acid-induced fatty liver in rats

Effect of chicory root extract (CRE) on the triglyceride metabolism in orotic acid (OA)-fed rats was investigated. Liver weights and hepatic triglyceride concentrations were markedly increased by OA-feeding rats. These results were attributed to the significant increase in the activity of hepatic phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and ratelimiting enzymes for triglyceride synthesis. Supplementation of CRE to OA did significantly reduced the hepatic triglyceride concentrations and DGAT activity without affecting PAP activity. Furthermore, OA treatment was significantly decreased plasma triglyceride (TG) and increased hepatic TG concentrations and reduced microsomal triglyceride transfer protein (MTP) activity without diminishing MTP mRNA expression in rats. However, hepatic TG concentration was significantly decreased and MTP activity was also reduced without diminishing MTP mRNA expression in rats fed simultaneous with OA and CRE diet. The hepatocytes in the OA-feeding rats contained numerous largely fat droplets, but CRE feeding prevented the OA-induced fat accumulation. Present study demonstrates that CRE reduces the liver TG accumulation by reduced DGAT and MTP activities without diminishing MTP mRNA expression by OA administration.     

Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats

In order to utilize yellowfin sole (Limanda aspera) frame protein, which is normally discarded as industrial waste in the process of fish manufacture, yellowfin sole frame protein was hydrolysed by α-chymotrypsin. Yellowfin sole frame protein hydrolysates (YFPHs) were fractionated into three ranges of molecular weight (YFPH-I, 30–10 kDa; YFPH-II, 10–5 kDa; YFPH-III, below 5 kDa) using an ultrafiltration (UF) membrane bioreactor system. Angiotensin I-converting enzyme (ACE) inhibitory activity was detected on YFPH-III, and the ACE inhibitory peptide (YFP) was purified from YFPH-III using consecutive chromatographic techniques. The YFP with a molecular mass of 1.3 kDa consisted of 11 amino acids, Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly-Pro-Glu-Leu, and its IC₅₀ value was 28.7 μg/ml. Lineweaver–Burk plots suggest that YFP acts as a non-competitive inhibitor to inhibit ACE. Antihypertensive effects of YFP on spontaneously hypertensive rats (SHR) following oral administration was determined as the blood pressure significantly decreased after peptide ingestion.     

A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter

The ability to monitor the development of a given cell lineage in a non-invasive manner by fluorescent markers both in vivo and in vitro provides a great advantage for the analysis of the lineage of interest. To date, a number of transgenic or knock-in mouse strains, in which developing germ cells are marked with fluorescent reporters, have been generated. We here describe a novel double transgenic reporter mouse strain that expresses membrane-targeted Venus (mVenus), a brighter variant of yellow fluorescent protein (YFP), under the control of Prdm1 (Blimp1) regulatory elements and enhanced cyan fluorescent protein (ECFP) under the control of Dppa3 (Stella/Pgc7). The double transgenic strain unambiguously marked Prdm1 expression in the lineage-restricted precursors of primordial germ cells (PGCs) in the proximal epiblast at embryonic day (E) 6.25 and specifically illuminated Prdm1- and Dppa3-positive migrating PGCs after E8.5. The double transgenic reporter also precisely recapitulated dynamic embryonic expression of Prdm1 outside the germ cell lineage. Moreover, we derived ES cells that bore both transgenes. These cells made a robust contribution both to the germ and somatic cell lineages in chimeras with accurate Prdm1-mVenus and Dppa3-ECFP expression. The transgenic strain and the ES cells will serve as valuable experimental materials not only for analyzing the origin and properties of the germ cell lineage in vivo, but also for establishing a culture system to efficiently induce proper germ cells with temporally coordinated Prdm1 and Dppa3 expression in vitro.     

Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.     

Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane-utilizing yeast Yarrowia lipolytica

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.