Cloning,nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110 016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5·5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.     

Glutamate dehydrogenases in the oleaginous yeast Yarrowia lipolytica

Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.     

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.     

Extracellular Protease from Pseudomonas marinoglutinosa: Some Properties and Its Action on Fish Actomyosin

A bacterium isolated from Indian mackerel (Rastrelliger kanagurta) and identified as Pseudomonas marinoglutinosa, was found to produce appreciable amounts of extracellular protease when grown in nutrient medium. This enzyme which degraded several proteins, was found to be most active against mackerel myofibrillar proteins. The optimum temperature and pH range for enzyme activity were 50°C and 7–8 respectively. Treatment of mackerel actomyosin with the protease at 0–2°C for 4 days resulted in degradation of the protein as assessed by release of tyrosine, loss in Mg⁺⁺-dependent ATPase activity and changes in SDS-polyacrylamide gel electrophoretic patterns.     

Optimisation of enzyme treatment for the degradation of feed proteins for an Escherichia coli auxotroph lysine availability assay

The lysine availability of animal feed sources is dependent upon the protein source and the processing treatment. Animal bioassays are the standard method for evaluating lysine availability. There is a need to improve in vitro assays for lysine availability to better reflect animal availability measures. Such assays could eventually replace the need for animal assays and substantially reduce assay time and costs. One in vitro assay for lysine availability that has been used previously measures the growth response of an Escherichia coli lysine auxotroph on a protein source. However, since E coli does not secrete proteolytic enzymes, it is necessary to predigest proteins to release free lysine or lysine peptides. The objective of this study was to determine the lysine availability of different protein sources using different enzyme pretreatments and varying the duration of digestion. Pepsin, pancreatin, protease and peptidase enzyme solutions were prepared as either single enzymes or enzyme combinations and subsequently added to various protein solutions. The lysine availability of the protein digests was assayed by measuring the growth response of the E coli lysine auxotroph in supplemented minimal medium. Lysine availability was significantly affected by the enzyme treatment (p < 0.01). The protease–peptidase enzyme combination had the most extensive digestion for all protein sources. There was no significant difference between 4 and 10 h digestion periods for the combination of protease–peptidase. The 4 h protease–peptidase treatment produced a digest with E coli-determined lysine availability highly correlated (Pearson correlation coefficient of 0.942) with animal availability values from previous work by others. Based on these results, it is possible to use a standard 4 h protease–peptidase enzyme treatment to predigest feed proteins to provide an accurate estimate of available lysine by an in vitro E coli lysine assay. © 1999 Society of Chemical Industry     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

Isolation and identification of a black Aspergillus strain and the effect of its novel protease on the aroma of Moutai‐flavoured liquor

Among Chinese traditional distilled liquors, Moutai‐flavoured liquor is the most famous, owing to its complicated process as it is derived from fermented sorghum coupled with the use of a Moutai‐flavoured Daqu. In this study, a novel isolate, belonging to a black Aspergillus, was obtained and identified as Aspergillus hennebergii by ITS‐5.8S rRNA sequencing analysis and conventional morphologic identification. The influences of initial pH, carbon source, nitrogen source and metal ions on the production of an A. hennebergii protease were studied. The results revealed that metal ions exerted a significant effect on enzyme production and activity. Additionally, the potential application of the protease from A. hennebergii was investigated. The enzymatic hydrolysis resulted in the identification and quantification of 42 compounds, including alcohols, aldehydes, pyrazines and esters. These volatile compounds exhibited special flavour properties. Significant differences were observed between the enzyme treatments and the control sample. Samples from the enzyme treatments led to the highest amounts of alcohols, pyrazines and aromatics. These results suggest that A. hennebergii, or its protease, may have some application values for the enhancement of the quality of Chinese liquor and for the improvement of the liquor production process. Copyright © 2014 The Institute of Brewing & Distilling     

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.     

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.     

EXTRACELLULAR PROTEASE ACTIVITY IN A STRAIN OF SACCHAROMYCES CEREVISIAE

The isolation and analysis of a strain of laboratory yeast with extracellular protease activity is described. The proteolytic activity found in culture supernatants exceeded the parental strain by at least an order of magnitude and apparently was due neither to cell lysis nor to increased cell wall permeability. The extracellular proteases were heterogeneous in composition, consisting of possibly 3 aberrantly secreted intracellular proteinases. This extracellular protease activity was conferred by a single recessive mutation (epr1.1) in a gene displaying classical Mendelian inheritance. EPR1 was tentatively assigned to chromosome XV with loose linkage to the HIS3 gene. Strains carrying the mutant epr1.1) allele also possessed increased levels of secreted invertase activity and it is proposed that EPR1 is closely involved in the intracellular protein translocation pathway of yeast.     

Effects of extracellular proteases and its inhibitors on the gel characteristics of soy protein induced by lactic acid bacteria

The purpose of this study was to analyse the relationship between extracellular protease activity and gel characteristics of soybean protein gel induced by lactic acid bacteria (LAB), the influence of protease inhibition on the gel characteristics was discussed through SDS-PAGE, rheological properties and microstructure. The results showed that the pH value of Lactobacillus casei decreased slowly, but the gel could be formed at 2 h, maybe its higher extracellular protease activity improved the gel characteristics. The addition of ethylenediaminetetraacetic acid (10 mmol L⁻¹) reduced the gel quality, when the protease activity was inhibited, the gel quality would decrease, even if there were macromolecular crosslinkers in the system. This study showed that extracellular protease had an important influence on gel characteristics and provided a theoretical basis for the application of protease producing LAB in fermentation-induced soybean gel foods.     

Predictive modelling of growth and enzymatic synthesis and activity by a cocktail of Yarrowia lipolytica,Zygosaccharomyces bailii and Pichia anomala

The aims of the investigations are the development and combination of predictive models for growth (increase in cellular number) and lipase synthesis and protease activity by a cocktail of yeast species comprising Yarrowia lipolytica, Zygosaccharomyces bailii and Pichia anomala. Determinations of yeast number and enzyme synthesis and activity were made within the three-dimensional matrix of environmental conditions incorporating temperatures between 2°C and 20°C, pH values between 4.0 and 7.5 and water activity (a_w) between 0.95 and 0.995. The microbiological data produced a reliable model, but it was not possible to use the curve-fitting function to fit the enzymatic data, since most enzyme synthesis and activity takes place during the stationary phase of growth. A predictive generic model for growth of yeasts is published and a database of systematic information describing enzyme synthesis and activity within the matrix of environmental conditions investigated will be made available.     

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.     

Role of O-acetylhomoserine sulfhydrylase in sulfur amino acid synthesis in various yeasts

Mutants defective in O-acetylhomoserine sulfhydrylase (OAH-SHLase) were obtained in five yeast strains representative of different yeast genera: Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe and Trichosporon cutaneum. In vitro, in all five strains, the enzyme also had O-acetylserine (OAS) sulfhydrylase activity so it is a ‘bifunctional’ OAH/OAS-SHLase (Yamagata, 1989). The enzyme was only found to be essential in S. cerevisiae (OAH SHLase-negative mutants are auxotrophs). Its impairment in K. lactis caused a slower growth rate and a decrease of the sulfur amino acid pool. In T. cutaneum only the pool was affected whereas in Y. lipolytica and S. pombe the lesion caused no change in the growth rate nor in the pool. In all strains where OAH SHLase-negative mutants were prototrophs, a monofunctional OAS sulfhydrylase was detected. The results indicate that OAH SHLase may play different physiological roles in various yeasts.