Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes. Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998     

Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases

Mechanisms regulating post-secretory limited proteolysis, carried out by the acid protease from Trichoderma reesei, were studied by following the release of α-galactosidase and multiple forms of cellobiohydrolase from this species. Both the rate of the proteolysis and the mode of action of the protease were affected by the pH of the culture medium, and only weakly depended on the amount of the enzyme. At pH between 2.7 and 3.5 the proteolytic reaction was limited, while at lower pH proteins were completely digested. Proteolysis depended on the degree of glycosylation of secreted enzymes. Inhibition of post-secretory deglycosylation decreased the rate of limited proteolysis in the culture medium in the course of fungal growth. Glucose and cellobiose, the main products of cellulose degradation carried out by the fungal cellulolytic complex, inhibited the proteolysis of the cellobiohydrolase in a concentration-dependent manner. A 32-kDa aspartic protease (EC 3.4.23.18) secreted by T. reesei was purified to homogeneity. The acid protease cleaved α-galactosidase and cellobiohydrolase into the same proteolytic fragments that had been isolated from the culture medium. Received: 4 December 1998 / Received revision: 22 February 1999 / Accepted: 5 March 1999     

Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae

 In order to direct the persistent expression of recombinant human serum albumin (HSA) from the GAL10 promoter in the yeast Saccharomyces cerevisiae, we carried out periodic feeding of galactose during shake-flask cultures. Unexpectedly, the recombinant protein secreted was observed to undergo rapid degradation, which was apparently accelerated by carbon-source feeding. The extracellular degradation of HSA occurred even in the strain deficient in the major vacuolar proteases PrA and PrB, and in the strain lacking the acidic protease Yap3p (involved in the generation of HSA-truncated fragments). Interestingly, the degradation correlated closely with the acidification of extracellular pH and thus was significantly overcome either by buffering the culture medium above pH 5.0 or by adding amino acid-rich supplements to the culture medium, which could prevent the acidification of medium pH during cultivation. Addition of arginine or ammonium salt also substantially minimized the degradation of HSA, even without buffering. The extracellular degradation activity was not detected in the cell-free culture supernatant but was found to be associated with intact cells. The results of the present study strongly suggest that the HSA secreted in S. cerevisiae is highly susceptible to the pH-dependent proteolysis mediated by cell-bound protease(s) whose activity and expression are greatly affected by the composition of the medium. Received: 23 August 1999 / Received revision: 8 November 1999 / Accepted: 12 December 1999     

Development of an arming yeast strain for efficient utilization of starch by co-display of sequential amylolytic enzymes on the cell surface

The construction of a whole-cell biocatalyst with its sequential reaction has been performed by the genetic immobilization of two amylolytic enzymes on the yeast cell surface. A recombinant strain of Saccharomyces cerevisiae that displays glucoamylase and α-amylase on its cell surface was constructed and its starch-utilizing ability was evaluated. The gene encoding Rhizopus oryzae glucoamylase, with its own secretion signal peptide, and a truncated fragment of the α-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast α factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The constructed fusion genes were introduced into the different loci of chromosomes of S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The glucoamylase and α-amylase activities were not detected in the culture medium, but in the cell pellet fraction. The transformant strain co-displaying glucoamylase and α-amylase could grow faster on starch as the sole carbon source than the transformant strain displaying only glucoamylase. Received: 16 June 1998 / Received last revision: 21 August 1998 / Accepted: 3 September 1998     

Expression of a catalytic domain of a Neocallimastix frontalis endoxylanase gene (xyn3) in Kluyveromyces lactis and Penicillium roqueforti

A cDNA fragment encoding the A catalytic domain of the Neocallimastix frontalis endoxylanase XYN3 was amplified and cloned by the polymerase chain reaction technique. The xyn3A DNA fragment was inserted between the Saccharomyces cerevisiae phosphoglycerate kinase gene promoter and terminator sequences on a multicopy episomal plasmid for Kluyveromyces lactis. The XYN3A domain was successfully expressed in K. lactis and functional endoxylanase was secreted by the yeast cells with the K. lactis killer toxin secretion signal. The XYN3A domain was also expressed in a strain of Penicillium roqueforti as a fusion protein (ShBLE::XYN3A) of the phleomycin-resistance gene product and the endoxylanase. Active endoxylanase was efficiently secreted from the fungal cells with the Trichoderma viride cellobiohydrolase (CBH1) secretion signal and processed by a related KEX2 endoprotease of the secretion pathway. Several differently glycosylated forms of the recombinant enzymes were secreted by the yeast and the filamentous fungus. Received: 10 November 1998 / Received revision: 8 March 1999 / Accepted: 14 March 1999     

Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

Active oxygen (AO) species generated in plants under stress conditions trigger degradation of Rubisco (EC 4.1.1.39). To find out whether AO species activate proteases or make the protein susceptible to proteolysis, purified and ¹⁴C-labelled Rubisco protein was incubated with stromal preparations obtained from barley (Hordeum vulgare L.) leaves. The protein was degraded into distinct fragments only after a treatment with AO. This result shows that AO-treated Rubisco has been modified to become a substrate for stromal protease(s) and dismisses the possibility of protease activation. Upon degradation, distinct fragments accumulated with time. The fragmentation pattern was indistinguishable from that obtained with intact chloroplasts subjected to oxidative conditions (cf. M. Desimone et al., 1996, Plant Physiol 111: 789–796). Degradation required ATP-hydrolysis, since AMP, ADP or non-hydrolysable ATP-analogs did not support proteolysis. The ClpP-deficient stromal preparations degraded AO-modified Rubisco, making the involvement of the ClpC/P protease unlikely. Received: 1 September 1997 / Accepted 15 November 1997     

Effect of xylitol and trehalose on dry resistance of yeasts

The effects of dehydration/rehydration on two strains of Saccharomyces cerevisiae: S600, a metabolically engineered xylose-utilising strain, and H158, the non-xylose-utilising host strain; and on the naturally xylose-utilising yeast Pachysolen tannophilus CBS 4044, were compared after glucose and xylose utilisation respectively. The yeast strains differed in their ability to excrete and accumulate intracellular xylitol. A high intracellular xylitol content before and after dehydration coincided with a higher viability after a dehydration/rehydration cycle. The intracellular trehalose content increased during dehydration in all three yeast strains, but this did not correspond to enhanced cell viability after dehydration/rehydration. The results are discussed in relation to the ability of xylitol and trehalose to structure water. Received: 9 July 1996 / Received revision: 29 October 1996 / Accepted: 2 November 1996     

Process development for high-level secretory production of carboxypeptidase Y by Saccharomyces cerevisiae

In order to develop a production process for carboxypeptidase Y (CPY, yeast vacuolar protease) secreted by Saccharomyces cerevisiae KS58-2D, medium composition, culture conditions, and expression systems were investigated. We found that the addition of histidine to thiamine-free medium, in which CPY production was almost negligible, raised the intracellular thiamine level, resulting in the increase of CPY production. On the basis of the choice of an expression system that uses an inducible GAL10 promoter, reassessment of histidine concentration in the medium, and optimization of the pH level during cultivation (pH 6.5), active CPY was secreted in a quantity of over 400 mg/l, which was more than tenfold that higher than that previously reported. The process developed could be easily scaled-up to industrial-scale fermentation. Received: 16 January 1998 / Received revision: 16 February 1998 / Accepted: 27 February 1998     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for l-lysine production in Corynebacterium glutamicum

The N-succinyl-ll-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the l-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE  ⁻ strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE  ⁻ strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions. Received: 20 May 1998 / Received revision: 12 August 1998 / Accepted: 3 September 1998     

Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp.

The marine photosynthetic bacterium Chromatium sp. successfully removed orthophosphate when grown phototrophically. The phosphate-uptake rate was almost constant at more than 5.0 mg- PO₄ ³⁻/l in synthetic medium. Addition of seawater causes flocculation of this strain. The successful use of seawater as an inexpensive source of magnesium could prove to be effective in the removal of photosynthetic bacterial cells from a medium. A semicontinuous culture system was used for the removal of low concentrations of phosphate and the phosphate-uptake activity of Chromatium sp. was maintained under 0.1 day⁻¹ dilution rate. This strain was also able to remove high concentrations of phosphate from domestic sewage. Received 24 May 1996 / Received revision: 5 August 1996 / Accepted: 6 September 1996     

Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp.

In our screening program for microorganisms that are able to metabolize eugenol, the main component of the essential oil of the clove tree Syzigium aromaticum (sy. Eugenia cariophyllus), we found a new Pseudomonas sp. that produces several substituted methoxyphenols when eugenol is fed to the culture. A taxonomic characterization of this new organism has been performed. Examples of the biotransformation products, produced in high amounts, were vanillic acid with 3.25 g/l within 99 h, ferulic acid with 5.8 g/l within 75 h and coniferyl alcohol with 3.22 g/l within 47.5 h. By changing the culture conditions the ratio of the different metabolites could be varied. Based on these results a scheme for the degradation of eugenol by this strain has been established. Received: 1 April 1996 / Received revision: 24 June 1996 / Accepted: 1 July 1996     

Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.     

Properties of a cold-active protease from psychrotrophic Flavobacterium balustinum P104

Protease activity was detected in the culture medium of Flavobacterium balustinum P104 grown at 10 °C, which was isolated from salmon (Oncorhynchus keta) intestine. The enzyme, designated as CP-70 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographyies. The molecular mass of the protease was 70 kDa, and its isoelectric point was close to 3.5. Maximal activity toward azocasein was observed at 40 °C and from pH 7.0 to 9.0. The activity was strongly inhibited by phenylmethylsulfonyl fluoride, suggesting that the enzyme is a serine protease. The n-terminal amino acid sequence was Asp-Thr-Arg-Gln-Leu-Leu-Asn-Ala-Asn-Ser-Asp-Leu-Leu-Asn-Thr-Thr-Gly-Asn-Val-Thr-Gly-Leu-Thr-Gly-Ala-Phe-Asn-Gly-Glu-Asn. A search through the database for sequence homology yielded no significant match. The initial cleavage sites for oxidized insulin B-chain were found to be the Glu13-Ala14 and Phe24-Phe25 bonds. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-70 protease has a broader specificity than the other cold-active proteases against the peptide substrate. Received: 17 April 1998 / Received last revision: 17 June 1998 / Accepted: 10 July 1998     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Production of xanthan gum and ice-nucleating material from whey by Xanthomonas campestris pv. translucens

Xanthomonas campestris pv. translucens IFO13599 could produce xanthan gum (18.5 mg/100 mg, lactose) with lactose as the growth substrate in spite of a low level of β-galactosidase. This productivity corresponded to one-fifth that with glucose. This strain could also produce ice-nucleating material having an ice-nucleating temperature, T ₅₀, of −2.8 °C with xanthan gum in the culture broth. We found that this strain produced both materials in whey medium from which the insoluble components had been removed. The production of xanthan with ice-nucleating material reached a maximum after cultivation for 168 h under optimum conditions. Furthermore, the xanthan obtained had a low viscosity because of its variant structure revealed, by TLC and HPLC analyses, to be lacking pyruvic acid. Furthermore, we concluded that this mixture had considerable potential as a regeneratic agent, when compared to other regeneratic agents such as carboxymethylcellulose. Received: 29 August 1997 / Received revision: 17 November 1997 / Accepted: 18 November 1997     

Extracellular laccase production during hyphal interactions between Trichoderma sp. and Shiitake, Lentinula edodes

Lentinula edodes (Berk.) Pegler was cultivated in liquid media containing malt and yeast extract. Extracellular laccase activity, measured in the culture fluids, was 5–18 times higher in cultures incubated for 29 days than in cultures incubated for 24 days. The addition of water-soluble lignin derivatives or Trichoderma sp. in cultures of L. edodes incubated for 11 days increased laccase activity 3- to 20 fold. The higher response was obtained with live mycelium of Trichoderma sp., but cell-free culture fluids of Trichoderma sp. in pure cultures were also effective. Trichoderma sp. induced changes in the laccase isoenzyme pattern as a result of the alteration of laccases secreted by L. edodes and not the induction of new isoforms. Received: 3 November 1997 /  Received revision: 19 January 1998 /  Accepted: 24 January 1998     

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine

A gram-negative, rod-shaped bacterium capable of utilizing l-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed l-asparaginase was detected and it deaminated l-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM l-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on l-fructose, d-galactose, saccharose, or maltose, and in cells cultured on l-asparagine as the sole nitrogen source. The pH and temperature optimum of l-asparaginase was 8.5 and 37–42 °C, respectively. The half-life of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively. Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998     

Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media

Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media. The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects. To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied. Received: 17 April 1998 / Received revision: 22 June 1998 / Accepted: 27 June 1998