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     

Chemostat study of xylitol production by Candida guilliermondii

The mechanism of production of xylitol from xylose by Candida guilliermondii was studied using chemostat cultures and enzymatic assays. The maximum dilution rate in aerobic conditions was 0.34 1/h. No xylitol was produced. Under oxygen-limited conditions xylose uptake was impaired and glycerol accumulated but no xylitol was detected. Under transient oxygen limitation, caused by a gradual decrease in the agitation rate, onset of xylitol, acetate and residual xylose accumulation occurred simultaneously when q _O2 dropped below 25 mmol/C-mmol cell dry weight (CDW) per hour. Ethanol and glycerol started to accumulate when q _O2 dropped below 20 mmol/C-mmol CDW per hour. The highest in vitro enzyme activities were found at the lowest dilution rate studied (0.091/h) under aerobic conditions. The amount of active enzymes or cofactor availability did not limit the rate of xylose consumption. Our results confirm that a surplus of NADH during transient oxygen limitation inhibited the activity of xylitol dehydrogenase which resulted in xylitol accumulation. Phosphoglucoisomerase (E.C. 5.3.1.9.) and glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) activities suggest re-shuttling of the metabolites into the pentose phosphate pathway. Received: 7 March 2000 / Received revision: 9 June 2000 / Accepted: 18 June 2000     

Cloning and expression of Candida guilliermondii xylose reductase gene (xyl1) in Pichia pastoris

A xylose reductase gene (xyl1) of Candida guilliermondii ATCC 20118 was cloned and characterized. The open reading frame of xyl1 contained 954 nucleotides encoding a protein of 317 amino acids with a predicted molecular mass of 36 kDa. The derived amino acid sequence of C. guilliermondii xylose reductase was 70.4% homologous to that of Pichia stipitis. The gene was placed under the control of an alcohol oxidase promoter (AOX1) and integrated into the genome of a methylotrophic yeast, Pichia pastoris. Methanol induced the expression of the 36-kDa xylose reductase in both intracellular and secreted expression systems. The expressed enzyme preferentially utilized NADPH as a cofactor and was functional both in vitro and in vivo. The different cofactor specificity between P. pastoris and C. guilliermondii xylose reductases might be due to the difference in the numbers of histidine residues and their locations between the two proteins. The recombinant was able to ferment xylose, and the maximum xylitol accumulation (7.8 g/l) was observed when the organism was grown under aerobic conditions. Received: 26 August 1997 / Received revision: 6 November 1997 / Accepted: 21 November 1997     

Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii

The bioconversion of xylose to xylitol by Candida guilliermondii FTI 20037 cultivated in sugar cane bagasse hemicellulosic hydrolyzate was influenced by cell inoculum level, age of inoculum and hydrolyzate concentration. The maximum xylitol productivity (0.75 g L⁻¹ h⁻¹) occurred in tests carried out with hydrolyzate containing 54.5 g L⁻¹ of xylose, using 3.0 g L⁻¹ of a 24-h-old inoculum. Xylitol productivity and cell concentration decreased with hydrolyzate containing 74.2 g L⁻¹ of xylose. Received 02 February 1996/ Accepted in revised form 15 November 1996     

Expression of different levels of enzymes from the Pichia stipitis XYL1 and XYL2 genes in Saccharomyces cerevisiae and its effects on product formation during xylose utilisation

Saccharomyces cerevisiae was transformed with the Pichia stipitis CBS 6054 XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) respectively. The XYL1 and XYL2 genes were placed under the control of the alcohol dehydrogenase 1 (ADH1) and phosphoglycerate kinase (PGK1) promoters in the yeast vector YEp24. Different vector constructions were made resulting in different specific activities of XR and XDH. The XR:XDH ratio (ratio of specific enzyme activities) of the transformed S. cerevisiae strains varied from 17.5 to 0.06. In order to enhance xylose utilisation in the XYL1-, XYL2-containing S. cerevisiae strains, the native genes encoding transketolase and transaldolase were also overexpressed. A strain with an XR:XDH ratio of 17.5 formed 0.82 g xylitol/g consumed xylose, whereas a strain with an XR:XDH ratio of 5.0 formed 0.58 g xylitol/g xylose. The strain with an XR:XDH ratio of 0.06, on the other hand, formed no xylitol and less glycerol and acetic acid compared with strains with the higher XR:XDH ratios. In addition, the strain with an XR:XDH ratio of 0.06 produced more ethanol than the other strains. Received: 12 March 1997 / Received revision: 17 April 1997 / Accepted: 27 April 1997     

Xylitol production by recombinant Saccharomyces cerevisiae expressing the Pichia stipitis and Candida shehatae XYL1 genes

The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate. Received: 23 December 1999 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998     

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [¹⁴C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42 % and 36 % respectively of the initial added [¹⁴C]TNT (100 μM corresponding to 4.75 μCi/l) to ¹⁴CO₂ within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [¹⁴C]TNT significantly. Received: 8 August 1996 / Received revision: 16 December 1996 / Accepted: 20 December 1996     

Improvement of tempe fermentations by application of mixed cultures consisting of Rhizopus sp. and bacterial strains

Tempe fermentations using mixed cultures of Rhizopus oligosporus MS5, R. oryzae EN, Citrobacter freundii, and Brevibacterium epidermidis were investigated. Consumption of 150 g tempe, produced with a pure fungal mixed culture out of strains MS5 and EN, is sufficient to cover the daily requirements of niacin, vitamin K, ergosterol, and tocopherol as well as half of the daily requirement of pyridoxine, riboflavin, and biotin. Moreover, one-fourth of the recommended amount of folate is supplied. Supplementation of the fungal inoculum with C. freundii results in tempe enriched with vitamin B₁₂. Menachinone was produced as a typical bacterial vitamin K derivative. Metabolic activity of C.␣freundii led to an additional decrease of the α-galactosides stachyose and raffinose compared to pure fungal fermentations. No bacterial formation of factor 2 could be observed. Received: 31 July 1996 / Received revision: 4 October 1996 / Accepted: 14 October 1996     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

Production of synthetic spider dragline silk protein in Pichia pastoris

The methylotrophic yeast Pichia pastoris was tested as a host for the production of long, repetitive protein polymers. Synthetic genes for a designed analog of a spider dragline silk protein were readily expressed at high levels under control of the methanol-inducible AOX1 promoter. Transformants containing multiple gene copies produced elevated levels of silk protein, but of a variety of altered sizes as a result of gene rearrangements at the time of transformation. Genes up to 3000 codons in length or longer could be expressed with no evidence of the prevalent truncated synthesis observed for similar genes in Escherichia coli, though genes longer than 1600 codons were expressed less efficiently than shorter genes. Silk-producing P. pastoris strains were stable without selection for at least 100 doublings. Received: 4 March 1996 / Received revision: 26 June 1996 / Accepted: 12 August 1996     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Biosynthesis and chemical reactions of poly(amino acid)s from microorganisms

The biosynthesis and chemical reactions of poly(amino acid)s produced by microorganisms are reviewed. A large amount of γ-poly(glutamic acid) (PGA) has been produced by Bacillus strains. ε-Polylysine (PL) has been produced by Streptomyces albulus. As a modification of PGA and PL, pH-sensitive hydrogels have been prepared by means of γ irradiation or the addition of a crosslinking agent to an aqueous solution of PGA and PL. Received: 4 September 1996 / Received revision: 27 January 1997 / Accepted: 28 January 1997     

An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose

The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l⁻¹ cellobiose to 3-day-old maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l⁻¹ cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l⁻¹ and 560 mg l⁻¹ vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation. Received: 24 July 1996 / Received revision: 29 November 1996 / Accepted: 29 November 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     

Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

Interspecific protoplast fusion between␣Aspergillus terreus, an itaconic acid producer, and A.␣usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10⁻⁵−10⁻⁴. The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70 % of that of A. terreus from glucose (120 mg/ml). Received: 28 June 1996 / Received revision: 3 September 1996 / Accepted: 29 September 1996     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Xylose utilisation by recombinant strains of Saccharomyces cerevisiae on different carbon sources

Autoselective xylose-utilising strains of Saccharomyces cerevisiae expressing the xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes of Pichia stipitis were constructed by replacing the chromosomal FUR1 gene with a disrupted fur1::LEU2 allele. Anaerobic fermentations with 80 g l⁻¹ d-xylose as substrate showed a twofold higher consumption of xylose in complex medium compared to defined medium. The xylose consumption rate increased a further threefold when 20 g l⁻¹ d-glucose or raffinose was used as co-substrate together with 50 g l⁻¹ d-xylose. Xylose consumption was higher with raffinose as co-substrate than with glucose (85% versus 71%, respectively) after 82 h fermentations. A high initial ethanol concentration and moderate levels of glycerol and acetic acid accompanied glucose as co-substrate, whereas the ethanol concentration gradually increased with raffinose as co-substrate with no glycerol and much less acetic acid formation. Received: 12 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996