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     

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     

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     

Xylitol and riboflavin accumulation in xylose-grown cultures of Pichia guilliermondii

Seven strains of Pichia guilliermondii (Candida guilliermondii, asexual state) from diverse isolation sources were examined for the production of xylitol and riboflavin in xylose-grown cultures. Under the conditions tested, all strains produced xylitol from xylose; conversion efficiencies varied, on a strain-specific basis, from 7% to 36% of the initial substrate. Four of seven strains metabolized xylitol immediately as xylose levels became depleted. The remaining three strains metabolized xylitol slowly and incompletely. Surprisingly, utilization of xylitol showed an apparent relationship with riboflavin production. Strains that readily metabolized xylitol produced at least threefold greater levels of riboflavin than did strains that used xylitol slowly. Moreover, riboflavin accumulation took place during xylitol consumption. P. guilliermondii strains that produced the highest levels of riboflavin on xylose produced significantly less riboflavin when grown on glucose or directly on xylitol. Received: 24 April 1996 / Received revision: 29 July 1996 / Accepted: 24 August 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     

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     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae

Respiratory and fermentative pathways co-exist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiaeURA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote. Received: 6 January 1998 / Received revision: 9 April 1998 / Accepted: 19 April 1998     

The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension

Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential. Received: 5 May 1999 / Received revision: 31 May 2000 / Accepted: 3 June 2000     

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 × 10² transformants/μg DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 μF, and 600 Ω. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10¹ transformants/μg DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained. Received: 17 March 1998 / Received revision: 17 August 1998 / Accepted: 26 August 1998     

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 7.5 g l⁻¹ and 0.36 g (g xylose consumed)⁻¹. These parameters improved to 13.4 g l⁻¹ and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l⁻¹ glucose and 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 29.8 g l⁻¹ and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l⁻¹ and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield. Received: 14 May 1999 / Received revision: 27 September 1999 / Accepted: 2 October 1999     

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg l⁻¹ of the fusion protein (420 mg l⁻¹ of the recombinant hCT precursor) within 14 h, reaching 45 g l⁻¹ cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor l⁻¹ h⁻¹) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-l scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g⁻¹ yeast extract). Received: 18 January 2000 / Received revision: 14 April 2000 / Accepted: 14 April 2000     

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the l-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more l-glutamate and l-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM l-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM l-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.     

Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production

Genetically engineered Escherichia coli KO11 is capable of efficiently producing ethanol from all sugar constituents of lignocellulose but lacks the high ethanol tolerance of yeasts currently used for commercial starch-based ethanol processes. Using an enrichment method which selects alternatively for ethanol tolerance during growth in broth and for ethanol production on solid medium, mutants of KO11 with increased ethanol tolerance were isolated which can produce more than 60 g ethanol L⁻¹ from xylose in 72 h. Ethanol concentrations and yields achieved by the LY01 mutant with xylose exceed those reported for recombinant strains of Saccharomyces and Zymomonas mobilis, both of which have a high native ethanol tolerance. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Efficient secretory production of alkaline phosphatase by high cell density culture of recombinant Escherichia coli using the Bacillus sp. endoxylanase signal sequence

New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG, most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90% of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins in E. coli by high cell density cultivation. Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000     

Cryopreservation of Doritaenopsis suspension culture by vitrification

 Cells of a suspension culture of Doritaenopsis cv. New Toyohashi were placed in a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at room temperature and then dehydrated with a vitrification solution (PVS2) for 1–3 h on ice and plunged into liquid nitrogen. The highest viability (64% by 2,3,5-triphenyltetrazolium chloride stainability) was obtained when the cells were precultured in liquid New Dogashima medium with 0.1 M sucrose and 1.0 mg/l abscisic acid for 1 week at 25  °C in the light. Dehydration by PVS2 was important for the cryopreservation of Doritaenopsis cells. Protocorm-like bodies were induced from cryopreserved cells without morphological variations. Received: 18 January 2000 / Revision received: 16 June 2000 / Accepted: 22 June 2000     

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5–7 mg/l. This recombinant thaumatin is sweet. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

Sequential administration of interferon γ and interleukin-2 in metastatic renal cell carcinoma: results of a phase II trial

Background: Because of the known efficacy of several cytokines in the treatment of advanced renal cell cancer (RCC), we have conducted a phase II trial of the efficacy and toxicity of subcutaneous interferon γ (IFNγ) and interleukin-2 (IL-2). Methods: 63 patients with progressive metastatic RCC were treated with 100 μg recombinant IFNγ1b administered three times weekly during weeks 1 and 2 and with 4.5 MU recombinant IL-2 administered on 4 consecutive days during weeks 3 and 4, every 6 weeks. Results: 11% of patients had an objective response (CR: 3%, PR: 8%), 33% had SD. Toxicity was generally mild. The median duration of remissions (CR + PR) was 9.6 months; the median duration of SD 8 months. A significant survival benefit was evident at a median observation time of 51 months for patients (44%) responding to therapy (P < 0.0001). Conclusions: we conclude that sequential treatment with IFNγ and IL-2 may prolong survival in patients with metastatic RCC responding to therapy. Received: 2 April 2000 / Accepted: 21 April 2000     

Genetic reidentification of the pectinolytic yeast strain SCPP as Saccharomyces bayanus var. uvarum

Using genetic hybridization analysis, pulsed-field gel electrophoresis of chromosomal DNA and PCR/RFLP analysis of the MET2 gene, we reidentified 11 Champagne yeast strains. Two of them, SCPP and SC4, were found to belong to Saccharomyces bayanus var. uvarum and the remaining strains to S. cerevisiae. Strain SCPP (CLIB 2025) of S. bayanus var. uvarum is known as a producer of three pectinolytic enzymes. Received: 28 April 2000 / Received revision: 20 July 2000 / Accepted: 25 July 2000     

Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids)

A gene transfer system for Rhodococcus opacus PD630 based on electroporation was established and optimized employing the Escherichia coli-Rhodococcus shuttle vectors pNC9501 and pNC9503 as well as the E. coli-Corynebacterium glutamicum shuttle vector pJC1 as suitable cloning vectors for R. opacus PD630, resulting in transformation efficiencies up to 1.5 × 10⁵ CFUs/μg plasmid DNA. Applying the optimized electroporation protocol to the pNC9501-derivatives pAK68 and pAK71 harboring the entire PHB synthesis operon from Ralstonia eutropha and the PHA synthase gene phaC1 from Pseudomonas aeruginosa, respectively, recombinant PHA biosynthesis was established in R. opacus PD630 and the TAG-negative mutant ROM34. Plasmid pAK68 enabled synthesis and accumulation of poly(3HB) in R. opacus PD630 and ROM34 during cultivation under storage conditions from 1% (w/v) gluconate, of poly(3HB-co-3HV) from 0.2% (w/v) propionate and of poly(3HV) from 0.1% (w/v) valerate. Under storage conditions, recombinant strains of PD630 and ROM34 harboring pAK71 were able to synthesize and accumulate PHA of the medium chain length hydroxyalkanoic acids 3HHx, 3HO, 3HD and 3HDD from 0.1% (w/v) hexadecane or octadecane and a copolyester composed of 3HHp, 3HN and 3HUD from 0.1% (w/v) pentadecane or heptadecane. In the recombinant strains of PD630 and ROM34, the thiostrepton-induced overexpression of a 20 kDa protein was observed with its N-terminus exhibiting a homology of 60% identical amino acids to TipA from Streptomyces lividans. Received: 13 March 1999 / Received revision: 18 May 1999 / Accepted: 21 May 1999