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     

Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins

A Pichia pastoris strain with stereoselective nitrile hydratase activity has been constructed by engineering the co-expression of three genes derived from Pseudomonas putida. Using a technique that could be widely applicable, the genes encoding nitrile hydratase α and β structural subunits and P14K accessory protein were first assembled as individual expression cassettes and then incorporated onto one plasmid, which was integrated into the P. pastoris chromosome. The resulting strain can be used as a catalyst for bioconversions requiring stereospecific nitrile hydrolysis. Received: 3 November 1998 / Received revision: 25 February1999 / Accepted: 14 March 1999     

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     

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 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     

Construction of a recombinant yeast strain converting xylose and glucose to ethanol

Candida shehatae gene xyl1 and Pichia stipitis gene xyl2, encoding xylose reductase (XR) and xylitol dehydrogenase (XD) respectively, were amplified by PCR. The genes xyl1 and xyl2 were placed under the control of promoter GAL in vector pYES2 to construct the recombinant expression vector pYES2-P12. Subsequently the vector pYES2-P12 was transformed into S. cerevisiae YS58 by LiAc to produce the recombinant yeast YS58-12. The alcoholic ferment indicated that the recombinant yeast YS58-12 could convert xylose to ethanol with the xylose consumption rate of 81.3%. __________ Translated from Microbiology, 2006, 33(3): 104–108 [译自:微生物学通报]     

Biosynthetic production of type II fish antifreeze protein: fermentation by Pichia pastoris

Sea raven type II antifreeze protein (SRAFP) is one of three different fish antifreeze proteins isolated to date. These proteins are known to bind to the surface of ice and inhibit its growth. To solve the three-dimensional structure of SRAFP, study its ice-binding mechanism, and as a basis for engineering these molecules, an efficient system for its biosynthetic production was developed. Several different expression systems have been tested including baculovirus, Escherichia coli and yeast. The latter, using the methylotrophic organism Pichia pastoris as the host, was the most productive. In shake-flask cultures the levels of SRAFP secreted from Pichia were up to 5 mg/l. The recombinant protein has an identical activity to SRAFP from sea raven serum. In order to increase yields further, four different strategies were tested in 10-l fermentation vessels, including: (1) optimization of pH and dissolved oxygen, (2) mixed feeding of methanol and glycerol with Mut^s clones, (3) supplementation of amino acid building blocks, and (4) methanol feeding with Mut⁺ clones. The mixed-feeding/Mut^s strategy proved to be the most efficient with SRAFP yields reaching 30 mg/l. Received: 19 November 1996 / Received revision: 29 January 1997 / Accepted: 7 March 1997     

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     

Heterologous protein production in methylotrophic yeasts

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past. Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 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     

Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase leads to squalene accumulation in yeast

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate-limiting enzyme in early sterol biosynthesis in eukaryotic cells. To eliminate this regulation in the yeast Saccharomyces cerevisiae, a truncated HMG1 gene, producing a form of the enzyme that lacks the membrane-binding region (i.e. amino acids 1–552), was constructed and overexpressed in this yeast. The transformed strains accumulated large amounts of the sterol precursor squalene, while the levels of ergosterol and a number of other sterol compounds were only slightly elevated. These findings suggest that HMG-CoA reductase is not the only rate-limiting step in sterol synthesis and its overexpression cannot significantly influence this pathway beyond the sterol precursor squalene. Received: 9 June 1997 / Received revision: 1 September 1997 / Accepted: 19 September 1997     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

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     

Cloning and characterization of the inulinase gene from a marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.     

Effect of glucose on xylose utilization in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> harboring the xylose reductase gene

We have constructed recombinant Saccharomyces cerevisiae JH1 harboring a xylose reductase gene (xyl1) isolated from Pichia stipitis. However, JH1 still utilizes glucose more easily than xylose. Therefore, in this study, we characterized the effect of a glucose supplement on xylose utilization, the expression level of xylose reductase as a recombinant gene in JH1, and the expression levels of two hexose transporters (Hxt4 and Hxt7) due to co-fermentation of different concentrations of glucose and xylose. Co-fermentation using 20 g/l of glucose increased xylose consumption up to 11.7 g/l, which was 7.9-fold that of xylose fermentation without a glucose supplement. In addition, we found xyl1 mRNA levels dramatically increased as cells grew under co-fermentation conditions with supplementary glucose; this result is consistent with a significant decrease in the xylose concentration 48 h after cultivation. In addition, the expression levels of Hxt4 and Hxt7 were strongly activated by the presence of glucose and xylose; in particular, Hxt7 showed a 2.9-fold increased expression relative to that of recombinant S. cerevisiae JHM with only a backbone vector, pYES2. The results of this study suggest that xylose utilization would be improved by activation of hexose transporters induced by glucose (rather than xylose) reductase expression.     

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica. Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997     

Cloning, expression in Streptomyces lividans and biochemical characterization of a thermostable endo-β-1,4-xylanase of Thermomonospora alba UL JB1 with cellulose-binding ability

Several thermophilic actinomycetes were isolated from urban solid waste. One of them, Thermomonospora alba ULJB1, showed a broad degradative activity on xylan, cellulose, starch and other polymers. Xylanase and cellulase activities were quantified and compared with those of Thermomonospora fusca. Genes encoding two different endo-β-1,4-xylanases were cloned from T.␣alba ULJB1. One of them, xylA, was sequenced, subcloned and overexpressed in Streptomyces lividans. It encodes a protein of 482 amino acids with a deduced molecular mass of 48 456 Da. The protein contains a 38-amino-acid leader peptide with six Arg⁺ residues in its amino-terminal end, a catalytic domain and a cellulose-binding domain connected by a linker region rich in proline and glycine. The XylA protein was purified to near homogeneity from S. lividans/xylA cultures. Two forms of the extracellular xylanase, of 48 kDa and 38 kDa, were produced that differed in their cellulose-binding ability. The 48-kDa protein showed a strong binding to cellulose whereas the 38-kDa form did not bind to this polymer, apparently because of the removal during processing of the cellulose-binding domain. Both forms were able to degrade xylans form different origins but not lichenam or carboxymethylcellulose. The major degradation product was xylobiose with traces of xylose. The xylanase activity was thermostable, showing a good activity up to 95 °C, and had broad pH stability in the range from pH 4.0 to pH 10.0. Received: 9 January 1997 / Received revision: 27 March 1997 / Accepted: 13 April 1997     

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     

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