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  • 1
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A two-stage, cyclic fed-batch bioprocess was designed, and its performance evaluated to improve rice α-amylase productivity by the yeast Yarrowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containing a replicative plasmid coding for a rice α-amlyase. Transcription of the recombinant gene is controlled by the XPR2 promoter. The first stage (or growth stage) was operated in the fed-batch mode, and the growth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optimal feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h−1). Typically, when the volume in the first stage reached a preset value, a portion of culture broth (i.e., 55%) was transferred to the second stage (or production stage). The remaining cells in the growth stage were then fed with fresh growth medium according to the bioprocess control strategy developed, while induction of α-amylase expression and its production was taking place in the second stage. The second stage was also operated in the fed-batch mode, and the production medium designed to maintain a constant high cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the specific cell growth rate at an optimal level. The volumetric α-amylase productivity achieved (1835 units l−1 h−1) from the two-stage, cyclic fed-batch culture process was twofold higher than that of the fed-batch culture process. The genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein.
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  • 2
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The dimorphic yeast Yarrowia lipolytica forms true hyphae in a medium containing N-acetylglucosamine. We made a new finding that serum is a very effective inducer of hypha formation of Y. lipolytica: serum induced its hyphal growth very quickly compared to N-acetylglucosamine (4 h vs. 10 h). Osmotic and oxidative stresses (0.2 M NaCl and 20 mM H2O2) inhibited the hypha formation induced by N-acetylglucosamine, but did not suppress the hypha formation triggered by serum. Serum-specific morphological mutants, which formed hyphae in the N-acetylglucosamine medium but not in serum medium, could be isolated. These results suggest that the signal triggered by serum may be transduced through a different pathway, at least in part, from that used for the N-acetylglucosamine signal in Y. lipolytica.
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The pmrA gene, a yeast PMR1 homologue, was isolated from Aspergillus niger. Sequence analysis of the pmrA cDNA and the genomic DNA revealed that two introns exist in the coding region, and that an open reading frame in the cDNA encodes a polypeptide of 1056 amino acids containing all the conserved regions present in P-type Ca2+-ATPases. The predicted pmrA protein exhibited a high degree of sequence similarity to the Pmr1 proteins from yeasts and mammalians (50–59% identity). The expression of the pmrA cDNA partially restored the growth defect of Yarrowia lipolytica pmr1 null mutant on EGTA-containing medium. This indicates that the A. niger pmrA gene encodes a functional homologue of the yeast P-type Ca2+-ATPase involved in the secretory pathway. An A. niger pmrA null mutant exhibited growth retardation on EGTA-containing medium and the growth defect was overcome by adding Ca2+ or Mn2+ into the medium. This suggests an involvement of the pmrA protein in Ca2+ and Mn2+ homeostasis in A. niger.
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  • 4
    ISSN: 0749-503X
    Keywords: yeast ; PMR1 ; Hansenula polymorpha ; Ca2+-ATPase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Hansenula polymorpha. The partial DNA fragment of the H. polymorpha homologue was initially obtained by a polymerase chain reaction and used to isolate the entire gene which encodes a protein of 918 amino acids. The putative gene product contains all ten of the conserved regions observed in P-type ATPases. The cloned gene product exhibits 60·3% amino acid identity to the S. cerevisiae PMR1 gene product and complemented the growth defect of a S. cerevisiae pmr1 null mutant in the EGTA-containing medium. The results demonstrate that the H. polymorpha gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+-ATPase. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession Number U92083. © 1998 John Wiley & Sons, Ltd.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1520-6033
    Source: ACS Legacy Archives
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As part of the process optimization of a two-stage continuous culture system, the effect of growth rate μ2app on the performance of the second stage (production stage) was studied in a recombinant Escherichia coli K12 (ΔH1Δtrp/pPLc23trpA1). Important parameters considered were specific gene expression rate, plasmid content, and plasmid stability, all of which were closely related to the cell growth rate and the production rate of the cloned gene product (trpα). When operating conditions were maintained constant (T1 = 35°C, D1 = 0.9 h-1, T2 = 40°C, and D2 = 0.7 h-1) and μ2app was varied, plasmid content in the second stage showed its maximum at μ2app = 0.4 h-1 and decreased thereafter. Specific gene expression rate linearly increased with increasing μ2app, while plasmid stability decreased. Optimum cell growth rate giving the maximum value in overall productivity was observed at around μ2app = 0.4 h-1. The contribution or role of the three parameters, specific gene expression rate, plasmid content, and plasmid stability in exhibiting the maximum productivity at the optimal μ2app is discussed.
    Additional Material: 18 Ill.
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  • 7
    ISSN: 0006-3592
    Keywords: PL promoter ; plasmid content ; 45S RNA ; gene expression ; continuous culture ; two-stage ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc-RP4.5) was constructed. The vector has PL promoter and lacZ as a structure gene 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two-stage continuous culture system in combination with a temperature-sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The result showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop-) and a strong promoter (PL) were used to express a heterologous protein in E. coli.
    Additional Material: 10 Ill.
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  • 8
    ISSN: 1573-6776
    Keywords: polyhydroxyalkanote ; poly(3HB-co-3HV) ; phaA ; phaB ; phaC ; Alcaligenes sp. SH-69
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Polyhydroxyalkanoate (PHA) biosynthesis genes were cloned and characterized from Alcaligenes sp. SH-69 which can synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a single carbon source. The DNA sequence analysis revealed two consecutive genes coding for PHA synthase and β-ketothiolase and the gene coding for acetoacetyl-CoA reductase located about 2-kbp downstream of the two genes. Recombinant Escherichia coli strains with the cloned PHA biosynthesis genes synthesized poly(3-hydroxybutyrate) in Luria-Bertani medium containing 2% glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in M9 minimal medium supplemented with 1% glucose, 1 mM valine, and 2 mM threonine, which demonstrates that the PHA biosynthesis genes of Alcaligenes sp. SH-69 are functional in E. coli. © Rapid Science Ltd. 1998
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  • 9
    ISSN: 0006-3592
    Keywords: recombinant Yarrowia lipolytica ; heterologous protein expression ; improvement of rice α-amylase productivity ; cyclic fed-batch culture ; bioprocess control strategy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A cyclic fed-batch bioprocess is designed and a significant improvement of rice α-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high α-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1,960 units/L · h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L · h). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:379-385, 1998.
    Additional Material: 4 Ill.
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