Anaerobic and aerobic continuous cultures of Saccharomyces cerevisiae : comparison of plasmid stability and EXG1 gene expression

Lú-Chau, Thelmo A.; Guillán, A.; Núñez, Marı́a José; Roca, Enrique; Lema, Juan M.

doi:10.1007/s00449-003-0344-y

Cited by 6 publications

(4 citation statements)

References 24 publications

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“…Continuous biofilm cultures for HPP have advantages with respect to chemostats for retention of plasmid-bearing cells. Significant loss of plasmid from cells cultured in the chemostat occurred, similar to the findings of other investigators (Lu-Chau et al, 2004;Ryan and Parulekar, 1991;Yazdani and Mukherjee, 2002). In contrast, highdensity fed-batch cultures have been effectively managed for HPP with minimal loss of plasmid (Hu et al, 2003;Lau et al, 2004).…”

Section: Discussionsupporting

confidence: 86%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.

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Section: Discussionsupporting

confidence: 86%

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

O’Connell

Niu

Gilbert

2006

Biotech & Bioengineering

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“…In fact, genetic instability is a major problem of prolonged continuous fermentations with recombinant microorganisms. For continuous cultures, further stability improvements may be achieved by changing culture conditions and operating strategies as described by others in regard to plasmid instability in S. cerevisiae continuous cultures (29).…”

Section: Discussionmentioning

confidence: 99%

Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger β-galactosidase production

Oliveira

Teixeira

Lima

et al. 2007

Journal of Bioscience and Bioengineering

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A flocculent Saccharomyces cerevisiae strain was engineered to stably secrete Aspergillus niger beta-galactosidase in a continuous high-cell-density bioreactor. The delta-sequences from the yeast retrotransposon Ty1 were used as target sites for the integration of the beta-galactosidase expression cassette. High-copy-number transformants were successfully obtained using the delta-integration system together with the dominant selection antibiotic, G418. The integration of multiple copies was confirmed by genomic Southern blot analysis. Integrants with the highest beta-galactosidase levels (approximately eight gene copies) had similar beta-galactosidase activities as a recombinant strain carrying the beta-galactosidase expression cassette in a YEp-based vector. The beta-galactosidase expression cassettes integrated into the yeast genome were stably maintained after eight sequential batch cultures in a nonselective medium. In continuous high-cell-density culture under the same operating conditions, the integrant strain was more stable than the plasmid-carrying strain. To our knowledge, this is the first study of multicopy delta-integrant stability in a continuous bioreactor operating at different dilution rates.

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“…The EXG1 gene shows high expression under its native promoter during exponential growth, but is repressed in low hypoxic conditions that occur during fermentation (Lu-Chau et al. 2004). Overexpression of the EXG1 gene in a Saccharomyces wine yeast strain under the constitutive ACT1 promoter showed 20- to 40-fold higher EXG activity and enabled release of significantly higher levels of terpenoids than with a control strain from glycoside-rich grapevine extract in Muscat wine fermentations (Gil et al.…”

Section: Biosynthesis and Occurrence Of Fruity And Floral Aroma Compomentioning

confidence: 99%

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages

Holt

Mikš

Carvalho

et al. 2018

FEMS Microbiology Reviews

187

125

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Aroma compounds provide attractiveness and variety to alcoholic beverages. We discuss the molecular biology of a major subset of beer aroma volatiles, fruity and floral compounds, originating from raw materials (malt and hops), or formed by yeast during fermentation. We introduce aroma perception, describe the most aroma-active, fruity and floral compounds in fruits and their presence and origin in beer. They are classified into categories based on their functional groups and biosynthesis pathways: (1) higher alcohols and esters, (2) polyfunctional thiols, (3) lactones and furanones, and (4) terpenoids. Yeast and hops are the main sources of fruity and flowery aroma compounds in beer. For yeast, the focus is on higher alcohols and esters, and particularly the complex regulation of the alcohol acetyl transferase ATF1 gene. We discuss the release of polyfunctional thiols and monoterpenoids from cysteine- and glutathione-S-conjugated compounds and glucosides, respectively, the primary biological functions of the yeast enzymes involved, their mode of action and mechanisms of regulation that control aroma compound production. Furthermore, we discuss biochemistry and genetics of terpenoid production and formation of non-volatile precursors in Humulus lupulus (hops). Insight in these pathways provides a toolbox for creating innovative products with a diversity of pleasant aromas.

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Anaerobic and aerobic continuous cultures of Saccharomyces cerevisiae : comparison of plasmid stability and EXG1 gene expression

Cited by 6 publications

References 24 publications

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger β-galactosidase production

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages

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