Effects of cultivation conditions on the production of heterologous ?- galactosidase by Kluyveromyces lactis

Hensing, M.C.M.; Bangma, K. A.; Raamsdonk, Léonie M.; Hulster, E. de; Dijken, Johannes P. van; Pronk, J. T.

doi:10.1007/s002530050370

Cited by 8 publications

(22 citation statements)

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“…Notably, this is true for many of the acidic metabolites that are generated by carbon catabolism and that reduce the external pH, creating spatial variations in pH across plates that depend on both distance to edge, genotype, and medium thickness ( Figure S16). Note that the spatial variation in pH is exacerbated by the common use of ammonium as a nitrogen source: ammonium is basic, meaning that the initial pH is high, and the pH drops rather drastically when ammonium is consumed as a function of growth (Hensing et al 1995). The acidification in unbuffered media can reach down to pH 2.5.…”

Section: Discussionmentioning

confidence: 99%

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Zackrisson

Hallin²,

Ottosson

et al. 2015

Preprint

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The capacity to map traits over large cohorts of individuals-phenomics-lags far behind the explosive development in genomics. For microbes, the estimation of growth is the key phenotype because of its link to fitness. We introduce an automated microbial phenomics framework that delivers accurate, precise, and highly resolved growth phenotypes at an unprecedented scale. Advancements were achieved through the introduction of transmissive scanning hardware and software technology, frequent acquisition of exact colony population size measurements, extraction of population growth rates from growth curves, and removal of spatial bias by reference-surface normalization. Our prototype arrangement automatically records and analyzes close to 100,000 growth curves in parallel. We demonstrate the power of the approach by extending and nuancing the known salt-defense biology in baker's yeast. The introduced framework represents a major advance in microbial phenomics by providing high-quality data for extensive cohorts of individuals and generating well-populated and standardized phenomics databases KEYWORDS

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Section: Discussionmentioning

confidence: 99%

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Zackrisson

Hallin²,

Ottosson

et al. 2015

Preprint

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“…Exponentially growing cells were harvested, transferred into shake flasks containing various stresses, and grown for approximately 18 h. Growth conditions included synthetic medium at pH 3 (negative control) and identical media containing 0.5 mM H 2 O 2 (positive control) or 500 mM lactic acid. Urea was used as a nitrogen source to prevent further acidification of the growth medium (20). After 18 h of growth, the pH of each flask was adjusted to 5.0 with KOH-H 2 SO 4 , and 30 ml of each cell suspension was then transferred to 30 ml of potassium phosphate buffer (pH 5.0).…”

Section: Methodsmentioning

confidence: 99%

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Abbott

Suir

Duong

et al. 2009

Appl Environ Microbiol

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Industrial production of lactic acid with the current pyruvate decarboxylase-negative Saccharomyces cerevisiae strains requires aeration to allow for respiratory generation of ATP to facilitate growth and, even under nongrowing conditions, cellular maintenance. In the current study, we observed an inhibition of aerobic growth in the presence of lactic acid. Unexpectedly, the cyb2⌬ reference strain, used to avoid aerobic consumption of lactic acid, had a specific growth rate of 0.25 h ؊1 in anaerobic batch cultures containing lactic acid but only 0.16 h ؊1 in identical aerobic cultures. Measurements of aerobic cultures of S. cerevisiae showed that the addition of lactic acid to the growth medium resulted in elevated levels of reactive oxygen species (ROS). To reduce the accumulation of lactic acid-induced ROS, cytosolic catalase (CTT1) was overexpressed by replacing the native promoter with the strong constitutive TPI1 promoter. Increased activity of catalase was confirmed and later correlated with decreased levels of ROS and increased specific growth rates in the presence of high lactic acid concentrations. The increased fitness of this genetically modified strain demonstrates the successful attenuation of additional stress that is derived from aerobic metabolism and may provide the basis for enhanced (micro)aerobic production of organic acids in S. cerevisiae.Lactic acid is an organic acid with a wide range of applications. In the food industry, lactic acid has traditionally been used as an antimicrobial as well as a flavor enhancer. Besides having applications in textile, cosmetic, and pharmaceutical industries (5), lactic acid has been applied for the manufacture of lactic acid polymers (11,40). These polymers have properties that are similar to those of petroleum-derived plastics. Skyrocketing oil prices caused by dwindling fossil fuel reserves coupled with pressures to tackle environmental issues are creating increased demand for bioderived, and often biodegradable, polymers, such as poly-lactic acid.Current industrial lactic acid fermentations are based on different species of lactic acid bacteria. These bacteria have complex nutrient requirements due to their limited ability to synthesize B vitamins and amino acids (8) and are intolerant to acidic conditions with a pH between 5.5 and 6.5 required for growth (40). Acidification of the growth medium during lactic acid fermentation is typically counteracted by the addition of neutralizing agents (e.g., CaCO 3 ), resulting in the formation of large quantities of insoluble salts, such as gypsum, during downstream processing.Saccharomyces cerevisiae has received attention as a possible alternative biocatalyst. This organism is relatively tolerant to low pH and has simple nutrient requirements. The production of lactic acid with metabolically engineered S. cerevisiae was achieved by introducing a NAD ϩ -dependent lactate dehydrogenase, leading to the simultaneous formation of both ethanol and lactate (1a, 12, 31, 32, 36). Further improvements were made by constr...

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“…In contrast to the aft1⌬ strain, the reference strain (CEN.PK 113-7D) did not exhibit a growth deficiency in the presence of lactic acid with the standard concentration of iron (Ⅺ). The pH of all shake flasks was set to 5, and urea was utilized as the nitrogen source to prevent acidification of the growth medium (21).…”

Section: Isu1mentioning

confidence: 99%

Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae

Abbott

Suir

Maris

et al. 2008

Appl Environ Microbiol

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Based on the high acid tolerance and the simple nutritional requirements of Saccharomyces cerevisiae, engineered strains of this yeast are considered biocatalysts for industrial production of high-purity undissociated lactic acid. However, high concentrations of lactic acid are toxic to S. cerevisiae, thus limiting its growth and product formation. Physiological and transcriptional responses to high concentrations of lactic acid were studied in anaerobic, glucose-limited chemostat cultures grown at different pH values and lactic acid concentrations, resulting in a 50% decrease in the biomass yield. At pH 5, the yield decrease was caused mostly by osmotically induced glycerol production and not by the classic weak-acid action, as was observed at pH 3. Cultures grown at pH 5 with 900 mM lactic acid revealed an upregulation of many genes involved in iron homeostasis, indicating that iron chelation occurred at high concentrations of dissociated lactic acid. Chemostat cultivation at pH 3 with 500 mM lactate, resulting in lower anion concentrations, showed an alleviation of this iron homeostasis response. Six of the 10 known targets of the transcriptional regulator Haa1p were strongly upregulated in lactate-challenged cultures at pH 3 but showed only moderate induction by high lactate concentrations at pH 5. Moreover, the haa1⌬ mutant exhibited a growth defect at high lactic acid concentrations at pH 3. These results indicate that iron homeostasis plays a major role in the response of S. cerevisiae to high lactate concentrations, whereas the Haa1p regulon is involved primarily in the response to high concentrations of undissociated lactic acid.

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Effects of cultivation conditions on the production of heterologous ?- galactosidase by Kluyveromyces lactis

Cited by 8 publications

References 0 publications

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Scan-o-matic: high-resolution microbial phenomics at a massive scale

Catalase Overexpression Reduces Lactic Acid-Induced Oxidative Stress in Saccharomyces cerevisiae

Physiological and Transcriptional Responses to High Concentrations of Lactic Acid in Anaerobic Chemostat Cultures of Saccharomyces cerevisiae

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