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

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

doi:10.1007/bf00170623

Cited by 25 publications

(15 citation statements)

References 25 publications

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“…1 g glucosamine contains 78 mg nitrogen. Based on the biomass composition of yeast 27 , 28 a yeast molar biomass formula can be written as C 3.8 N 0.61 O 2.1 H 7 29 , therefore 190 mg dry yeast contain only 17 mg of nitrogen. This shows that the yeast growing in glucosamine has taken in an excess of nitrogen that needs to be discarded.…”

Section: Resultsmentioning

confidence: 99%

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source

Flores

Gancedo

2018

Sci Rep

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Saccharomyces cerevisiae can transport and phosphorylate glucosamine, but cannot grow on this amino sugar. While an enzyme catalyzing the reaction from glucosamine-6-phosphate to fructose-6-phosphate, necessary for glucosamine catabolism, is present in yeasts using N-acetylglucosamine as carbon source, a sequence homology search suggested that such an enzyme is absent from Saccharomyces cerevisiae. The gene YlNAG1 encoding glucosamine-6-phosphate deaminase from Yarrowia lipolytica was introduced into S. cerevisiae and growth in glucosamine tested. The constructed strain grew in glucosamine as only carbon and nitrogen source. Growth on the amino sugar required respiration and caused an important ammonium excretion. Strains overexpressing YlNAG1 and one of the S. cerevisiae glucose transporters HXT1, 2, 3, 4, 6 or 7 grew in glucosamine. The amino sugar caused catabolite repression of different enzymes to a lower extent than that produced by glucose. The availability of a strain of S. cerevisiae able to grow on glucosamine opens new possibilities to investigate or manipulate pathways related with glucosamine metabolism in a well-studied organism.

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Section: Resultsmentioning

confidence: 99%

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source

Flores

Gancedo

2018

Sci Rep

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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

Hallin

Ottosson

et al. 2016

G3 Genes|Genomes|Genetics

129

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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

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“…Unless stated otherwise, yeast cultures were grown in 500-mL shake-flasks containing 100 mL synthetic medium with 20 g/L glucose as the sole carbon and energy source (SMG), at 30 °C and 200 rpm. Synthetic medium was prepared according to Verduyn et al [ 46 ], with the following modifications: ammonium sulphate (5 g/L) was replaced by 2.3 g/L urea to prevent acidification due to nitrogen source consumption and 6.6 g/L K 2 SO 4 was added [ 47 ]. The anaerobic growth factors ergosterol and Tween-80 were added to all cultures in this study as previously described [ 48 ].…”

Section: Methodsmentioning

confidence: 99%

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

González-Ramos

Vries

Grijseels

et al. 2016

Biotechnol Biofuels

111

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BackgroundAcetic acid, released during hydrolysis of lignocellulosic feedstocks for second generation bioethanol production, inhibits yeast growth and alcoholic fermentation. Yeast biomass generated in a propagation step that precedes ethanol production should therefore express a high and constitutive level of acetic acid tolerance before introduction into lignocellulosic hydrolysates. However, earlier laboratory evolution strategies for increasing acetic acid tolerance of Saccharomyces cerevisiae, based on prolonged cultivation in the presence of acetic acid, selected for inducible rather than constitutive tolerance to this inhibitor.ResultsPreadaptation in the presence of acetic acid was shown to strongly increase the fraction of yeast cells that could initiate growth in the presence of this inhibitor. Serial microaerobic batch cultivation, with alternating transfers to fresh medium with and without acetic acid, yielded evolved S. cerevisiae cultures with constitutive acetic acid tolerance. Single-cell lines isolated from five such evolution experiments after 50–55 transfers were selected for further study. An additional constitutively acetic acid tolerant mutant was selected after UV-mutagenesis. All six mutants showed an increased fraction of growing cells upon a transfer from a non-stressed condition to a medium containing acetic acid. Whole-genome sequencing identified six genes that contained (different) mutations in multiple acetic acid-tolerant mutants. Haploid segregation studies and expression of the mutant alleles in the unevolved ancestor strain identified causal mutations for the acquired acetic acid tolerance in four genes (ASG1, ADH3, SKS1 and GIS4). Effects of the mutations in ASG1, ADH3 and SKS1 on acetic acid tolerance were additive.ConclusionsA novel laboratory evolution strategy based on alternating cultivation cycles in the presence and absence of acetic acid conferred a selective advantage to constitutively acetic acid-tolerant mutants and may be applicable for selection of constitutive tolerance to other stressors. Mutations in four genes (ASG1, ADH3, SKS1 and GIS4) were identified as causative for acetic acid tolerance. The laboratory evolution strategy as well as the identified mutations can contribute to improving acetic acid tolerance in industrial yeast strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0583-1) contains supplementary material, which is available to authorized users.

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

Cited by 25 publications

References 25 publications

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source

Construction and characterization of a Saccharomyces cerevisiae strain able to grow on glucosamine as sole carbon and nitrogen source

Scan-o-matic: High-Resolution Microbial Phenomics at a Massive Scale

A new laboratory evolution approach to select for constitutive acetic acid tolerance in Saccharomyces cerevisiae and identification of causal mutations

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