Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

Knudsen, Jan; Johanson, Ted; Lantz, Anna Eliasson; Carlquist, Magnus

doi:10.1016/j.btre.2015.06.001

Cited by 7 publications

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“…In accordance with previous studies [ 21 , 28 ] the rate of sugar consumption differed significantly between the gpd1 Δ gpd2 Δ strain and the control strain (Fig. 3 a; Table 3 ).…”

Section: Resultssupporting

confidence: 93%

“…This has been considered a limiting factor for the exploitation of the strain for biocatalytic purposes. However, recent findings indiciate that the aerobic growth limitatation can be overcome by applying high aereration rates [ 28 ]. This allows for an efficient cultivation of the biocatalyst and thus opens further exploitation of the strain.…”

Section: Discussionmentioning

confidence: 99%

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Increased availability of NADH in metabolically engineered baker’s yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

et al. 2016

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BackgroundSaccharomyces cerevisiae can be engineered to perform a multitude of different chemical reactions that are not programmed in its original genetic code. It has a large potential to function as whole-cell biocatalyst for one-pot multistep synthesis of various organic molecules, and it may thus serve as a powerful alternative or complement to traditional organic synthetic routes for new chemical entities (NCEs). However, although the selectivity in many cases is high, the catalytic activity is often low which results in low space-time-yields. In the case for NADH-dependent heterologous reductive reactions, a possible constraint is the availability of cytosolic NADH, which may be limited due to competition with native oxidative enzymes that act to maintain redox homeostasis. In this study, the effect of increasing the availability of cytosolic NADH on the catalytic activity of engineered yeast for transamination-reduction coupled asymmetric one-pot conversion was investigated.ResultsA series of active whole-cell biocatalysts were constructed by over-expressing the (S)-selective ω-transaminase (VAMT) from Capsicum chinense together with the NADH-dependent (S)-selective alcohol dehydrogenase (SADH) originating from Rhodococcus erythropolis in strains with or without deletion of glycerol-3-phosphate dehydrogenases 1 and 2 (GPD1 and GPD2). The yeast strains were evaluated as catalysts for simultaneous: (a) kinetic resolution of the racemic mixture to (R)-1-phenylethylamine, and (b) reduction of the produced acetophenone to (S)-1-phenylethanol. For the gpd1Δgpd2Δ strain, cell metabolism was effectively used for the supply of both amine acceptors and the co-factor pyridoxal-5′-phosphate (PLP) for the ω-transaminase, as well as for regenerating NADH for the reduction. In contrast, there was nearly no formation of (S)-1-phenylethanol when using the control strain with intact GPDs and over-expressing the VAMT-SADH coupling. It was found that a gpd1Δgpd2Δ strain over-expressing SADH had a 3-fold higher reduction rate and a 3-fold lower glucose requirement than the strain with intact GPDs over-expressing SADH.ConclusionsOverall the results demonstrate that the deletion of the GPD1 and GPD2 genes significantly increases activity of the whole-cell biocatalyst, and at the same time reduces the co-substrate demand in a process configuration where only yeast and sugar is added to drive the reactions, i.e. without addition of external co-factors or prosthetic groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0430-x) contains supplementary material, which is available to authorized users.

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“…In accordance with previous studies [ 21 , 28 ] the rate of sugar consumption differed significantly between the gpd1 Δ gpd2 Δ strain and the control strain (Fig. 3 a; Table 3 ).…”

Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Increased availability of NADH in metabolically engineered baker’s yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

et al. 2016

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“…Reporter strains have already been proven to be useful tools in numerous studies of population heterogeneity in lab-scale simulating industrial scale bioprocesses. Examples are investigation of nutrient and oxygen limitation, study of the redox balance or identification of the most robust and best producing subpopulation of cells by integrating a fluorescent protein whose fluorescence output is proportional to product formation [24][25][26][27].…”

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confidence: 99%

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

et al. 2020

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Background: Today there is an increasing demand for high yielding robust and cost efficient biotechnological production processes. Although cells in these processes originate from isogenic cultures, heterogeneity induced by intrinsic and extrinsic influences is omnipresent. To increase understanding of this mechanistically poorly understood phenomenon, advanced tools that provide insights into single cell physiology are needed. Results: Two Escherichia coli triple reporter strains have been designed based on the industrially relevant production host E. coli BL21(DE3) and a modified version thereof, E. coli T7E2. The strains carry three different fluorescence proteins chromosomally integrated. Single cell growth is followed with EmeraldGFP (EmGFP)-expression together with the ribosomal promoter rrnB. General stress response of single cells is monitored by expression of sigma factor rpoS with mStrawberry, whereas expression of the nar-operon together with TagRFP657 gives information about oxygen limitation of single cells. First, the strains were characterized in batch operated stirred-tank bioreactors in comparison to wildtype E. coli BL21(DE3). Afterwards, applicability of the triple reporter strains for investigation of population heterogeneity in bioprocesses was demonstrated in continuous processes in stirred-tank bioreactors at different growth rates and in response to glucose and oxygen perturbation simulating gradients on industrial scale. Population and single cell level physiology was monitored evaluating general physiology and flow cytometry analysis of fluorescence distributions of the triple reporter strains. Although both triple reporter strains reflected physiological changes that were expected based on the expression characteristics of the marker proteins, the triple reporter strain based on E. coli T7E2 showed higher sensitivity in response to environmental changes. For both strains, noise in gene expression was observed during transition from phases of non-growth to growth. Apparently, under some process conditions, e.g. the stationary phase in batch cultures, the fluorescence response of EmGFP and mStrawberry is preserved, whereas TagRFP657 showed a distinct response. Conclusions: Single cell growth, general stress response and oxygen limitation of single cells could be followed using the two triple reporter strains developed in this study. They represent valuable tools to study population heterogeneity in bioprocesses significantly increasing the level of information compared to the use of single reporter strains.

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“…Yeasts can be used to obtain products that range from therapeutic proteins and industrial enzymes and various metabolites, just give conditions to the yeasts for biosynthesis to occur, as stated by Knudsen et al (2015), when using S. cerevisiae, nutrients and an assimilable carbon source are needed to obtain a specific product such as ethanol or in a spectrum of products such as ethanol and by-products such as glycerol and other commercially valuable alcohols (Nandy & Srivastava , 2018).…”

Section: Resultsmentioning

confidence: 99%

Analysis of fermentative parameters and the importance of Saccharomyces cerevisiae in the development of goods and services

Batistote

2020

RSD

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In the fermentation process, yeasts need to adapt to the environmental changes that occur during the production process. Responses to these adjustments can alter biochemical routes and the amount of metabolites produced. Thus, the objective was to analyze the fermentative parameters of industrial yeast strains in different growing conditions, as well as to evaluate the its applicability in different sectors of goods and services. A pre-inoculum was performed with the YPSAC 5% medium for the activation of the yeasts Catanduva-1 and Fleischmann that remained incubated for 24 hours at 30 °C at 250 rpm. After the cells were recovered by centrifugation and inoculated in the fermentation medium based on sugarcane juice at 15 °Brix at temperatures of 30 and 40 °C. Aliquots were removed for the analysis of the fermentative parameters. Concomitantly, a survey was carried out regarding the use of yeasts in the process of preparing goods and services. The data show that the best yeast fermentation performance occurred at 30 °C in 10 hours. In addition, yeasts have the ability to produce, under ideal conditions, metabolites that can be used in different biotechnological processes.

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Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae

Cited by 7 publications

References 56 publications

Increased availability of NADH in metabolically engineered baker’s yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

Increased availability of NADH in metabolically engineered baker’s yeast improves transaminase-oxidoreductase coupled asymmetric whole-cell bioconversion

Development and characterization of Escherichia coli triple reporter strains for investigation of population heterogeneity in bioprocesses

Analysis of fermentative parameters and the importance of Saccharomyces cerevisiae in the development of goods and services

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