Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Inicio
1. Yeasts in Bioeconomy: an introductory narrative, Carlos Gancedo and CarmenLisset Flores
2. Yeast
as
the
Formula
One
of
novel
bioprocesses:
the
case
of
microencapsulation, Sebastián Chávez, Lidia Delgado-Ramos, Sonia Montero,
Juan Carlos Rodríguez-Aguilera, María Flores-Mosquera, Xenia Peñate
3. Exploring and exploiting natural variation in Saccharomyces yeasts, Ed Louis
4. Yeast can help to solve oenological problems caused by global climate
changes, Amparo Querol
5. Current and potential applications of nitrate assimilatory yeasts, José M.
Siverio
6. Biotechnological
production
of
sphingoid
bases
with
the
yeast
Wickerhamomyces (Pichia) ciferrii, Eckhard Boles
7. Potential and actual applications of Yarrowia lipolytica, as wild-type yeast or
recombinant strains expressing heterologous proteins, Catherine Madzak
8. Cold is cool: how studies in yeast help to understand membrane properties
regulation, Francisca Randez-Gil, Isaac Córcoles-Sáez, Sara García-Marqués,
Lidia Ballester-Tomás y Jose A Prieto.
9. Coordination of yeast metabolism through transcription and enzyme
phosphorylation, Uwe Sauer
10. Using Engineered yeasts to perform biological computations, Francesc Posas
11. Systems metabolic engineering for protein production in yeast, Pau Ferrer
12. Development of CBP Biocatalysts for Industrial Ethanol Production, John
McBride
13. Laboratory evolution and reverse engineering of evolved phenotypes in
Saccharomyces cerevisiae, Jack T. Pronk
14. Better, stronger, faster: Synthetic biology and genomic approaches for
optimizing isoprenoid production by S. cerevisiae, Kirsten R. Benjamin
15. Yeast assays: simple solutions to complex problems, María Molina
16. Beer, bread and now the brain: Yeast models for protein folding diseases,
Joris Winderickx
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Yeasts in Bioeconomy: an introductory narrative, Carlos Gancedo and Carmen-Lisset
Flores
Yeasts have accompanied humans for millennia. Their contribution to every day life has
been important but their economic impact only increased significantly since the mid of the
nineteenth century when new technological and basic knowledge allowed a great
expansion of classical fermentation industries. The advent of new techniques and of the
concept of bioeconomy or “knowledge-based bio-economy” has prompted the use of these
organisms in previously unexpected fields. One of the main goals of the bioeconomy is to
produce commodities derived from renewable sources using organisms adequately
equipped with new activities. The knowledge accumulated about yeasts in different areas
including physiology, classical genetics, and molecular biology has situated these
organisms in a privileged position to be used effectively for that purpose.
In this presentation we will try to provide a framework to situate yeasts, both conventional
and non-conventional, in current bioeconomy. A short overview will be presented showing
some uses of yeasts as models or tools before the concept of "knowledge based bioeconomy" came into wide use. This will be followed by some examples of successful
biotechnological application of yeasts together with some considerations on the difficulties
to translate results from the laboratory bench to the industrial viable production.
Public appreciation of the contribution of yeasts to economy is scarce, this needs to be
approached by the yeast community to get more attention from science policy makers.
Some aspects of bioeconomy may require an extensive public debate as they may
implicate genetic modified organisms. It is of paramount importance that scientists contact
with the public in a clear language explaining the risks-benefits balance and avoiding to
raise unrealistic expectations.
Acknowledgements.-We thank Juana M. Gancedo (IIBM, Madrid, Spain) and Hans Van
Dijken (The Netherlands) for discussions about the topics considered. Work in the authors
laboratory is partially supported by grant BFU2010-19628-CO2-O2 from the Spanish
Ministerio de Economía y Competitividad
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FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Yeast as the Formula One of novel bioprocesses: the case of microencapsulation,
Sebastian Chávez, Lidia Delgado-Ramos, Sonia Montero, Juan Carlos RodríguezAguilera, María Flores-Mosquera, Xenia Peñate
Yeasts are eukaryotic microorganisms that have made important contributions to biological
research since Pasteur’s time. Saccharomyces cerevisiae and Schizosaccharomyces
pombe are two of the main model organisms in modern biology, given their wide
possibilities for genetic analysis and molecular manipulation. Several Saccharomyces
species and other related yeasts are also essential characters in food biotechnology and
manufacturing.
As a consequence of this central location in the R&D scenario, yeasts have been usually
chosen as initial organisms for the design of novel experimental approaches and for the
development of new bioprocesses. Microencapsulation is a good example of it. Initially
conceived as a strategy for the preservation of drugs and macromolecules,
microencapsulation is now extended to cells, and is becoming an important ingredient of
different technological fields, from cell therapy to the probiotics industry.
We will describe the potential of yeast in the application of cell microencapsulation and we
will illustrate it with two examples, in which yeast cells were manipulated using the flow
focusing technology. First, we will show results demonstrating the efficiency of core-shell
microcapsules for extending shelf life of probiotics. In this case concentric flow focusing
was used, followed by spray drying.
We will also show our results combining single-cell microencapsulation with large-object
flow cytometry, and its application for detecting and quantifying proliferation of the different
components of a cell population. In this case, the Cellena technology, also based in flow
focusing, was utilized.
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Exploring and exploiting natural variation in Saccharomyces yeasts, Ed Louis
Recent surveys of Saccharomyces sensu stricto yeasts, from a variety of locations and
niches, have revealed a great deal of genetic variation as well as a significant amount of
hybridization within and between the species. The laboratory and commercial strains
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
currently in use, utilise only a fraction of the genetic variation available in the species.
Through genetic crosses and hybridisation, we have found that the large genetic variation
can provide a great deal of phenotypic variation, beyond that of existing strains. This
means that it is possible to improve many traits of interest in fermentation for food and
beverages as well as for industrial purposes through modern breeding with a wider variety
of strains.
It is possible to generate a very large number of new genotypes by interbreeding
diverged strains, both within and between species. These new genotypes reveal
phenotypic variation beyond that of the input strains. Selection for improved growth,
increased ethanol production, resistance to various conditions and inhibitors, and other
properties of interest results in novel strains of potential economic value. Not only are
these novel strains created, it is possible using modern genomics to map the underlying
quantitative trait loci responsible for the improvements with high precision and sensitivity.
This knowledge helps inform the improvements to existing industrial and commercial
strains.
It is entirely possible to undertake this programme of programme of improvement
and genetic analysis without any genetic modification, which allows for the ‘natural’
production of desired products. Many economic uses of yeasts may allow for genetic
modification, in which case improvements can be further refined and may be quicker to
attain, particularly in a desired starting strain already in use. We have used breeding and
genetic
analysis
to
determine
the
underlying
genetic
variation
of
oenological
characterisitics in a wine strain, generate new yeasts resistant to high levels of ethanol,
high temperatures and inhibitors encountered in 2nd generation bioethanol production. The
potential for building better yeast is great for both commercial as well as academic uses.
VOLVER/RETURN
Yeast can help to solve oenological problems caused by global climate changes,
Amparo Querol
The wine fermentation is a complex process produced as a result of the activities of a
succession of microorganisms, being Saccharomyces yeasts (mainly S. cerevisiae) the
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
responsible for the alcoholic fermentation. Although S. cerevisiae is the most frequent
species in wines, and the subject of most studies, S. bayanus var. uvarum, S. eubayanus
and S. paradoxus strains and natural hybrids between Saccharomyces species such as S.
cerevisiae x S. kudriavzevii and S. cerevisiae x S. bayanusvar. uvarum are also involved in
wine fermentations and can be preponderant in certain wine regions.
Studies performed in our laboratory, which compared the properties of natural wine
hybrids with their parent species, showed that strains of non-conventional Saccharomyces
species, such as S. bayanus var. uvarum, S. eubayanus, S. kudriavzevii and the hybrids
exhibit physiological properties of potential interest in enology because they can respond
to the new demands of the wine industry, such as their ability to ferment at low
temperatures, their increased production of glycerol, their lower ethanol yield and their
higher assimilation of fructose.
Several of these characteristics such as low sugar/alcohol rate or an increment in glycerol
production might be of special interest to solve the problems in the oenological sector
caused by the climatic change. The increase of glycerol can conceal the astringency
caused by tannins in younger grapes and lowering the sugar/alcohol ratio is already a
requirement of the sector independently of the climatic change.
Finally, the study of natural hybrids present in fermentation processes opens a new
strategy for the development of novel industrial yeasts: the generation of artificial hybrids.
Producing hybrids between S. cerevisiae wine strains and other species could be a useful
strategy to develop industrial yeasts with novel enological characteristics.
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Current and potential applications of nitrate assimilatory yeasts, José M. Siverio
The yeast Hansenula polymorpha (Ogataea polymorpha) Candida utilis (Lindnera jadinii)
and Arxula adeninivorans have the capacity of assimilate nitrate, are amenable to gene
manipulation and in the case of the two first the genome has been sequenced and the
access is free. Nitrate assimilation consists in the transport of nitrate into the cell and
consecutive reduction to nitrite and ammonium by nitrite and nitrate reductase. In lower
eukaryotes, unlike plants, genes involved in nitrate assimilation are clustered.
These
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
genes are induced by nitrate and repressed
by reduced
ammonium, glutamine, glutamate, etc. H. polymorpha is
nitrogen sources such as
a methylotrophic and
termotolerant yeast with the best characterization of the nitrate assimilation pathways
Nitrate (NO-3) and nitrite (NO-2) present in water and food can cause health problem and
also eutrophication of lakes and rivers. The H. polymorpha nitrate reductase is produced
in Pichia pastoris and commercialized
as reagent to test nitrate. Plant crops takes
nitrogen mainly as nitrate, in this framework maize expressing a functional version of the
H. polymoprha
nitrate transporter (YNT1) have been constructed and patented. The
authors claims a yield increase in poor and normal nitrogen content soils.
Dekkera
bruxellensis is also able to assimilate nitrate, it can outcompete Saccharomyces cerevisiae
in ethanol production from sugar cane juice The capacity to use and eliminate nitrate even
in the presence of preferred nitrogen sources could explain in part this, since sugar cane
juice can contain high levels nitrate and nitrite which results toxic for S. cerevisiae.
In the past the main aim of our group was to study the regulatory network of nitrate
assimilatory genes in H. polymorpha. So we found three GATA factors, Ure2 and the
calcineurin pathway involved in the derepression of these genes while nitrate induction
required two Zn(2)-Cys(6) binuclear zinc cluster factors Yna1 and Yna2. More recently we
shown unequivocally that nitrate acts as inducer once it enters the cell and that at least
two excretion systems are involved in regulating its intracellular levels. Using this
information we have constructed an expression system based in a integrative vector
containing the promoter and terminator of the nitrate reductase gene and as host mutant
presenting a high expression of YNR1 (nitrate reductase gene) in response to nitrate. This
expression system has been successfully used to functionally express heterologous nitrate
transporters in H. polymorpha. Nowadays we make effort in collaboration with Proviovet
S.L. to express different polipeptides to be used as animal vaccines. Currently we are
focusing on the role of yeast arrestin in the regulation of nitrate transporter (Ynt1) and we
hope to find inputs in yeast bioeconomy derived from this.
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FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Biotechnological production of sphingoid bases with the yeast Wickerhamomyces
(Pichia) ciferrii, Eckhard Boles
Sphingolipids are essential components of biological membranes and also play numerous
other vital functions in living cells. Moreover, they are major constituents of the outermost
layer of human epidermis which acts as permeability barrier of the skin. Therefore, they
have a high potential to be used in a wide variety of application fields. Sphingolipids are
composed of an aminoalcohol, called sphingoid base or long-chain base, a fatty acid and,
in case of more complex sphingolipids, a headgroup. Their chemical synthesis is a
complex and cost-intensive process. As the yeast Wickerhamomyces (Pichia) ciferrii has
been found to be a natural producer of acetylated sphingoid bases, it provides a promising
alternative for their biotechnological synthesis. In the last years, we have established this
yeast by classical strain improvements as well as modern genetic engineering for the
industrial production of tetraacetyl phytosphingosine (TAPS). We improved precursor
availability by blocking degradation of L-serine which is condensed with palmitoyl-CoA in
the first committed step of sphingolipid biosynthesis. Moreover, genetic engineering of the
sphingolipid pathway further increased secretion of TAPS. The final recombinant W. ciferrii
strain produced up to 199 mg(TAPS) * g-1(cdw) with a titer of about 2 g * L-1. Moreover, routes
for the synthesis of sphinganine and sphingosin have been implemented (1). In our talk,
we will summarize the current knowledge about biosynthesis of sphingoid bases, genetic
engineering of W. ciferrii for their biotechnological production as well as their applications
in cosmetic formulations.
(1) reviewed in Appl Microbiol Biotechnol. 2013, 97:4301-8.
This work has been supported by a grant of the German Federal Ministry of Education and
Research (BioIndustrie 2021: FKZ 0315191).
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Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Potential and actual applications of Yarrowia lipolytica, as wild-type yeast or
recombinant strains expressing heterologous proteins, Catherine Madzak
Yarrowia lipolytica is an oleaginous hemiascomycetous yeast exhibiting remarkable
lipolytic and proteolytic activities. Its natural occurrence appears rather ubiquitous,
including soils, marine waters, mycorrhizae and a variety of foods (particularly dairy
products, including numerous cheeses, and meat). Since more than 50 years, this nonconventional yeast has promoted interest for industrial applications, firstly oriented towards
the production of metabolites with commercial value, and secondly aimed at producing
heterologous proteins. Yarrowia lipolytica is considered as a biosafety class 1
microorganism, which means that it is ‘‘safe-to-use’’. Consequently, several of its
applications have obtained a ‘‘Generally Regarded as Safe’’ (GRAS) status (US FDA).
The industrial use of Yarrowia lipolytica was pioneered by British Petroleum (UK), during
the search for microorganisms able to produce, on a large-scale, some high-quality protein
products for nutritional supply, which started in the 1950s. In addition to this production of
‘‘single-cell protein”, wild-type or engineered Yarrowia lipolytica cells are used to produce
several metabolites: different organic acids (e.g. citric acid - Pfizer Inc. and ADM, USA),
‘‘single-cell oil” enriched in polyunsaturated fatty acids (DuPont, USA), erythritol (used as
food additive - Baolingbao Biology Co., China), or carotenoids (DuPont and Microbia,
USA). The Polish company Skotan SA has started to commercialise Yarrowia lipolytica
biomass for use as fodder yeast in Europe, and is developing prebiotic and probiotic
applications of this yeast for food industry. Other potential applications include biofuel
production and bioremediation (Artechno, Belgium).
Since several industrial (Pfizer Inc., USA; Novozymes, Denmark) or academic laboratories
have developed genetic and molecular tools, Yarrowia lipolytica emerged as an efficient
heterologous production host for pharmaceutical or industrial proteins and enzymes. A
state-of-the-art of Yarrowia lipolytica technology will be exposed. Expression/secretion
vectors and optimized strains developed in our laboratory were included into the YLEX™
Expression Kit, commercialized by Yeastern Biotech Co. (Taiwan). Since 25 years, more
than 100 heterologous proteins, from more than 60 species, have been successfully
produced in this yeast. The relatively recent development of surface display systems
allowed using Yarrowia lipolytica as arming yeast, increasing further its possibilities.
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
However, despite numerous scientific publications concerning the use of engineered
Yarrowia lipolytica as whole cells or as recombinant protein producer, most of the
described applications remain only potential, their development rarely passing the
exploratory stage. In addition, it remains critical to know if the use of whole Yarrowia
lipolytica cells, as a genetically modified microorganism, will be acceptable. Up to now,
only a few projects using engineered Yarrowia lipolytica are on the edge to the marketing
stage: Oxyrane (UK) uses optimized strains to produce human lysosomal enzymes, for
enzyme replacement therapies of lysosomal storage diseases, and Mayoly Spindler
(France) uses an overexpressed homologous Yarrowia lipolytica lipase to cure exocrine
pancreatic insufficiency (clinical trials stage in both cases). We can hope that such
examples will be more numerous in a close future.
- Bankar AV, Kumar AR, Zinjarde SS. (2009). Environmental and industrial applications of
Yarrowia lipolytica. Appl Microbiol Biotechnol 84:847–65.
- Groenewald M, Boekhout T, Neuvéglise C, Gaillardin C, van Dijck PW, Wyss M. (2013).
Yarrowia lipolytica: Safety assessment of an oleaginous yeast with a great industrial
potential. Crit Rev Microbiol. [Epub ahead of print]
- Madzak C, Gaillardin C, Beckerich JM. (2004). Heterologous protein expression and
secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–
81.
- Madzak C, Beckerich JM. (2013). Heterologous protein expression and secretion in
Yarrowia lipolytica. In : Yarrowia lipolytica: Biotechnological Application, Microbiology
Monographs Vol. 25. Ed: G. Barth, Springer, Heidelberg, Germany. pp. 1-76.
VOLVER/RETURN
Cold is cool: how studies in yeast help to understand membrane properties
regulation, Francisca Randez-Gil, Isaac Córcoles-Sáez, Sara García-Marqués, Lidia
Ballester-Tomás y Jose A Prieto.
The development of yeast strains providing better fermentative capability or to be applied
under new industrial conditions, is a growing demand. In particular, the development of
commercial yeast with enhanced cold tolerance and freeze resistance has a great
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
economic interest. Hence, a better understanding of the principles underlying the
adaptation and tolerance mechanisms to this stress is a key factor for industrial strain
selection. Results from our group indicate that exposure to low temperatures in S.
cerevisiae activates signaling cascades that modify processes and cellular structures (1,
2). Among them, the plasma membrane is a primary target of stress-induced damage and
consequently, the regulation of its composition and organization determines the
functionality of many membrane-anchored proteins. Studies in our laboratory have
revealed the existence in S. cerevisiae of a Plc1p-inositol phosphate-mediated signaling
mechanism that detects changes in membrane fluidity, transmits this signal and triggers an
adaptive response by regulating the synthesis of lipids (3). Furthermore, we have identified
a novel regulator of the Pkh-Fpk kinases module, which controls the activity of the
flippases Dnf1p and Dnf2p, and thus contributes to maintain the membrane phospholipids
asymmetry (4). These findings highlight the importance of cold stress to uncover pathways
and actors implied in membrane homeostasis and the possibility to develop molecules and
processes of biotechnological and pharmaceutical relevance.
1.- Panadero et al. (2006) A downshift in temperature activates the high osmolarity
glycerol (HOG) pathway, which determines freeze tolerance in Saccharomyces cerevisiae.
J Biol Chem.281(8):4638-45.
2.- Córcoles-Sáez et al. (2012) Low temperature highlights the functional role of the cell
wall integrity pathway in the regulation of growth in Saccharomyces cerevisiae.Biochem J.
446(3):477-88.
3.-
Córcoles-Sáez
et
al.
(submitted)
Phosphatidylinositol
4,5-bisphosphate
diphosphoinositol phosphates play a role in fluidity signaling and homeostasis
and
by
regulating lipid synthesis
4.- García-Marqués et al. (submitted) Sng1 regulates the sphingolipid-responsive Pkh-Fpk
kinases module and influences membrane properties and cold growth in Saccharomyces
cerevisiae
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FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Coordination
of
yeast
metabolism
through
transcription
and
enzyme
phosphorylation, Uwe Sauer
Ultimately, metabolic engineering requires a massive redirection of cellular fluxes. Albeit
many successful metabolic engineering applications were reported in the past 2 decades,
it is a common experience in the field that our engineering approaches often do not yield
the anticipated result – the cells appear to be resilient to our efforts. One reason is
probably that we do not properly understand how cells actually make metabolic decisions.
While our knowledge on regulation events is steadily increasing, we are much less
informed about the functionality of individual regulation events and their quantitative
relevance for controlling a given biological function. For metabolism this function is the flux
of small molecules that can be quantified network-wide through methods of
13C-flux
analysis (1). This ability to quantify metabolic function allows us to investigate the question
which of the multiple overlapping regulation mechanisms are employed by microbial cells
to manage their small molecule traffic (2, 3). Even seemingly simple environmental
adaptions typically lead to extensive transcriptional responses, although often only very
few of these co-occurring regulation events are required for the specific adaptation (4). By
combining various omics methods with computational analysis, we delineate regulation
events that actively control metabolic flux coordination from the much larger number of cooccurring regulation events. Here I will discuss that our results strongly suggest that
transcriptional regulation plays a much less important role than previously thought. For
yeast we find instead that enzyme phosphorylation plays a much more important in
controlling central metabolic fluxes (5, 6), and I will present several newly discovered
phosphoenzyme functionalities.
References:
1.
Sauer. Mol. Sys. Biol. 2: 62 (2006)
2.
Heinemann & Sauer. Curr. Opin. Microbiol. 13: 337 (2010)
3.
Gerosa & Sauer. Curr. Opin. Biotechnol. 22:566 (2011)
4.
Büscher et al. & Sauer. Science 335: 1099-1103 (2012)
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
5.
Oliveira & Sauer. FEMS Yeast Res. 12: 104
6.
Oliveira et al. Mol. Sys. Biol. 8: 623. (2012)
VOLVER/RETURN
Using Engineered yeasts to perform biological computations, Francesc Posas
Engineering approaches to synthetic biology have shown that there are a number of
strategies allowing to build complex functional constructs with computational abilities.
There are a number of efforts towards building artificial computational devices that could
be used for a wide range of applications, including bioremediation, food production or
biomedicine. Ideally, these designed networks should be scalable and reusable. Using
yeast as a model organism we have been able to implement complex circuits by
distributing computation within cellular consortia. This approach to biological computation
has opened the possibility to develop a novel method of properly design general purpose,
LEGO-like multicellular systems, which can be combined in multiple ways to create
complex computational circuits.
Systems metabolic engineering for protein production in yeast, Pau Ferrer
Since the implementation of S. cerevisiae as an expression platform for heterologous
proteins in the early 80’, yeasts have been widely used for the production of recombinant
proteins. Moreover, a range of alternative yeast expression systems have emerged since
then, playing an increasing role in the biopharmaceutical market (1).
Among these
alternative cell factories, the methylotrophic yeast Pichia pastoris is widely used as a
production platform, as well as model organism for peroxisome and secretory organelle
proliferation (2). Several products from P. pastoris are marketed, in the clinical trial pipeline
or in development (1).
Despite the increasing biotechnological impact of P. pastoris, there still are a number of
physiological limitations that constrain the potential of the production strains and process
development.
For instance, it is well established that recombinant overproduction of
proteins may lead to an overload of the folding and secretory pathways, resulting in the
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
accumulation of unfolded proteins in the endoplasmatic reticulum, thereby triggering the
unfolded protein stress response. Moreover, protein production is also interconnected with
different cellular processes caused by stress factors such as temperature, pH, osmolarity
or oxygen, which are highly relevant from the fermentation process point of view.
Accordingly, most of the 1st generation metabolic engineering approaches for strain
improvement targeted at the improvement of protein folding and secretion.
The genome sequence of P. pastoris has been published recently (4-5), boosting the
development and application of omics analytical platforms (transcriptomics, proteomics,
metabolomics, fluoxomics, etc) and systems biology tools (genome-scale models) to the
genetic and physiological research of this yeast. Such systems-level approach is enabling
an important progress on fundamental understanding of physiological processes and,
subsequently, rapid development of production strains. Notably, it is allowing the rational
design of strain improvement strategies targeting cellular processes beyond the protein
folding and secretion pathways.
References:
1.
Corchero JL et al. (2013) Unconventional microbial systems for the cost-efficient
production of high-quality protein therapeutics. Biotechnol Adv. 31:140-53.
2.
Gasser B et al. (2013) Pichia pastoris: protein production host and model organism
for biomedical research. Future Microbiol. 8:191-208.
3.
De Schutter K et al. (2009) Genome sequence of the recombinant protein
production host Pichia pastoris. Nat Biotechnol. 27:561-566.
4.
Mattanovich M et al. (2009) Genome, secretome and glucose transport highlight
unique features of the protein production host Pichia pastoris. Microb Cell Fact 8:29
5.
Baumann et al. (2010) A multi-level study of recombinant Pichia pastoris in different
oxygen conditions. BMC Syst Biol. 4:141.
6.
Jordà et al. (2012) Metabolic flux profiling of recombinant protein secreting Pichia
pastoris growing on glucose:methanol mixtures. Microb Cell Fact. 11:57.
VOLVER/RETURN
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Development of CBP Biocatalysts for Industrial Ethanol Production, John McBride
Mascoma Corporation, a Lebanon, NH, based biotechnology company, has developed a
suite of technologies implemented in Saccharomyces cerevisiae to improve the economics
of industrial ethanol production. We have focused on bringing together the secretion of
enzymes important for the breakdown of polymeric carbohydrates found in biomass, with
the necessary metabolic pathways to ferment monomeric sugars derived from that
biomass to ethanol in high yield in robust strain backgrounds.
Several strains of yeast
have been developed for the U.S. based corn ethanol industry that secrete amylase
enzyme to reduce exogenously added enzyme requirements, and reduce the formation of
the byproduct glycerol. In cooperation with Lallemand, Inc., we have deployed our strains
to more than 10% of the U.S. corn ethanol industry and produced in excess of 600 million
gallons (~2.2 billion liters) of ethanol with these strains to date.
Mascoma has also
developed strains of yeast targeted towards the emerging 2 nd generation ethanol industry.
Technologies for extremely robust and rapid xylose fermentation, high level cellulase and
hemicellulase secretion, and glycerol reduction will be discussed. The combination of
these technologies allows for increased yields and/or decreased enzyme requirements for
the processing of lignocellulosicfeedstocks, and can represent value creation in excess of
$0.40/gallon of ethanol.Taken together, our developments have shown that consolidated
bioprocessing (CBP) via S. cerevisiae is not only feasible, but the preferred biotechnology
solution for biomass conversion.
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Laboratory
evolution
and
reverse
engineering
of
evolved
phenotypes
in
Saccharomyces cerevisiae, Jack T. Pronk
Laboratory evolution is a tremendously powerful tool to improve aspects of the
performance of industrial micro-organisms that can be directly linked to specific growth
rate or survival. Although design of selective strategies is intellectually stimulating, the
evolved strains long remained ‘black box’ systems with respect to the responsible
mutations were concerned. Still, knowledge on the molecular basis for improved
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
performance of microbial strains obtained by laboratory evolution can contribute to
improved understanding as well as to the reverse engineering of specific traits in other
strain backgrounds and/or species.
For a number of years, microarray-based transcriptome analysis was the only affordable
genome-wide tool available for the analysis of evolved strains of the yeast Saccharomyces
cerevisiae. However, context-dependency of transcriptome analysis, the frequent
generation of indirect transcriptional responses and constraints in experimental design limit
the power of transcriptomics for identifying the molecular basis of improved performance in
evolved strains.
In this lecture, I will give an overview of the experiences with laboratory evolution and, in
particular, of our experiences in reverse engineering of the evolved phenotypes. To this
end, I will discuss several case studies on phenotypes that we intentionally and, in one
case not so intentionally, selected by long-term cultivation under a specific selective
pressure. These cases illustrate that whole-genome sequencing, by now an affordable
technique for yeast research, is a true game changer in the analysis and reverse
engineering of evolved yeast phenotypes, especially when combined with classical yeast
genetics methods and applied to parallel, independent evolution experiments.
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Better, stronger, faster: Synthetic biology and genomic approaches for optimizing
isoprenoid production by S. cerevisiae, Kirsten R. Benjamin
To provide stable, low-cost, environmentally sustainable supplies of many chemicals
needed for an expanding worldwide middle-class population, we need to develop new biobased sources of hydrocarbons and related molecules. At Amyris, we engineer microbial
factories and manufacture isoprenoid products via fermentation and downstream
chemistry. The isoprenoid biosynthetic pathway is used by all classes of living organisms
for creation of over 30,000 known compounds, including molecules that are currently or
might soon be used for biofuels, flavors, fragrances, steroids, sterols, emollients, coloring
agents, pharmaceuticals, nutraceuticals, and monomers for polymer production. Synthetic
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
biology approaches at Amyris have accelerated creation and improvement of S. cerevisiae
strains that make high levels of isoprenoids. This presentation will summarize the
approaches, progress, and discoveries within the Amyris strain improvement program. A
key component isour Automated Strain Engineering technology and its application for
combinatorial exploration of thousands of complex genotypes including many genes
imported from other organisms. Complementary approaches include high-throughput
screening, classical mutagenesis, mating and meiosis, computational modeling, systems
biology, genomic approaches, and detailed physiological characterization of strains. By
developing and implementing 21st Century technologies for industrial strain improvement,
Amyris has rapidly improved its isoprenoid-producing strains sufficiently to begin
manufacturing several commercial products.
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Yeast assays: simple solutions to complex problems, María Molina
Working with the yeast Saccharomyces cerevisiae has many advantages. Indeed, this
unicellular eukaryotic organism is considered both an excellent tool and an ideal model for
biological research owing to its ease of use for biochemical, genetic and genomic studies.
For many years, such studies have produced a cumulative bulk of data on biological
processes that are highly conserved along the evolutionary scale. This yeast is also readily
amenable for synthetic biology approaches that open the possibility to create new
synthetic pathways and functionalities with specific applications in biomedicine or
biotechnology (1). For example, the heterologous expression of proteins involved in
human diseases that couple to or interfere with yeast physiology allows the development
of easily manageable assays just relying on the detection of yeast growth. Such simple
yeast-based bioassays are very useful for structure-function analysis and adaptable to
high throughput screening (HTS) technologies for drug discovery. Importantly, these
bioassays provide an in vivo eukaryotic environment and, at the same time, are really
inexpensive as compared to enzymatic or receptor-ligand in vitro assays or even to
mammalian cell culture assays (2). Moreover, they may help to overcome ethical and
experimental constraints that apply to higher eukaryotes, for example, concerning toxicity
assays.
FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
In this line, our research group has developed several yeast bioassays to study diseaserelated proteins based on the reconstitution of the oncogenic PI3K-PTEN-Akt human
pathway (3), as well as on the use of the yeast cell as a host for bacterial virulence
proteins that target conserved eukaryotic cellular processes (4,5). When the expression of
a bacterial effector leads to a discernible phenotype in yeast, it is possible to test
hypotheses regarding its role in pathogenesis, to perform mutagenesis analyses to identify
functional domains, and eventually to validate its use as a drug target and develop a
bioassay for primary HTS.
(1) Furukawa K, Hohmann S. 2013. Synthetic biology: lessons from engineering yeast
MAPK signalling pathways. Mol Microbiol. 88:5-19.
(2) Fernández-Acero T, Rodríguez-Escudero I, Vicente F, Monteiro MC, Tormo JR,
Cantizani J, Molina M, Cid VJ. 2012. A yeast-based in vivo bioassay to screen for class
I phosphatidylinositol 3-kinase specific inhibitors. J Biomol Screen. 17:1018-29.
(3) Cid VJ, Rodríguez-Escudero I, Andrés-Pons A, Romá-Mateo C, Gil A, den Hertog
J, Molina M, Pulido R. 2008. Assessment of PTEN tumor suppressor activity in
nonmammalian models: the year of the yeast. Oncogene. 27:5431-42.
(4) Rodríguez-Escudero I, Ferrer NL, Rotger R, Cid VJ, Molina M. 2011. Interaction of
the Salmonella Typhimurium effector protein SopB with host cell Cdc42 is involved
in intracellular replication. Mol Microbiol. 80:1220-40.
(5) Fernandez-Piñar P, Alemán A, Sondek J, Dohlman HG, Molina M, Martín H. The
Salmonella Typhimurium effector SteC inhibits Cdc42-mediated signaling through
binding to the exchange factor Cdc24 in Saccharomyces cerevisiae. 2012. Mol Biol Cell.
23:4430-43.
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FUNDACIÓN RAMÓN ARECES
Simposio Internacional: Las levaduras en Bioeconomía
International Symposium: Yeast in Bioeconomy
Madrid, 7 y 8 de noviembre de 2013
Madrid, November 7-8, 2013
Beer, bread and now the brain: Yeast models for protein folding diseases, Joris
Winderickx
The budding yeast Saccharomyces cerevisiae has contributed significantly to our current
understanding of eukaryotic cell biology. It served as a tool and model to elucidate the
molecular basis of a wide variety of cellular phenomena, which appeared to be conserved
in other organisms as well. Because of this conservation, the budding yeast became an
attractive cellular and biological relevant model to investigate disease-related proteins,
even when the yeast genome does not encode for an apparent homologous counterpart.
These so-called humanized yeast models hold great promise for the dissection of diseaserelated molecular processes and the discovery of novel medicinal compounds. A good
example are the yeast models used to clarify the biochemical and cytotoxic properties of
proteins associated to neurodegenerative disorders like Parkinson’s, Huntington’s and
Alzheimer’s disease, which are commonly classified as protein folding disorders. Studies
with these models not only provided fundamental insight on the interplay of protein quality
control mechanisms and how failure of these systems result in cytotoxicity and eventually
cell death, but also led to the identification of novel players in disease etiology and the
validation of prognostic biomarkers that formed the basis for the development of improved
diagnostic assays. In addition, these humanized yeast models offered an unsurpassed
performance in phenotypic and chemo-genetic screenings aiming to select and study the
mode-of-action of lead compounds with promising therapeutic activity.
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