New, enabling tools for broad utilization of cell factories in sustainable production of chemicals and fuels

By Jussi Jäntti and Dominik Mojzita

Irrespective of the development of oil prices and our technical abilities to retrieve oil from the ground, it is inevitable that in order to fight climate change there is a need to reduce our dependency on fossil fuels and develop sustainable, environmentally friendly production processes using renewable raw materials.

The world we live in has 119 million identified inorganic and organic chemical substances that are either natural or man-made (www.cas.org). The global chemical industry produces roughly 70,000 different chemicals. Oil consumption in Finland is 5.5 liters/day/Finn of which 1 liter/day/Finn contributes raw material for production of chemicals and lubricants (www.oil.fi). Globally, roughly 2 liters/day/person of oil is consumed (www.iea.org).

One way to reduce our oil dependency  is to harness the diversity of biology, especially in the form of microbes to convert waste streams such as pulp and paper industry wood hydrosylates to valuable compounds in bioreactors. This can be achieved when the vast selection of chemical reactions that microbes carry out naturally is combined with novel, designed biochemical pathways. Such pathways can be engineered in an increasing number of microbes using modern genome engineering technologies.

The modified microbes generated in this way can be considered as microbial cell factories that convert a biological raw material into designed end products such as polymer precursors and fuels.

Novel technologies accelerate the development

Significant advances have taken place in our abilities to generate enzymatic production pathways for more and more complex chemicals in microbes due to development of  novel genome engineering techniques (e.g. CRISPR/Cas9), the wealth of DNA sequence data of life on earth, and improved methods in sequence analysis and mathematical modelling of reaction pathways. The first wave of new technologies has already now significantly reduced the time needed for engineering of an industrial, microbial production host. For example, the genome engineering of an industrial yeast strain can now be done over 10 times faster than before. At the same time, these technologies enable engineering of an increasingly broad spectrum of organisms.

Components of a biological system need to be expressed typically at defined levels in order to obtain optimal functionality. This can be important for example for metabolic pathway engineering, where individual genes encoding a production pathway need to be expressed (and the corresponding enzymes produced) in balanced ratios to ensure optimal metabolic flux towards a desired product. Thus, one of the challenges in microbial production host engineering is how to establish specific levels of expression of multiple genes. Furthermore, for many potentially interesting, but currently little used industrial microbes, there are very limited or non-existing tools and/or methods to accomplish controlled expression of heterologous genes. This prohibits efficient use of these hosts in industrial applications.

Need for predictable, stable expression of target genes

An additional level of complexity for the production host engineering comes from the need to maintain predictable and stable gene expression in diverse cultivation conditions or stages of growth. Often, in currently available gene expression systems specific inducing conditions need to be used to achieve desirable expression of target genes. This leads to specific requirements for growth media composition or downstream processing approaches that ultimately result in increased production costs.

In order to achieve predictable and stable expression of target genes in a production organism it is important that the expression of these genes is minimally affected by the intrinsic regulatory mechanisms of the organism. This can be achieved by the use of non-native components (such as promoters and transcription factors) in the engineered gene expression system.

Modular expression system from VTT

We have established a modular expression system that is independent from externally added compound(s) and that enables tight control over a wide range of gene expression levels in the yeast S. cerevisiae (Rantasalo et al. 2016 http://dx.doi.org/10.1371/journal.pone.0148320). A basal, low level expression of a synthetic transcription factor (sTF) is ensured by a core promoter which expression activity is not affected by growth conditions. The sTF is composed of modular DNA binding and gene transcription activating parts that can be used to drive expression of any target gene at tunable levels simply by varying the number of sTF binding sites within the promoter that regulates the expression of the gene of interest.

stf
Importantly, subsequent work has enabled us to extend the published system with a set of novel core promoters and sTFs that can be used to achieve highly tunable expression of target genes, e.g. for optimized metabolic pathways or hydrolytic enzyme mixtures, in a broad range of industrially relevant microbes from various yeasts to filamentous fungi (patent pending).

These systems provide us with novel tools and abilities to engineer eukaryotic microbes for designed purposes using a few, well defined genetic elements. Thus, this system enables us to expand the selection of production organisms that can be effectively engineered for sustainable production of chemicals, fuels and proteins.

If you want to learn more, contact Senior Research Scientist Dominik Mojzita (dominik.mojzita@vtt.fi) or Research Team Leader Jussi Jäntti (jussi.jantti@vtt.fi).

 

jussiDr. Jussi Jäntti leads the Production Host Engineering research team. His team focuses on developing engineering tools for a broad range of eukaryotic microbes. These tools are used to generate cell factories for the sustainable production of chemicals and fuels. jussi.jantti@vtt.fi

 

 

dominikDr. Dominik Mojzita has a strong background in molecular biology and genetic engineering of yeast and filamentous fungi. He has worked on the identification and characterization of novel genes and metabolic pathways, transcriptional and metabolic regulation, genome-wide and gene-specific expression analysis, the production of organic compounds, single-cell analysis, development of synthetic expression tools and the establishment of synthetic control circuits in S. cerevisiae and A. niger. dominik.mojzita@vtt.fi

Novel regulatory factors can help to improve enzyme production in Trichoderma reesei

By Tiina Pakula

In this blog post we describe how we used a systems biology approach to discover new tools for strain improvement in Trichoderma reesei. As a saprophytic fungus by origin living on plant debris, it produces a large repertoire of enzyme activities degrading lignocellulosic material. Genomic analyses have revealed over 200 genes encoding carbohydrate active enzymes, over 60 of them predicted to encode cellulolytic or hemicellulolytic activities.

Based on literature, T. reesei is the most efficient organism for production of enzymes at industrial scale. Optimisation of culture conditions and systematic strain development has made it possible to produce secreted proteins even as much as over 100 g/l.

The enzyme industry exploits T. reesei widely in enzyme manufacturing for e.g. pulp and paper, food, feed, and textile applications. In the recent years the importance of this production host has increased greatly along with the rising trend of producing fuels and chemicals from lignocellulosic feedstocks in second generation biorefineries. We have long experience in developing T. reesei as a producer of lignocellulose degrading enzymes and recombinant proteins. This production platform is the flagship of VTT Expression Service providing custom recombinant protein production.

Group

VTT’s Protein Production team

Understanding of regulatory mechanisms is the key for the enhancement of enzyme production

Production of extracellular enzymes by the fungus is modulated by a multitude of environmental and physiological conditions, the most important determinant being the carbon source available for the fungus.  Hydrolysis of complex plant-derived material requires a synergistic action of different enzymatic activities, and therefore it is beneficial for the fungus to be able to adjust the enzyme production according to the need and to produce suitable combination of enzymes in the presence of the particular material.

Novel biotechnical applications aim at the utilisation of different types of bio-based raw materials in production of useful compounds, and tailor-made enzyme cocktails are needed for efficient utilisation of the material. Understanding of the regulatory mechanisms in production of the enzymes provides tools to enhance the overall production as well as to engineer enzyme cocktails for specific raw materials.

We have applied transcriptomics analysis to examine expression of genes encoding hydrolytic enzymes in the presence of different carbon sources, ranging from defined oligosaccharides to complex plant-derived raw materials such as bagasse, spruce and wheat straw.  Groups of co-regulated genes encoding hydrolytic enzymes were identified on the different carbon source materials, suggesting a potentially important role for them in utilisation of the material.  Furthermore, the analyses revealed novel candidate regulatory genes encoding transcription factors and other regulatory proteins that had a similar expression patterns with the genes encoding the hydrolytic enzymes (Fig. 1).

Fig 1

Figure 1. Heatmap presentation of expression patterns of selected genes in the presence of different lignocellulosic substrates. Red colour in the heatmap indicates induction of the gene by the specific substrates (shown on the bottom). Genes encoding hydrolytic enzymes are shown by green colour and candidate regulatory genes by red on the right.

A set of 28 such candidate regulatory genes were selected for further studies to reveal their possible role in regulation of enzyme production. These genes were first over-expressed in T. reesei with a constitutive promoter.   Seven of the genes tested were shown to increase cellulase and/or xylanase production when overexpressed in the fungal cells (Fig. 2), and some genes appeared to have a negative effect.

Fig 2

Figure 2. Production of (A) xylanase and (B) cellulase activity by strains overexpressing the candidate regulatory genes as compared to the parental strain (parental level shown by red line).

A part of the genes had an effect on production of one particular enzyme activity whereas others appeared to have an effect on production of a broader set of activities. Further analysis of one of the genes, designated ace3, showed that it is essential for cellulase production by the fungus. Overexpression of ace3 (pMH15 in Figure 2) clearly enhanced cellulase and xylanase production, whereas deletion of the gene abolished cellulase production completely and reduced xylanase production significantly (Figure 3).

Fig 3

Figure 3. Production of cellulase and xylanase activity by strains deleted for ace3 and by the parental strain

The multitude of environmental factors affecting production of hydrolytic enzymes and the very large number of enzymes produced by T. reesei suggest that a complex regulatory and signalling network exists in order to maintain coordinated expression of the genes and production of the enzymes. Several regulator genes affecting the expression of cellulase and hemicellulase genes have been identified by us and others over the years, such as the positively acting xyr1 and ace2, and the negatively acting cre1 and ace1. Our results have brought new insights in the understanding of the regulation of the (hemi)cellulolytic system of T. reesei. Novel regulators have been identified, including essential positively acting regulators that can be utilised in further improvement of the production host organism.

Ongoing work focusing on the interplay of the known regulatory factors will lead to better understanding of the regulatory network involved in cellulase gene regulation. This offers tools for the generation of more efficient and robust, production strains of enzymes.

Tiina Pakula

Dr. Tiina Pakula (Principal Scientist) has worked at VTT Technical Research Centre of Finland Ltd since 1994. Her current research topics are related to protein production in microbial host organisms, especially in the filamentous fungus Trichoderma reesei. Application of systems biology approaches in order to understand the cellular processes and to improve the properties of the organism are the main topics of interest. tiina.pakula@vtt.fi

 

 

Additional information and reading:

Home page of VTT Expression Service

Häkkinen, M., Arvas, M., Oja, M., Aro, N., Penttilä, M., Saloheimo, M., and  Pakula, T. 2012. Re-annotation of the GAZy genes of Trichoderma reesei and transcription in the presence of lignocellulosic substrates.Microbial Cell Factories 11, 134.

Häkkinen, M.,  Valkonen, M. J., Westerholm-Parvinen, A., Aro, N., Arvas, M., Vitikainen, M., Penttilä, M., Saloheimo, M. and Pakula, T. M. 2014. Screening of candidate regulators for cellulase and hemicellulase production in Trichoderma reesei and identification of a factor essential for cellulase production.  Biotechnology for Biofuels 7,14.

Häkkinen, M.,  Sivasiddhartan, D., Aro, N., Saloheimo, M. and Pakula, T. M. 2015. The effects of extracellular pH and of the transcriptional regulator PACI on the transcriptome of Trichoderma reesei. Microbial Cell Factories  14, 63.

 

Bringing Mould to Work: Upgrading Citrus Peel and Sugar Beet Pulp

by Peter Richard

For regular households, mould in the food is hardly considered good news. For us research scientists it’s another story. Researching the mould on your onion can open the way to produce chemicals from agricultural wastes that can replace chemicals made from fossil oil.

115 million tons of citrus fruit and 270 million tons of sugar beet are produced annually worldwide which leaves a lot of citrus peel and sugar beet pulp. These raw materials are rich in pectin but of not much use. Sugar beet pulp is dried and used as animal feed. It has to be dried since it otherwise would rot; however drying is very energy consuming. Citrus peel can also be used as animal feed but is often just dumped which is problematic because of the odour. The pectin from citrus peel is sometimes extracted and used as a gelling agent in food, such as jams or marmalades. For this application only 40 – 50 thousand tons are used, i.e. there is a lot of pectin in the world that is unused.

OnionThe mould Aspergillus niger, the black mould, is a common contaminate of foods. If you find black spots on the onion in your kitchen, this is most likely Aspergillus niger. This mould is however also a useful microorganism that is used to produce citric acid. Since the 1930’s citric acid is produced in industrial scale using Aspergillus niger. It is used in food as an acidifier and especially much in soft drinks. Almost all the commercial citric acid, which is currently about 1.6 million tons per year is produced by this mould.

Aspergillus niger ferments also very efficiently pectin which is a polymer made out of D-galacturonic acid monomers. The mould produces pectinase enzymes that hydrolyse the pectin to D-galacturonic acid (oxidised sugar) which is then used as an energy source for growth. We thought that if we want to make something useful out of pectin or D-galacturonic acid, this is a suitable organism. We succeeded to genetically modify the mould so that it would convert D-galacturonic acid to different products, for example galactaric acid which is also called mucic acid.

With the engineered mould the process to produce galactaric acid is very simple. Galactaric acid is not soluble in water at acidic pH which facilitates the downstream processing. The substrates are all soluble and the mould operates well at acidic conditions.

Currently galactaric acid is produced chemically by oxidising the sugar D-galactose using nitric acid. This process is environmentally challenging. Galactaric acid is currently used in skincare products.  In the past, i.e. in the 1930’s it was also used as a food component in self-rising flour. In Mobile, a city in the south of the USA, D-galactose from a wood processing plant was oxidised chemically to produce about 600 tonnes galactaric acid per year.

Besides these applications galactaric acid can be converted chemically to other compounds like adipic acid or furan dicarboxylic acid, FDCA. Adipic acid is used for Nylon. FDCA can also be polymerised. The polymer has good gas barrier properties and can be used for bottles for carbonated drinks. This could replace the PET bottles that are currently used.

Reference: Mojzita et al.Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate. Appl Environ Microbiol. 2010, 76(1):169-75.

100_0046If you want more information about the fungal pathways and D-galacturonic acid catabolism contact the author Dr. Peter Richard. He works at VTT Technical Research Centre of Finland Ltd. as Principal Scientist in Metabolic Engineering research team. His main interests are metabolic engineering of yeast and mould for the production of fuels and chemicals. peter.richard@vtt.fi

A Fresh Look at Old Beer

by Brian Gibson

Archaeological brewing at VTT

The work of an archaeologist is complicated by the fact that the materials under investigation are invariably imperfect. Metal artefacts rust, wood decomposes, DNA degrades, bones and clay vessels shatter. Time leaves only a suggestion of the original materials’ properties and the archaeologist is left to conclude as much as possible from the limited information available. However difficult, with the right approach one can learn much from ancient materials regardless of the size or condition. In this post I write about two of our recent archeological brewing projects: shipwreck beer and sahti.

Shipwreck beer

Shipwreck bottle C49How to interpret information from compromised samples was an issue faced by a team at VTT after receiving two bottles from the ‘Champagne wreck’ – the remains of a schooner found on the sea bed near the Åland Islands in the Baltic sea. Archaeological analysis dated the wreck to sometime in the 1840s. The bottles appeared to contain beer but sensory analysis was impossible due to extensive bacterial spoilage of the beer. Trained beer tasters described the contents in less than favourable terms: rancid, goaty, sulphuric. With such degraded samples it was difficult to imagine how the beer would have originally tasted or how it could be recreated.

The first challenge of the VTT team, led by Dr John Londesborough, was to determine if the contents were, in fact, beer. This was done through sugar analysis which revealed traces of maltose and maltotriose, which are the main sugars present in beer. Further confirmation came from collaboration with partners at the University of Munich who showed unequivocally that the samples contained hops, one of the main ingredients of beer and one which is not found in other beverages. Having established that the samples were beer, it was necessary to determine the level of sample degradation. In particular, it was crucial to determine the level of seawater contamination. The bottles recovered were sealed with corks, which offered only limited protection against the entry of seawater. By determining the concentration of sodium in the beer it was possible to calculate the level of NaCl (salt) and therefore seawater contamination, which in both bottles was approx. 30%.

Vrak Öl Sample collectionAnalysis in Munich  showed that the hop components were extensively degraded, but the concentrations and kinds of degradation compounds could be used to determine what the original compounds were. The presence of hop iso-α-acids indicated that the wort had been boiled prior to fermentation.  A relatively high level of β-acids showed that the hops were less refined than today’s varieties (β-acids have been bred out of modern hop varieties because of their harsh taste). The ethanol concentration (correcting for seawater-contamination) was a relatively mild 4.5% alcohol by volume (ABV). The fruit and floral flavours characteristic of beer were in the normal range, except that the concentrations of 2-phenylethanol were relatively high. This would have imparted a touch of rose aroma to the beers. The presence of a volatile phenol giving a slight clove aroma was also detected. This suggests that the beer may have been ale, as such flavours are not produced by lager yeast.

Detailed microbiological analysis failed to recover any viable yeast cells from the beer, though microscopy revealed abundant dead cells. Likewise, any yeast DNA that may have been present originally was absent – probably degraded enzymatically by the bacteria that appear to have been much more tolerant of the conditions in the bottles during their long storage.

Despite the many challenges faced by the VTT team that analysed the bottles, recreation of the beer, based on the physical and chemical profiling carried out at VTT (and some creative licence) was possible, and Stallhagen’s 1843 beer can now be purchased in Finnish supermarkets.  More detailed results of the study can be found in the Journal of Agriculture and Food Chemistry:

Londesborough, J., Dresel, M., Gibson, B., Holopainen, U., Mikkelson, A., Seppänen-Laakso, T., Viljanen, K., Virtanen, H., Wilpola, A., Kivilompolo, M., Hoffmann, T. & Wilhelmson, A. (2015) Analysis of beers from an 1840s’ shipwreck. Journal of Agriculture and Food Chemistry, DOI: 10.1021/jf5052943.

Sahti: a part of Finland’s living heritage

In 1938, fishermen off the coast of South Africa hauled up an unusual fish. This find was exceptional not because the fish was unknown to science but rather because the species was known only from the fossil record. It had been believed that this fish, the coelacanth Latimeria chalumnae had become extinct 65 million years previously. The coelacanth is one of a number of species that seem to have resisted the effects of evolution over a huge stretch of time. The gingko tree is another notable example which has survived, essentially in the same form, for 170 million years. Darwin, in the Origin of Species wrote:

“…these anomalous forms may almost be called living fossils; they have endured to the present day, from having inhabited a confined area, and from having thus been exposed to less severe competition.”

Such species are uniquely valuable as they provide a direct link to the past. One does not need to base conclusions on scraps of information from long dead and degraded material but can study the real thing in detail. Such studies provide not just a glimpse into the past but also a clearer understanding of how evolutionary forces have shaped modern species; relatives of the lobe-finned coelacanth eventually became the first land animals.  The concept of the living fossil is not necessarily restricted to biological species but can also be applied to the cultural sphere; many traditions, customs, languages, celebrations, etc. have survived unchanged over long periods and offer a unique glimpse into the past. One can even apply this concept to brewing of beer. One relevant example is Finland’s own sahti beer, which appears to have been brewed in more or less the same way for hundreds of years.

Sahti GlassSahti is one of the few ‘ancient’ beer styles still produced in Europe and differs in many respects to modern beers. The main difference is that hops are not an essential ingredient in sahti. Hops have been used to add bitterness and aroma to beer since around 800 AD. Prior to this, a diverse range of ingredients were utilized to add bitterness and balance to beer flavour. These included anise, heather, cinnamon, clove, coriander, liquorice and, as in the case of sahti, juniper. Sahti’s method of production pre-dates the use of hops and is often described as an ancient beer style for exactly this reason. In the EU, sahti is protected by ‘Traditional Speciality Guaranteed’ label, implying that commercial use of the name is restricted to beer produced according to the traditional, registered production method. But what exactly is sahti and how does it differ from modern beers? These questions motivated scientists at VTT to carry out the first comprehensive physical and chemical study of sahti beer.

By analysing samples collected throughout the traditional sahti brewing region of southern and south-western Finland, the researchers showed that, in every respect, sahti was different to commercially available lager, wheat and porter beers. All 12 sahti beers were strong with average alcohol levels of 8% ABV but also with high levels of residual sugars, in some cases more than 100g per litre. Sahti is typically brewed with baker’s yeast rather than brewer’s yeast and this imparts specific flavour characteristics to the beer. In particular, sahti beers contain a trace of clove aroma, which is normally only found in wheat beers. Like English ales, sahti beers have little or no foam – in this case probably because of the high sugar concentration and the absence of wort boiling, both of which are expected to limit foam stability. Other unique features of sahti are a very low level of bitterness due to the absence of hops and an intense fruit flavour due to esters produced by the yeast during fermentation. In some cases the concentrations of fruit compounds were more than ten times higher than in commercial beers. These compounds give sahti its intense banana aroma for example.

As sahti wort is not boiled and contains little or no hops (a natural antimicrobial agent), sahti beer tends to stay fresh for only a short period. The ephemeral nature of the product has probably contributed to its limited geographic distribution and this may, as Darwin contended for biological species, explain why this beer has not ‘evolved’ in the same way as other European beer styles.

Our study has emphasised the uniqueness of sahti beer compared to modern beers and has introduced the international brewing research community to a very individual style of beer and an important part of Finland’s living heritage. Results appear in an upcoming issue of the Journal of the Institute of Brewing:

Jukka Ekberg, Brian Gibson, Jussi Joensuu, Kristoffer Krogerus, Frederico Magalhães, Atte Mikkelson, Tuulikki Seppänen-Laakso & Arvi Wilpola (2015) Physicochemical characterization of sahti, an ‘ancient’ beer style indigenous to Finland. J Inst Brew. In press.

Brian GibsonDr. Brian Gibson has worked at VTT Technical Research Centre of Finland, Ltd. as Senior Scientist and Project Manager since 2009. Brian is responsible mainly for projects relating to brewing yeast biology, fermentation efficiency and beer quality. Current topics of interest include improvement of yeast performance through hybridization and/or adaptive evolution as well as optimization of process conditions. The underlying mechanisms (genetic, molecular, physiological) that govern yeast performance are a main topic of interest. brian.gibson@vtt.fi

Link to Brian’s publications

 

 

 

CoReCo – New metabolic modelling tool for the production strain development

by Mikko Arvas

 
Metabolic engineering is required to make a microbe to produce a new chemical or to improve the production of an existing product. But how to select the right genes and pathways to be engineered?

 
Stoichiometric metabolic modelling encompasses numerous techniques to make these selections using state-of-the-art computational tools and databases of chemical reactions and compounds. At the heart of stoichiometric metabolic modelling are the metabolic models of organisms. In order to model the metabolism of an organism and hence to select the required genetic modifications a metabolic model for that organism is required.

 
Our task at VTT is to develop products, production strains and production processes for the biotechnology industry using microbial production systems. Our focus is on the production of bulk chemicals (for example polymer precursors or biofuels) and proteins (for example biomass degrading enzymes such as cellulases).

 
In collaboration with Aalto University and University of Helsinki, we have developed a novel tool, CoReCo (Comparative ReConstruction), to reconstruct genome wide metabolic models from genome sequence alone (Figure). Unlike previous tools it takes into account information from related species through a phylogenetic approach and verifies the correctness of reactions by atom-maps.

FigureFigure. CoReCo (Comparative ReConstruction) process. Sequence homology searches (InterProScan, Blast and GTG) are carried out for a set of genomes i.e. a genome of interest and some related genomes. A probabilistic model is built for the presence of each enzyme in the set of species and their ancestors. After that atom mapped, electron and element balanced reactions are taken from a reaction database (for example KEGG) to reconstruct a metabolic network of the reactions that the enzymes can carry out. The end product of the process is a Systems Biology Markup Language – model which contains the stoichiometric matrix required for stoichiometric modelling.

 
We have demonstrated the functionality and usability of the reconstructed models with computational steady-state biomass production experiments (Pitkänen et al. (2014) PLoS Computational Biology, 10(2), e1003465). For example, we show that functional models can be built for species that are very distant from major model organisms such as baker’s yeast and for incomplete genome sequences. After the publication we have carried out extensive development of bacterial and fungal reaction databases and also made algorithmic improvements.

 
With novel long read sequencing techniques such as PacBIO, purchasing a high quality genome for a micro-organism starts to be very cost efficient. For example, a yeast genome costs around 5 – 10 000 €. This opens up efficient genetic modifications techniques and now also, with CoReCo, metabolic modelling for any cultivable micro-organism.

 
Well-established microbial production organisms, such as baker’s yeast, are heavily patented and only represent a tiny fraction of natural variability of metabolism. Therefore, exploration of novel production organisms utilizing CoReCo represents considerable opportunities for the industrial biotechnology sector to create new production strains and IPR.

Mikko photoAsk more information about CoReCo and metabolic modelling from the author Senior Scientist Dr Mikko Arvas. He has background in genetics, but for the last ten years he has concentrated on computational genome analysis of fungi for the needs of industrial biotechnology.

Great future ahead with the help of Plant Molecular Farming!

by Anneli Ritala and Suvi T. Häkkinen

Green

Roughly a year ago, we wrote a commentary “Molecular pharming in plants and plant cell cultures – a great future ahead?” to Pharmaceutical Bioprocessing [1]. And now a huge breakthrough has been made with the help of Plant Molecular Pharming: Two Ebola patients were saved with a plant-made antibody that is still in the experimental phase.

Pharming or Farming?

The term Molecular Pharming is used to highlight the production of protein-based biopharmaceuticals, which contribute to the sustainable production of drugs that promote human and animal wellbeing. It also applies to the production of valuable secondary metabolites such as the analgesic drug morphine and the anticancer drugs paclitaxel, vincristine and vinblastine which are far too complex molecules to be synthetized in an economically feasible way.

In broader perspective, term Molecular Farming can be used in the context of utilization of the versatility of plants and plant cells to produce diverse valuable proteins and other compounds for any applications. Thus Molecular Farming covers also other fields than pharmaceuticals and describes better the approach taken at VTT Ltd.

Biopharmaceuticals

Biopharmaceuticals are on the commercial forefront of the pharmaceutical sector and roughly 30% of the new drugs under development belong to this class. The biopharmaceutical market has been steadily rising and reached total cumulative sales of US$ 140 billion in 2013 [2].

The FDA (The US Food and Drug Administration) and EMA (The European Medicines Agency) are already familiar with the two major biopharmaceutical production systems:

  • Microbes – mainly Escherichia coli and different yeast hosts
  • Mammalian cells such as the Chinese hamster ovary (CHO) platform,

and standard protocols can be followed to ensure the approval of new products.

Currently, the equivalent protocols are emerging for plant-based production systems, and there is one plant-derived biopharmaceutical protein on the market: Elelyso™ (taliglucerase alfa). It is produced in carrot cells by the Israeli company Protalix Biotherapeutics and licensed to Pfizer Inc., and is used for the treatment of the life-threatening lysosomal storage disorder, Gaucher’s disease. The recombinant product gained FDA approval in 2012 and the product is currently for sale in USA and Israel.

Advantages of plant-based production systems

Picture2The plant-based systems are starting to compete with the above mentioned established biopharmaceutical production systems, and on a technological basis plant-based systems have the advantage in following areas:

 

  • Speed
  • Scalability
  • Improved product quality

In need-for-speed situations, like in case of epidemic diseases as Ebola and bioterrorist threats, the transient plant expression systems benefit from the rapid onset of recombinant protein production. The plant material is propagated before the introduction of foreign DNA, allowing plants to be grown in the open or in greenhouse conditions after which the plant material is moved into contained, GMP-compliant facilities for protein production.

The greatest advantage of intact plants that are stably transformed to produce a target protein is their unparalleled scalability. For biopharmaceutical products, manufacturing will probably be restricted to greenhouses and other closed environments to ensure product safety and batch-to-batch consistency when production is carried out under controlled conditions. The Canadian company SemBioSys developed a safflower-based production system for insulin before filing for bankruptcy in 2012. The SemBioSys platform was so outstandingly efficient that theoretically 16 mid-sized Canadian farms could have produced enough insulin to meet the entire exponentially growing global demand. At VTT we have taken an initiative in producing food allergen specific antibodies for diagnostic and safety verification purposes. The barley-produced antibody can recognize and precipitate beta-lactoglobulin, which is the major allergen in cow´s milk. The established platform has potential in development of hypoallergenic products for milk allergic patients [3].

The high product quality is gained with the use of plant cell suspension cultures for Molecular Pharming as well as Farming purposes. At VTT we have harnessed the traditional microbial bioreactors to cultivate plant cells at the 600-litre scale [3]. We are currently working on a pharmaceutical target, Transferrin, in a project getting financial support from the Academy of Finland. We also only recently got funding from ERA-Anihwa, and we are entering with our plant cell culture expression system on fish vaccine production which is a very relevant target for Plant Molecular Pharming . The annual loss in aquaculture caused by viral diseases is remarkable and in order to be able to keep the fast-growing aquaculture industry ecologically, environmentally and ethically sustainable, good health for farmed aquaculture organisms is essential.

Plant Molecular Farming provides a safe and sustainable platform for the production of valuable proteins and other compounds – the great future is here and we are happy to be part of it!

References

  1. Ritala A, Häkkinen ST, Schillberg S. 2014. Molecular pharming in plants and plant cell cultures: a great future ahead? Pharm. Bioprocess. 2:223-226.
  2. Walsh G. 2014. Biopharmaceutical benchmarks 2014. Nat. Biotechnol. 32:992-1000.
  3. Ritala A, Leelavathi S, Oksman-Caldentey KM, Reddy VS, Laukkanen ML. 2014. Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, ß-lactoglobulin. Transgen. Res. 23:477-487.
  4. Reuter LJ, Bailey MJ, Joensuu JJ, Ritala A. 2014. Scale-up of hydrophobin-assisted recombinant protein production in tobacco BY-2 suspension cells. Plant Biotech. J. 12:402-410.

AnneliThe author Dr. Anneli Ritala, Principal Scientist (PhD Pharm., Docent in Pharmaceutical Biology) has special expertise in production of recombinant proteins and small molecules in plant cell cultures. She has over 20 years´ experience on plant biotechnology, especially genetic and metabolic engineering of plants and plant cell cultures including barley, oats, tobacco and other medicinal plants. anneli.ritala@vtt.fi

SuviThe author Dr. Suvi T. Häkkinen, Senior Scientist (D.Sc.(Tech)) has special expertise in medicinal plants and natural compound research. She obtained her doctoral degree for her work related to alkaloid biosynthesis and she has over 15 years´ experience on plant biotechnology including metabolic engineering, recombinant protein production and plant cell culture technology. suvi.hakkinen@vtt.fi

 

 

How new yeast species are inspiring a revolution in brewing

by Kristoffer Krogerus

Lager beers – sometimes crisp & light pilsners, sometimes dark & malty doppelbocks, have a common denominator: They are all produced using the lager yeast Saccharomyces pastorianus, the workhorse of the lager brewing industry. This yeast is known for its tolerance to lower temperatures, and brewers take advantage of this when producing lager beers.

These beers typically have a ‘clean’ flavour profile (i.e. lack of yeast character) you see, and by fermenting the beer at colder temperatures, the yeast produces less flavour-active by-products.

Blog Image 5

Recent analysis of lager brewing yeast genomes has revealed that the many hundreds of strains used in the brewing industry are, in fact, all closely related – more like multiple variants of the same strain than individual strains. Brewers have essentially been using the same strain to brew lager beers for probably 500 years. This is in stark contrast to the other fermented beverage industries, ale, whiskey, wine, cider and so on, where a rich and diverse collection of individual yeast strains is taken for granted.

Therefore, there is huge potential for introducing diversity into the lager brewing industry by generating new strains of lager yeast.

But before one can create new lager yeast it is important to understand what exactly the lager yeast is…

It has been known for some time that lager yeast is actually a hybrid species – more like a mule than the proverbial workhorse. It was clear that one parent was the well-known ale yeast Saccharomyces cerevisiae. It wasn’t until recently that the other side of the family, Saccharomyces eubayanus, was discovered. This discovery has allowed for the improved characterization of lager yeasts, and also opened up the possibility to create new tailor-made lager yeast strains. This is possible through mating of selected strains from the two parent species. These new strains could, e.g. produce unique flavours or ferment the beer more efficiently.

This is exactly what has been the focus of our ongoing research projects at VTT.

Blog Image 3The research team. From left to right: Brian Gibson, Kristoffer Krogerus, Virve Vidgren and Frederico Magalhães in VTT’s pilot brewery.

Screening perfect parents to mate

There are four main challenges in generating new lager yeasts: To select suitable parent strains. To get the parents to mate. To separate the hybrid cells from the parents. And finally, to confirm that they actually are hybrids.

We began by screening a range of ale yeast strains, from both VTT’s Culture Collection and commercial yeast suppliers, for beneficial fermentation properties. Once suitable parent ale yeast strains had been identified, the next step was to try to mate them with a strain of S. eubayanus, the other parent of lager yeast.

Before mating, the parent strains still had to be modified with selection markers, so that any hybrid cells could be isolated from the population. We did this by selecting spontaneous auxotrophic mutants of the parent strains, i.e. cells that weren’t able to grow on media lacking certain amino acids. This meant the hybrid cells could be selected by their ability to grow on media lacking these certain amino acids. Mating was then attempted by simply mixing populations of both parent strains, and letting them grow for a couple of days.

After isolating some potential hybrid cells, their hybrid status was confirmed through various PCR tests, which showed whether DNA from both parent strains was present in them. After confirmation that we had produced our own lager yeast hybrids, we wanted to compare them to the parent strains in an actual wort fermentation.

To our pleasant surprise, all hybrid strains performed better than both parent strains, fermenting faster and reaching higher ethanol contents!

The hybrid strains also inherited beneficial properties from both parent strains, such as strong flocculation, cold tolerance and maltotriose utilization.

These first results suggest that this technique is suitable for producing new lager yeast strains with unique properties. These new strains also have the benefit of being non-GMO, which currently at least remains a necessity for brewers.

We are continuing our attempts to find and create perfect lager yeast hybrids at VTT. Our research will especially pay attention to flavour formation and determining how their genetic composition is reflected in their physiology.

Our work will show, for the first time, that such hybrids can be created and how they can be applied in the brewing industry. The results will appear shortly in the Journal of Industrial Microbiology and Biotechology: Krogerus, K., Magalhães, F., Vidgren, V. & Gibson, B. (2015) New lager yeast strains generated by interspecific hybridization. Journal of Industrial Microbiology and Biotechnology, in press. DOI:10.1007/s10295-015-1597-6.

Maybe someday also you have an opportunity to enjoy these new tasty lager beers in your local pub. Cheers!

Blog Image 2

The author Kristoffer Krogerus is first-year PhD student working at VTT Industrial Biotechnology. His supervisor Dr. Brian Gibson is Senior scientist and project manager with responsibility for projects relating to brewing yeast physiology and fermentation.

 

ext-kristoffer.krogerus@vtt.fi , brian.gibson@vtt.fi

www-pages of VTT’s brewing yest development