Anneli Ritala

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T0), and four of the 90 T0 spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T1). The four transgenic shoots of Toivo (the T0 plant) produced 25 plantlets as T1 progeny. Altogether fifteen of these T1 plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T2 progeny.

Recent advances in the malting and brewing industry

Brewing is often mentioned as a typical example of traditional or old biotechnology, because of its extremely long history. However, the modern malting and brewing industry applies a whole spectrum of new technical, biochemical, microbiological and genetic inventions. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Malted barley contains all the enzymes needed for all-malt brews. Exogenous microbial enzymes may be needed when using high amounts of cereal adjuncts. Transgenic barleys and proper starter cultures in malting offer interesting new possibilities to ensure balanced enzyme activities and to avoid harmful Fusarium contaminations. High gravity brewing, automated mash filters and hop extracts produced with super-critical or liquid carbon dioxide have been adopted by the industry. Continuous bioreactors with immobilized yeast are already used for maturation of beer. The residence time in the bioreactor is only 2 h, whereas several weeks are needed for traditional lagering. Continuous main fermentation with immobilized yeast is the next step. Several genetically modified brewers yeasts have been constructed, e.g. yeasts encoding α-acetolactate decarboxylase and super-flocculating yeasts. The brewing industry is now waiting to be assured of consumer approval.Abstract Brewing is often mentioned as a typical example of traditional or old biotechnology, because of its extremely long history. However, the modern malting and brewing industry applies a whole spectrum of new technical, biochemical, microbiological and genetic inventions. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Malted barley contains all the enzymes needed for all-malt brews. Exogenous microbial enzymes may be needed when using high amounts of cereal adjuncts. Transgenic barleys and proper starter cultures in malting offer interesting new possibilities to ensure balanced enzyme activities and to avoid harmful Fusarium contaminations. High gravity brewing, automated mash filters and hop extracts produced with super-critical or liquid carbon dioxide have been adopted by the industry. Continuous bioreactors with immobilized yeast are already used for maturation of beer. The residence time in the bioreactor is only 2 h, whereas several weeks are needed for traditional lagering. Continuous main fermentation with immobilized yeast is the next step. Several genetically modified brewers yeasts have been constructed, e.g. yeasts encoding α -acetolactate decarboxylase and super-flocculating yeasts. The brewing industry is now waiting to be assured of consumer approval.

Review articleRecent advances in the malting and brewing industry1

Brewing is often mentioned as a typical example of traditional or old biotechnology, because of its extremely long history. However, the modern malting and brewing industry applies a whole spectrum of new technical, biochemical, microbiological and genetic inventions. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Malted barley contains all the enzymes needed for all-malt brews. Exogenous microbial enzymes may be needed when using high amounts of cereal adjuncts. Transgenic barleys and proper starter cultures in malting offer interesting new possibilities to ensure balanced enzyme activities and to avoid harmful Fusarium contaminations. High gravity brewing, automated mash filters and hop extracts produced with super-critical or liquid carbon dioxide have been adopted by the industry. Continuous bioreactors with immobilized yeast are already used for maturation of beer. The residence time in the bioreactor is only 2 h, whereas several weeks are needed for traditional lagering. Continuous main fermentation with immobilized yeast is the next step. Several genetically modified brewers yeasts have been constructed, e.g. yeasts encoding α-acetolactate decarboxylase and super-flocculating yeasts. The brewing industry is now waiting to be assured of consumer approval.Abstract Brewing is often mentioned as a typical example of traditional or old biotechnology, because of its extremely long history. However, the modern malting and brewing industry applies a whole spectrum of new technical, biochemical, microbiological and genetic inventions. Examples of contemporary achievements can be found along the whole production chain from barley to beer. Malted barley contains all the enzymes needed for all-malt brews. Exogenous microbial enzymes may be needed when using high amounts of cereal adjuncts. Transgenic barleys and proper starter cultures in malting offer interesting new possibilities to ensure balanced enzyme activities and to avoid harmful Fusarium contaminations. High gravity brewing, automated mash filters and hop extracts produced with super-critical or liquid carbon dioxide have been adopted by the industry. Continuous bioreactors with immobilized yeast are already used for maturation of beer. The residence time in the bioreactor is only 2 h, whereas several weeks are needed for traditional lagering. Continuous main fermentation with immobilized yeast is the next step. Several genetically modified brewers yeasts have been constructed, e.g. yeasts encoding α -acetolactate decarboxylase and super-flocculating yeasts. The brewing industry is now waiting to be assured of consumer approval.

Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts

SummaryProtoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NPTII activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.

Production of a recombinant industrial protein using barley cell cultures.

The use of recombinant DNA-based protein production using genetically modified plants could provide a reproducible, consistent quality, safe, animal-component free, origin-traceable, and cost-effective source for industrial proteins required in large amounts (1000s of metric tons) and at low cost (below US

Plant cells as pharmaceutical factories.

100/Kg). The aim of this work was to demonstrate the feasibility of using barley suspension cell culture to support timely testing of the genetic constructs and early product characterization to detect for example post-translational modifications within the industrial protein caused by the selected recombinant system. For this study the human Collagen I alpha 1 (CIa1) chain gene encoding the complete helical region of CIa1 optimized for monocot expression was fused to its N- and C-terminal telopeptide and to a bacteriophage T4 fibritin foldon peptide encoding sequences. The CIa1 accumulation was targeted to the endoplasmic reticulum (ER) by fusing the CIa1 gene to an ER-directing signal peptide sequence and an ER retention signal HDEL. The construct containing the CIa1 gene was then introduced into immature barley half embryos or barley cells by particle bombardment. Transgenic barley cells resulting from these transformations were grown as suspension cultures in flasks and in a Wave bioreactor producing CIa1 similar to CIa1 purified from the yeast Pichia pastoris based on Western blotting, pepsin resistance, and mass spectroscopy analysis. The barley cell culture derived-CIa1 intracellular accumulation levels ranged from 2 to 9 microg/l illustrating the need for further process improvement in order to use this technology to supply material for product development activities.

Production of a recombinant full-length collagen type I α-1 and of a 45-kDa collagen type I α-1 fragment in barley seeds

Molecules derived from plants make up a sizeable proportion of the drugs currently available on the market. These include a number of secondary metabolite compounds the monetary value of which is very high. New pharmaceuticals often originate in nature. Approximately 50% of new drug entities against cancer or microbial infections are derived from plants or micro-organisms. However, these compounds are structurally often too complex to be economically manufactured by chemical synthesis, and frequently isolation from naturally grown or cultivated plants is not a sustainable option. Therefore the biotechnological production of high-value plant secondary metabolites in cultivated cells is potentially an attractive alternative. Compared to microbial systems eukaryotic organisms such as plants are far more complex, and our understanding of the metabolic pathways in plants and their regulation at the systems level has been rather poor until recently. However, metabolic engineering including advanced multigene transformation techniques and state-of-art metabolomics platforms has given us entirely new tools to exploit plants as Green Factories. Single step engineering may be successful on occasion but in complex pathways, intermediate gene interventions most often do not affect the end product accumulation. In this review we discuss recent developments towards elucidation of complex plant biosynthetic pathways and the production of a number of highvalue pharmaceuticals including paclitaxel, tropane, morphine and terpenoid indole alkaloids in plants and cell cultures.

Molecular Farming in Tobacco Hairy Roots by Triggering the Secretion of a Pharmaceutical Antibody

Recombinant DNA technology can be used to design and express collagen and gelatin-related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full-length collagen type I alpha1 (rCIa1) and a related 45-kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45-kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen-related structural proteins. The 45-kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin, seed endosperm-specific rice glutelin and germination-specific barley alpha-amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the lowest accumulation was obtained with the alpha-amylase promoter. To induce homozygosity for stable 45-kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45-kDa rCIa1 expression levels achieved from the best DH lines were 13 mg/kg dry seeds under the ubiquitin promoter and 45 mg/kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post-translational modifications.

Metabolic engineering of plant secondary products: which way forward?

Recombinant pharmaceutical proteins expressed in hairy root cultures can be secreted into the medium to improve product homogeneity and to facilitate purification, although this may result in significant degradation if the protein is inherently unstable or particularly susceptible to proteases. To address these challenges, we used a design of experiments approach to develop an optimized induction protocol for the cultivation of tobacco hairy roots secreting the full‐size monoclonal antibody M12. The antibody yield was enhanced 30‐fold by the addition of 14 g/L KNO3, 19 mg/L 1‐naphthaleneacetic acid and 1.5 g/L of the stabilizing agent polyvinylpyrrolidone. Analysis of hairy root cross sections revealed that the optimized medium induced lateral root formation and morphological changes in the inner cortex and pericycle cells, indicating that the improved productivity was at least partially based on the enhanced efficiency of antibody secretion. We found that 57% of the antibody was secreted, yielding 5.9 mg of product per liter of induction medium. Both the secreted and intracellular forms of the antibody could be isolated by protein A affinity chromatography and their functionality was confirmed using vitronectin‐binding assays. Glycan analysis revealed three major plant complex‐type glycans on both forms of the antibody, although the secreted form was more homogeneous due to the predominance of a specific glycoform. Tobacco hairy root cultures therefore offer a practical solution for the production of homogeneous pharmaceutical antibodies in containment. Biotechnol. Bioeng. 2014;111: 336–346.

Metabolic flux phenotype of tobacco hairy roots engineered for increased geraniol production

Secondary products are small molecular weight compounds produced by secondary metabolic pathways in plants. They are regarded as non-essential for normal growth and development but often confer benefits such as defense against pathogens, pests and herbivores or the attraction of pollinators. Many secondary products affect the survival and/or behavior of microbes, insects and mammals and they often have useful pharmacological effects in humans. Most secondary products can only be obtained as extracts from medicinal plants, many of which grow slowly and are difficult to cultivate. Chemical synthesis, although possible in principle, is often impractical or uneconomical due to the complexity of their molecular structures. The large scale production of secondary products by metabolic engineering has therefore been investigated in a number of heterologous systems including microbes, plant cell/organ cultures, and intact plants. In this critical review of production platforms for plant secondary products, we discuss the advantages and constraints of different approaches and the impact of post-genomics technologies on gene discovery and metabolite analysis. We highlight bottlenecks that remain to be overcome before the routine exploitation of secondary products can be achieved for the benefit of mankind.