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Preface
The incidental use of yeast for fermented products can be traced back to about 6,000 years ago. However, the definition of yeast as living organism and as responsible for sugar fermentation became clear only in the 1800s, when considerable attention was paid—especially for economic reasons—to the study of fermentation, aiming at preventing spoilage of wines and other alcoholic beverages. Those studies have been seminal for a better understanding of the fermentation process and of the role of yeast, and further steps forward have been made with the discovery and isolation at the beginning of the 1900s of different yeast species and strains with peculiar properties. From those years and on, the science of yeast has never stopped. Thanks to the development of novel molecular biology techniques and the availability of the complete genome sequence of Saccharomyces cerevisiae, yeast has been used both as a model organism for higher eukaryotes and as a work horse microorganism for diverse industrial productions, ranging from proteins to metabolites with diverse applications. The branch of technologies and techniques that have brought the use of yeast to several production processes goes under the name of Metabolic Engineering, whose aim is to modify and tune yeast metabolism according to the production target.
Several publications already exist on this topic, but technologies and methods are continuously being developed and improved. Therefore, this volume is intended to provide an overview of the widely established basic tools used in yeast metabolic engineering; while describing in deeper detail novel and innovative methods and protocols that have a valuable potential to improve metabolic engineering strategies aiming at industrial biotechnology applications.
With this perspective, the first part of the volume tries to give an overview of the basic tools existing for S. cerevisiae metabolic engineering, such as selection markers and engineered promoters, aiming to give the reader a sort of compendium that collects such tools which will always remain fundamental in the field. On the other hand, novel metabolic engineering techniques and technologies, such as the use of RNA switches and the generation of arming yeasts, are described in the form of detailed protocols, as they are not commonly established yet and their potential might be great for certain applications.
Although S. cerevisiae is the species to which the word “yeast” is commonly referred to, other yeast genera and species are receiving increasing interest thanks to their peculiar features conferring them high potential for specific biotechnological applications. Therefore, particular focus is given to protocols that can be used when dealing with metabolic engineering of Komagataella spp. (formerly known as Pichia spp.), Hansenula polymorpha, and Zygosaccharomyces bailii.
The reader familiar with laboratory practices is also aware of the fact that often the protocols developed for the so-called laboratory yeast strains are not easily transferable to wild or industrial yeasts, which are known to be genetically more complex. For this reason, a few chapters provide protocols for the engineering of industrial strains also presenting an innovative protocol for the optimization of fed-batch fermentations with Pichia pastoris.
While the first section provides the tools for engineering yeasts, the second section (Tools and technologies for investigation and determination of yeast metabolic features)
provides detailed protocols established to identify and evaluate the actual metabolic changes generated through genetic engineering. In particular, a protocol for metabolic flux analysis is described using the yeast P. pastoris as a case study, and a specific metabolite profiling method is reported also providing a summary of existing methodologies for yeast metabolome analysis. Since one of the most challenging steps in metabolome studies is the analysis of the resulting huge amount of data, it has been considered worthwhile to dedicate one full chapter to a novel bioinformatics tool for processing and understanding metabolome data.
Along the bioinformatics line, the third section of the volume deals with Metabolic models for yeast metabolic engineering, which are more and more popular for the initial definition and the improvement of metabolic engineering strategies. The two chapters focusing on this topic provide an overview on how genome-scale metabolic models are constructed and show a metabolic engineering application that has been developed exploiting yeast metabolic models and the related bioinformatics tools.
Since the topics in this volume have been treated giving considerable relevance to the industrial application of the metabolically engineered yeasts, the editor thought that some space, though little, could be given to the patenting practice as conclusion of the volume. It might not look a proper conclusion in a book of methods and protocols, but the editor’s personal opinion is that knowing the fundamental principles of patenting the products resulting from laboratory investigation can be extremely useful also in guiding the choice of the methods that the researchers intend to use in their research.
In conclusion, I would like to thank all the researchers and authors who contributed with enthusiasm, patience, and professionalism to this volume, willing to share the protocols they developed and the knowledge they hold with the scientific community. It has been a real pleasure dealing with such people. Furthermore, last but not least, I would like to thank Dr. John Walker, the Editor-in-Chief of the Methods in Molecular Biology series, for his continued trust and support.
Gothenburg, Sweden
Valeria Mapelli
Preface .
Contributors.
PART I MOLECULAR TOOLS AND TECHNOLOGY FOR YEAST ENGINEERING
1 An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae
Verena Siewers
2 Natural and Modified Promoters for Tailored Metabolic Engineering of the Yeast Saccharomyces cerevisiae 17
Georg Hubmann, Johan M. Thevelein, and Elke Nevoigt
3 Tools for Genetic Engineering of the Yeast Hansenula polymorpha .
Ruchi Saraya, Loknath Gidijala, Marten Veenhuis, and Ida J. van der Klei
4 Molecular Tools and Protocols for Engineering the Acid-Tolerant Yeast Zygosaccharomyces bailii as a Potential Cell Factory.
Paola Branduardi, Laura Dato, and Danilo Porro
5 Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris
Michael Felber, Harald Pichler, and Claudia Ruth
6 Methods for Efficient High-Throughput Screening of Protein Expression in Recombinant Pichia pastoris Strains.
Andrea Camattari, Katrin Weinhandl, and Rama K. Gudiminchi
8 Generation of Arming Yeasts with Active Proteins and Peptides via Cell Surface Display System: Cell Surface Engineering, Bio-arming Technology
Kouichi Kuroda and Mitsuyoshi Ueda
9 Genetic Engineering of Industrial Saccharomyces cerevisiae Strains Using a Selection/Counter-selection Approach.
Dariusz R. Kutyna, Antonio G. Cordente, and Cristian Varela
10 Evolutionary Engineering of Yeast.
Ceren Alkım, Burcu Turanlı-Yıldız, and Z. Petek Çakar
11 Determination of a Dynamic Feeding Strategy for Recombinant Pichia pastoris Strains.
Oliver Spadiut, Christian Dietzsch, and Christoph Herwig
PART II TOOLS AND TECHNOLOGIES FOR INVESTIGATION AND DETERMINATION OF YEAST METABOLIC FEATURES
12 Yeast Metabolomics: Sample Preparation for a GC/MS-Based Analysis . . . . . . 197
Sónia Carneiro, Rui Pereira, and Isabel Rocha
13 13C-Based Metabolic Flux Analysis in Yeast: The Pichia pastoris Case.
Pau Ferrer and Joan Albiol
209
14 Pathway Activity Profiling (PAPi): A Tool for Metabolic Pathway Analysis. . . . 233
Raphael B.M. Aggio
15 QTL Mapping by Pooled-Segregant Whole-Genome Sequencing in Yeast. . . . 251
Thiago M. Pais, María R. Foulquié-Moreno, and Johan M. Thevelein
PART III METABOLIC MODELS FOR YEAST METABOLIC ENGINEERING
16 Genome-Scale Metabolic Models of Yeast, Methods for Their Reconstruction, and Other Applications.
Sergio Bordel
17 Model-Guided Identification of Gene Deletion Targets for Metabolic Engineering in Saccharomyces cerevisiae
Ana Rita Brochado and Kiran Raosaheb Patil
PART IV PATENTING AND REGULATIONS
18 Patents: A Tool to Bring Innovation from the Lab Bench to the Marketplace
Z. Ying Li and Wolfram Meyer
Index
Contributors
RAPHAEL B.M. AGGIO • Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
JOAN ALBIOL • Department of Chemical Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain
CEREN ALKIM • Department of Molecular Biology and Genetics, Faculty of Science and Letters, Dr. Orhan Öcalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University, Istanbul, Turkey
SERGIO BORDEL • Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
PAOLA BRANDUARDI • Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
ANA RITA BROCHADO • Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany
Z. PETEK ÇAKAR • Department of Molecular Biology and Genetics, Faculty of Science and Letters, Dr. Orhan Öcalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University, Istanbul, Turkey
ANDREA CAMATTARI • Graz University of Technology, Graz, Austria
SÓNIA CARNEIRO • Center of Biological Engineering, IBB Institute for Biotechnology and Bioengineering, University of Minho, Braga, Portugal
ANTONIO G. CORDENTE • The Australian Wine Research Institute, Adelaide, SA, Australia
LAURA DATO • Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
CHRISTIAN DIETZSCH • Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria
MICHAEL FELBER • Austrian Centre of Industrial Biotechnology, Graz, Austria
PAU FERRER • Department of Chemical Engineering, Escola d’Enginyeria, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Spain
MARÍA R. FOULQUIÉ-MORENO • Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium; Department of Molecular Microbiology, VIB, Flanders, Belgium
LOKNATH GIDIJALA • Molecular Cell Biology, Kluyver Centre for Genomics of Industrial Fermentation, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
RAMA K. GUDIMINCHI • Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria
CHRISTOPH HERWIG • Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria
GEORG HUBMANN • Molecular Systems Biology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium; Department of Molecular Microbiology, VIB, Flanders, Belgium
IDA J. VAN DER KLEI • Molecular Cell Biology, Kluyver Centre for Genomics of Industrial Fermentation, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
KOUICHI KURODA • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
DARIUSZ R. KUTYNA • The Australian Wine Research Institute, Adelaide, SA, Australia
Z. YING LI • Ropes & Gray LLP, New York, NY, USA
WOLFRAM MEYER • European Patent Office, Munich, Germany
JOSHUA K. MICHENER • Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA
ELKE NEVOIGT • School of Engineering and Science, Jacobs University gGmbH, Bremen, Germany
THIAGO M. PAIS • Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium; Department of Molecular Microbiology, VIB, Flanders, Belgium; Instituto de Ciências da Saúde, Universidade Federal de Mato Grosso – UFMT, Sinop, MT, Brazil
KIRAN RAOSAHEB PATIL • Structural and Computational Biology, European Molecular Biology Laboratory, Heidelberg, Germany
RUI PEREIRA • Center of Biological Engineering, IBB Institute for Biotechnology and Bioengineering, University of Minho, Braga, Portugal
HARALD PICHLER • Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria; Austrian Centre of Industrial Biotechnology, Graz, Austria
DANILO PORRO • Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy
ISABEL ROCHA • Center of Biological Engineering, IBB Institute for Biotechnology and Bioengineering, University of Minho, Braga, Portugal
CLAUDIA RUTH • Austrian Centre of Industrial Biotechnology, Graz, Austria
RUCHI SARAYA • Molecular Cell Biology, Kluyver Centre for Genomics of Industrial Fermentation, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
VERENA SIEWERS • Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden
CHRISTINA D. SMOLKE • Department of Bioengineering, Stanford University, Stanford, CA, USA
OLIVER SPADIUT • Research Area Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria
JOHAN M. THEVELEIN • Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Flanders, Belgium; Department of Molecular Microbiology, VIB, Flanders, Belgium
BURCU TURANLI-YILDIZ • Department of Molecular Biology and Genetics, Faculty of Science and Letters, Dr. Orhan Öcalgiray Molecular Biology, Biotechnology and Genetics Research Center (ITU-MOBGAM), Istanbul Technical University, Istanbul, Turkey
MITSUYOSHI UEDA • Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
CRISTIAN VARELA • The Australian Wine Research Institute, Adelaide, SA, Australia
MARTEN VEENHUIS • Molecular Cell Biology, Kluyver Centre for Genomics of Industrial Fermentation, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
KATRIN WEINHANDL • Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria
Part I
Molecular Tools and Technology for Yeast Engineering
Chapter 1
An Overview on Selection Marker Genes for Transformation of Saccharomyces cerevisiae
Verena Siewers
Abstract
For genetic manipulation of yeast, numerous selection marker genes have been employed. These include prototrophic markers, markers conferring drug resistance, autoselection markers, and counterselectable markers. This chapter describes the different classes of selection markers and provides a number of examples for different applications.
Key words Auxotrophy, Autoselection, Drug resistance, Counterselection, Marker loop-out
1 Introduction
Deletion of endogenous genes, introduction of new features into the yeast genome, as well as transformation with centromeric or episomal plasmids require the use of marker genes in order to be able to select for transformation events. While after genomic integration the new properties are usually stably inherited and the strain can be cultivated under nonselective conditions, selective conditions will in most cases have to be maintained after transformation with a non-integrative plasmid in order to avoid plasmid loss. The first marker genes used for yeast transformation were endogenous prototrophic markers, which were later complemented by dominant (mainly drug-resistance) markers and autoselection systems. In the following subchapters, different types of marker genes with their potential applications, advantages, and disadvantages are introduced.
Prototrophic marker genes are probably the most commonly used selection markers. They are usually derived from either amino acid (e.g., LEU2 , TRP1 ) or nucleotide base (e.g., URA3 , ADE2 ) biosynthesis pathways and require the availability of an auxotrophic host strain carrying a nonfunctional version or a deletion of the respective gene. Further examples are listed in Table 1.
Apart from using endogenous genes, it is also possible to complement auxotrophies in S. cerevisiae with heterologous genes. Examples that have shown sufficient activity to be used as selection markers are the URA3 gene of Kluyveromyces lactis [1] and the Schizosaccharomyces pombe his5 + gene [2], equivalents of S. cerevisiae HIS3
Some prototrophic markers allow for additional genotype screenings based on colony color. Strains carrying an inactive ade1 or ade2 allele result in red colonies due to the vacuolar accumulation of purine biosynthetic pathway precursors; adenine prototrophic colonies in contrast appear white [3]. Another example are methionine-auxotrophic met15 strains, which become black when grown in the presence of divalent lead ions (Pb2+), while their prototrophic counterparts stay white [4, 5].
3
C/N Source-Related Markers
Several genes that confer the ability to grow on certain carbon or nitrogen sources have been used as selection markers (Table 2). S. cerevisiae cells expressing FCY1 encoding cytosine deaminase and GAP1 encoding a general amino acid permease can be selected on medium containing cytosine and L-citrulline, respectively, as sole nitrogen source [16, 17]. Since both genes are present in a wild-type strain, in analogy to auxotrophic markers, the availability of a background strain carrying the respective deletion is required. On the other hand, the LAC4/LAC12 and LSD1 genes, which allow for growth on lactose and dextran as sole carbon sources, respectively, are derived from different species and do not have any equivalents in the S. cerevisiae genome [18, 19]; i.e., they represent dominant marker genes, and this feature makes them ver y attractive markers for the transformation of industrial strains.
All marker genes discussed so far rely on the use of chemically defined media for selection. When selective conditions are required for stable maintenance of centromeric or episomal plasmids, chemically defined media might not represent an obstacle for small-scale fermentations. They are however not practical for long-term plas mid maintenance in industrial processes that are normally based on complex media. Here, autoselection systems can serve as an alternative.
Gene
[ 6 ]
[ 5 ]
[ 7 ]
[ 8 ]
[ 6 ]
[ 9 ]
[ 9 ]
[ 7 ]
[ 10 ]
([ 4 , 5 ])
[ 9 ]
[ 9 ]
Table 1
Prototrophic markers
Gene
N-succinyl-5-aminoimidazole-4-carboxamide ribotide synthetase involved in purine biosynthesis w/o a adenine
Phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis w/o adenine
Phosphoribosyl-glycinamide transformylase involved in purine biosynthesis w/o adenine
Ketopantoate hydroxymethyltransferase involved in pantothenic acid biosynthesis w/o pantothenic acid
Histidinolphosphatase involved in histidine biosynthesis w/o histidine
Imidazoleglycerol-phosphate dehydratase involved in histidine biosynthesis w/o histidine
β -Isopropylmalate dehydrogenase involved in leucine biosysthesis w/o leucine
α -Aminoadipate reductase involved in lysine biosynthesis w/o lysine
Phosphopantetheinyl transferase involved in lysine biosynthesis w/o lysine
In an autoselection system (Table 3), the marker gene is essential for the viability of the cell under any (or almost any) growth condition. Thus, selection pressure can be maintained even in complex media. Furthermore, there is little risk of cross-feeding, which when using prototrophic markers even under selective conditions can lead to subpopulations of cells that have lost the marker gene while living on metabolites provided by the marker gene-carrying cells [20].
The URA3 system (see above) was modified by using a background strain, in which not only pyrimidine biosynthesis is inhibited by a ura3 mutation, but even the pyrimidine salvage pathway is inactivated through a fur1 urk1 double mutation. External supplementation with uracil, uridine, cytosine, or cytidine does therefore not enable growth in the absence of the URA3 gene, and URA3-bearing plasmids are stably maintained [21].
In several examples, glycolytic pathway genes such as FBA1, TPI1 (derived from either S. cerevisiae or a heterologous host), and PGI1 were used as marker genes and shown to provide stable plasmid maintenance in complex media [22–24]. A second group of genes used as autoselection markers are essential cell division cycle genes such as CDC4, CDC9, and CDC28 [23, 25, 26].
The construction and maintenance of the host strain used in an autoselection system can however require a special procedure, since an essential gene needs to be deleted. One possibility is the use of a strain that is still viable under specific conditions. For example, a strain carrying the srb1-1 allele, a mutation in PSA1 encoding GDP-mannose pyrophosphorylase involved in cell wall synthesis, is nonviable in the absence of osmotic stabilizers but can
POT Schizosaccharomyces pombe triose phosphate isomerase [24]
TPI A. nidulans triose phosphate isomerase [23]
PGI1 Phosphoglucose isomerase [23]
CDC4 F-box protein [23]
CDC9 DNA ligase [25]
CDC28 Catalytic subunit of the main cell cycle cyclin-dependent kinase [26]
MOB1 Component of the mitotic exit network [26]
PSA1 (SRB1) GDP-mannose pyrophosphorylase [27]
be maintained by the addition of sorbitol to the medium [ 27 ]. A second option is the use of a maintenance plasmid carrying the essential gene that can be exchanged against the target plasmid in a plasmid-shuffling procedure [26].
5 Resistance Markers
If the host strain does not contain the appropriate mutant allele required for the use of a prototrophic or an autoselection marker— as it is often the case for industrial strains—a (semi)dominant marker needs to be employed. Two examples for dominant markers (LAC4/LAC12 and LSD1) based on carbon source utilization have already been mentioned above. Most (semi)dominant markers however confer resistance to various growth-inhibitory or toxic compounds (Table 4). These can be divided into three groups:
1. Endogenous genes, which confer resistance to specific agents when overexpressed either by introduction of multiple copies or by expression from a strong promoter: There are many examples of such genes in the literature, but only those specifically tested as marker genes are listed in Table 4. For instance, expression of formaldehyde dehydrogenase encoding SFA1 from the strong GPD1 promoter allowed cells to grow at up to 7 mM formaldehyde [28].
2. Mutant alleles of endogenous genes: These may encode proteins with a lower affinity for an inhibitory drug such as a ribosomal
Verena Siewers
Table 4
Resistance markers
Gene
Endogenous genes
CUP1 Metallothionein conferring resistance to copper and cadmium 1–14 mM CuSO4 [34, 35]
2 mg/ml D-serine 5 mg/ml L-proline [45] hph Klebsiella pneumoniae phosphotransferase conferring resistance to hygromycin B
kan Tn 903 phosphotransferase conferring resistance to G418
300 μg/ml hygromycin B [46]
200 mg/l G418 [33] (continued)
Selection Markers
Table 4 (continued) Gene
mdr3 Mus musculus P-glycoprotein conferring resistance to FK520
nat1
pat
Streptomyces noursei acetyltransferase conferring resistance to nourseothricin
Streptomyces viridochromogenes acetyltransferase conferring resistance to bialaphos
R · dhfr E. coli dihydrofolate reductase conferring resistance to methotrexate
100 μg/ml FK520 [47]
100 μg/ml nourseothricin [46]
200 μg/ml bialaphos [46]
10 μg/ml methotrexate 5 mg/ml sulfanilamide [32]
protein with reduced affinity for cycloheximide [29] or mutated transcription factors that increase the expression of drug exporters such as Fzf1-4 and Pdr3-9 [30, 31].
3. Heterologous genes: Drug resistance in these cases can, e.g., be based on the expression of an enzyme which, in contrast to the native one, is not susceptible to an inhibitor such as E. coli dihydrofolate reductase, which unlike the homologous yeast enzyme is insensitive to methotrexate [32]. Other mechanisms are based on drug inactivation, e.g., by binding (ble, [13]) or enzymatic inactivation (kan, [33]).
It should be noted that the selection conditions given in Table 4 represent indicative values derived from the cited examples. Drug concentrations suitable for selection may need to be adapted depending on the natural resistance/sensitivity of the desired host strain, the expression level of the resistance gene (strong vs. weak promoter and single-copy vs. multi-copy vector) and the medium composition (defined vs. complex medium). The use of some resistance marker genes comes along with very specific medium requirements. As chloramphenicol only acts on mitochondrial and not on cytoplasmic ribosomes, it should be used in combination with non-fermentable carbon sources whose assimilation is dependent on intact mitochondria. Another example is G418, which has a reduced activity in ammonium sulfate-containing media. When using minimal medium for G418-based selection, ammonium sulfate as nitrogen source should therefore be replaced with glutamate.
In many cases, it has been shown to be beneficial to incubate the transformed cells for ca. 2 up to 18 h in nonselective medium before plating them on selective plates in order to allow for the resistance gene to be expressed.
Verena Siewers
6
Marker Reuse and Counterselection
If a yeast strain needs to be cured of a plasmid, e.g., during plasmid shuffling, or an integrated marker is to be eliminated, e.g., for later reuse in an iterative gene deletion or gene integration approach, counterselectable markers can be of great benefit. These allow selecting for their absence in a cell. Several selectable markers presented so far are at the same time counterselectable, the most widely used system being the URA3 marker, which does not allow for growth in the presence of 5-fluoroorotic acid [48] (Table 5). Other markers do not allow for positive selection but only counterselection. An example is PKA3 (TPK2) encoding the catalytic subunit of cAMP-dependent protein kinase, which becomes toxic to
Table 5 Counterselectable markers Gene name
amdS A. nidulans acetamidase conferring sensitivity to fluoroacetamide
FCY1 FCA1 Cytosine deaminase conferring sensitivity to 5-fluorocytosine (5-FC) 100 μM–1 mM 5-FC [16]
GAP1 General amino acid permease conferring sensitivity to D-histidine
CAN1 Arginine permease conferring sensitivity to L-canavanine
GIN11M86 (Modified version of a subtelomeric, growth-inhibitory sequence)
PKA3 (=TPK2)
Catalytic subunit of cAMP-dependent protein kinase
1.6 g/l D-histidine 1 g/l L-proline [17]
100 μg/ml L-canavanine [58]
Induced overexpression [59]
Induced overexpression [14]
cells when overexpressed. By setting this gene under the control of a strong conditional promoter, marker gene loss can be initiated through promoter induction [14].
When eliminating an integrated marker, the aim is usually to select for complete deletions and not for random mutations: a common approach is to flank the marker gene with direct repeats, as homologous recombination between these repeats will lead to a marker gene “loop-out” [49]. While being rather simple, this method has the disadvantage that it leaves one copy of the relatively large repeat sequence in the genome (usually at least about 150 bp, [50]), which can lead to unwanted recombination events in case the same marker gene cassette is used multiple times. To avoid any sequence remnants, the so-called delitto perfetto approach has been developed. Here, the marker gene is removed in a second transformation step using oligonucleotides homologous to the sequences flanking the integration site [51]. The efficiency of both methods has been improved through the introduction of I-SceI recognition sites into the marker gene cassette and inducible expression of the corresponding endonuclease to generate doublestrand breaks [52, 53].
An additional possibility is recombination between direct repeats, which represent recognition sites for specific recombinase enzymes. With about 34 bp, these are much shorter than the direct repeats employed for recombination via the yeast DNA repair system as described above. Both the bacteriophage-derived Cre/loxP system as well as the yeast 2 μm plasmid-based Flp/FRT system have been applied for marker gene excision [54, 55]. An advantage is the high efficiency of this approach, which therefore does not necessarily require the use of a counterselectable marker.
7 Marker Modifications
When introducing marker genes into S. cerevisiae, it is in general desirable to reduce the amount of sequences with a high degree of homology to yeast genome sequences in order to prevent unwanted recombination events. One possibility is to use the entire marker gene cassettes consisting of promoter, gene, and terminator derived from a different organism such as in the case of the KlURA3 cassette (i.e., the URA3 gene from K. lactis) [1]. In the ideal situation, this organism should have enough sequence dissimilarity to avoid recombination but still be related closely enough for the promoter and terminator to function in S. cerevisiae. In other cases, marker genes from non-related organisms are combined with a promoter and terminator of fungal origin other than S. cerevisiae. Very commonly used are the so-called MX cassettes containing the Ashbya gossypii (Ag) TEF promoter and terminator (e.g., [33, 46]). It has however recently been discovered that the AgTEF promoter can be toxic to S. cerevisiae at high copy numbers [60].
Verena Siewers
A second advantage for using heterologous promoters can be a low activity in S. cerevisiae. If the transcription level is not high enough to sustain cell growth, this may be compensated for by increasing the copy number. This can be exploited when selecting for cells with multiple integration events or increased copy numbers of episomal plasmids. An extreme example is probably the utilization of the neo gene conferring resistance to G418 without any eukaryotic promoter to select for multiple genomic integrations [61]. In other studies, marker genes have been put under the control of a regulatable [22] or a truncated promoter, such as in the widely used LEU2-d and URA3-d alleles in order to increase episomal plasmid copy number [62, 63]. Additional approaches to promote high plasmid copy number are the use of temperaturesensitive marker gene alleles [25] or the use of markers with a reduced protein half-life [64].
8 Concluding Remarks
It may have become clear from the above explanations that a marker gene needs to be selected based on the specific application. The first decisive element is the background strain: if it does not contain the appropriate genotype, e.g., an auxotrophy that allows for the use of a recessive marker allele, a dominant marker gene has to be chosen. This is often the case when transforming wild or industrial strains. The marker gene will also have an influence on transformation efficiency. Especially the use of drug resistance markers requires the optimization of selection conditions to obtain sufficient amounts of transformants while avoiding the growth of non-transformed background colonies. For certain drugs, spontaneous resistant mutants may appear. Of crucial importance is the marker choice when plasmids need to be maintained in the cell. In fact, a specific medium composition in order to maintain selection pressure can represent an important cost factor especially for the use of antibiotics and in large-scale fermentations. In addition, the marker will influence the stability but also the copy number of an episomal plasmid.
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Natural and Modified Promoters for Tailored Metabolic Engineering of the Yeast Saccharomyces cerevisiae
Georg Hubmann, Johan M. Thevelein, and Elke Nevoigt
Abstract
The ease of highly sophisticated genetic manipulations in the yeast Saccharomyces cerevisiae has initiated numerous initiatives towards development of metabolically engineered strains for novel applications beyond its traditional use in brewing, baking, and wine making. In fact, baker’s yeast has become a key cell factory for the production of various bulk and fine chemicals. Successful metabolic engineering requires fine-tuned adjustments of metabolic fluxes and coordination of multiple pathways within the cell. This has mostly been achieved by controlling gene expression at the transcriptional level, i.e., by using promoters with appropriate strengths and regulatory properties. Here we present an overview of natural and modified promoters, which have been used in metabolic pathway engineering of S. cerevisiae. Recent developments in creating promoters with tailor-made properties are also discussed.
Key words Promoter, Yeast, Saccharomyces cerevisiae, Engineering, Metabolic engineering
1 Introduction
Many initial metabolic engineering approaches established new pathways and/or restructured the cell’s metabolic network by either strong, constitutive expression or entire deletion of genes encoding appropriate enzymes, transporters, or regulatory proteins. However, these extreme types of modifications often cause “metabolic burden,” particularly when the modification affects the cell’s energy and redox metabolism or when the final product or one or more pathway intermediates are toxic. It became obvious that metabolic engineering approaches targeting the central carbon and energy metabolism require fine-tuning of pathways to ensure a good balance between smooth flux towards the final product and the basic metabolic requirements of the cell. A recent example of such an attempt has been provided by the study of Hubmann et al. [1]. In some cases, it might even be necessary to uncouple biomass pr oduction from product formation, allowing a period of cell growth without the burden caused by product formation.
In this case, the metabolic flux towards the product is only switched on in a later phase of the process, i.e., after sufficient biomass has been formed. This latter scenario has traditionally been applied when engineering cell factories for heterologous protein production. There are manifold molecular levels at which metabolic fluxes (i.e., the involved protein activities) in a cell can be modified. Gene copy number, transcription efficiency, mRNA stability, translation efficiency, protein stability, and allosteric control are the most obvious set screws. In cell factory design, control of transcription efficiency has been most popular. This is certainly due to the fact that cellular regulation at this molecular level is relatively well understood and, thus, easy to modify by targeted genetic modifications. The transcription of genes is mainly controlled by regions upstream of the coding sequences and appropriate protein factors (global and gene-specific transcription factors) which bind to these regions and recruit the RNA polymerase. These sequences are referred to as promoters. They control how often a gene is transcribed in a given period of time. They also allow the cell to regulate transcription efficiency as a function of the external and internal conditions. High diversity in promoter structure within a genome allows for very complex gene- and condition-dependent transcriptional regulation. In prokaryotes, genes encoding enzymes, transporters, and regulatory proteins involved in a certain metabolic pathway are often clustered as an operon in the genome, i.e., transcribed as one single polycistronic mRNA and thus concertedly regulated at this level. Eukaryotic organisms, such as S. cerevisiae, usually lack these gene modules, and each coding sequence has its own upstream sequence for initiation (promoter) and a downstream sequence for termination of transcription (terminator). In the context of engineering complex metabolic pathways and regulatory networks of S. cerevisiae, the natural lack of polycistronic gene expression requires precisely coordinating transcription of each individual gene in terms of strength and timing [2]. Nevertheless, there have been attempts of obtaining polycistronic gene expression in eukaryotic organisms including the yeast S. cerevisiae by using viral ribosome entry sequences [3].
A large body of knowledge exists regarding the initiation and control of transcription in S. cerevisiae due to the fact that this yeast has become a major eukaryotic model organism in fundamental research. Various well-characterized endogenous as well as synthetic promoters of different strength and regulation have been identified or developed in S. cerevisiae in order to precisely control gene expression at the transcriptional level. This review first introduces the general structure of S. cerevisiae promoters and the basic principle of the transcription initiation process. Afterwards, natural and modified promoters as well as promoter collections which have been used in yeast metabolic engineering are described, and methods to obtain promoters with tailor-made properties are also addressed. Finally, the review discusses the limitations of promoter modifications in the context of metabolic engineering.
2 Promoter Structure of Protein-encoding Genes in Saccharomyces cerevisiae
2.1 Structure of Yeast Core Promoters
In eukaryotes, transcription of protein-encoding genes is performed by RNA polymerase II. The complex process of transcription generally occurs in two steps. First, the basic transcription machinery composed of RNA polymerase II and general (basal) transcription factors are assembled at the core promoter forming the pre-initiation complex (PIC). The core promoter responsible for basal transcription is located directly upstream of an open reading frame (Fig. 1). The polymerase is subsequently released from the PIC and starts the gene transcription at the transcriptional start site (TSS) (Fig. 1). The general transcription factors remain bound at the core promoter and facilitate re-initiation of transcription of the same gene in consecutive rounds by recruiting free RNA polymerase II. Therefore, the core promoter is indispensible for significant transcription of any gene. For a comprehensive review on transcription initiation in S. cerevisiae the reader is referred to Hahn and Young [4].
In general, the initiation at the TSS can occur either within a single nucleotide referred to as focused transcription initiation or within a cluster of a broader range of 50–100 bp nucleotides, containing several weak start points referred to as dispersed transcription initiation [5]. Focused and dispersed transcription initiation correlate with the two distinct types of promoters commonly found in yeast [6]; i.e., focused transcription is typically associated with regulated promoters, whereas dispersed transcription is commonly found in constitutive promoters [5].
Fig. 1 Frequent promoter motifs of (a) metazoan and (b) Saccharomyces cerevisiae promoters. The metazoan promoter contains several conserved motifs, including the TATA box, the transcriptional start site (TSS; +1) at the initiator (INR), recognition elements of the transcription factor IIB (BRE elements), and downstream regulatory elements, such as the motif 10 element (MTE) and core-promoter element (DPE). Compared to metazoan promoters, only the TATA box has been identified as a similar conserved motif in about 20 % of all S. cerevisiae core promoters. The cis-acting elements in S. cerevisiae, which are recognized by specific transcription activators or repressors, are located further upstream of the core promoter in the upstream activator or repressor sequences (UAS/URS)
Georg Hubmann et al.
Both RNA polymerase II and general transcription factors occupy at least 60 bp of the core promoter DNA [4]. In fact, they interact with several core promoter motifs, which are located from around 40 bp upstream to 40 bp downstream, relative to the TSS. In general, core promoters contain several functional DNA motifs, referred to as core promoter elements (Fig. 1). Metazoan core promoter elements include (1) the TATA box located at −30 relative to the TSS; (2) the initiator (INR) element located at, or immediately adjacent to, the TSS; (3) the TFIIB recognition element (BRE) immediately flanking the TATA box; as well as (4) two further elements located downstream of the TSS, i.e., the downstream promoter element (DPE) centered at +30 and the motif 10 element (MTE) (Fig. 1). Notably, most of these core promoter motifs are degenerated in S. cerevisiae, and their occurrence is remarkably variable in different promoters. In fact, out of the above metazoan core promoter motifs, only the TATA motif is clearly discernible in approximately 20 % of all yeast genes [7, 8]. The location of the TATA box in S. cerevisiae is variable between 40 and 120 bp upstream of the TSS, while it is fixed at 25–30 bp upstream of the transcription start site in metazoan promoters (Fig. 1). The TATA box synergistically acts together with the TSS to determine the direction of the transcription. As comprehensively reviewed by Rando and Winston [9], the TATA box is found in regulated S. cerevisiae promoters, i.e., those which are highly controlled by external and internal conditions. This is in contrast to constitutive promoters found upstream of housekeeping genes, whose expression level is basically the same even under different environmental conditions. According to the authors, genes with and without TATA box are referred to as stress and growth genes, respectively.
Interestingly, those S. cerevisiae promoters which carry a TATA box are associated with an atypical chromatin structure, exhibiting dense occupation of the core promoter by nucleosomes instead of the usual nucleosome depletion at the TSS in constitutive promoters. Thus, yeast promoters with a TATA box show chromatin structure-dependent expression, resulting in a higher expression variability compared to TATA box-less promoters [10].
Transcription is most commonly initiated at an adenosine site within the TSS, referred to as the “+1” position. A frequently appearing TSS motif was identified with the consensus sequence “PuPuPyPuPu”—i.e., a pyrimidine nucleotide (Py) flanked on either side by two purine nucleotides (Pu) [11, 12]. Other not yet identified TSS motifs might exist: this is supported by the fact that promoters deficient in the latter motif only show slightly reduced or even unaffected expression strength. As a consequence of variability in TSS motifs, the transcription process in S. cerevisiae might even start at several heterogeneous positions within the core promoters [11].
Other frequently appearing motifs in the core promoter in S. cerevisiae are AT-rich sequences, which usually consist of short
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E A .
As originally erected in the Zoological Gardens, Regent's Park
I A
May be taken as suggestive for the construction or appropriation of rooms for the larger Apiaries in summer-houses or other out-buildings.
THE NEW BOTTLE-FEEDER.
It has long been acknowledged that the best mode of feeding bees is through an opening at the top of the stock-hive. The new bottle-feeder is a simple and good means of administering food when a stock requires help in that way, as bees can take the food
from it without leaving the hive. Any kind of hive that has an opening at the top may thus be fed. Another important feature is the cleanliness with which liberal feeding can be accomplished; and few operations require more care than does feeding. If liquid sweet is left hanging about the hive, it tempts robber-bees; and when once the bees of an apiary have had a taste, there is no knowing where their depredations will stop: they resolutely attack and endeavour to rob other hives, fighting and killing one another to a considerable extent. Even if no hives be completely destroyed, weakness from loss of numbers will be the portion of most, if not of all, the hives in the garden.
The morals of our favourites are here a good deal at fault, for the stronger hives, when their inordinate passion is thus stirred up by the carelessness or want of knowledge of the bee-keeper, attack and prey upon the weaker ones. To be "forewarned is to be forearmed"— and "prevention is better than cure." We strongly recommend closely covering up the feeder; one of the middle-size bell glasses put over it makes a close-fitting cover, should the regular cover to the hive not be sufficiently tight. When bees are not kept in a bee-house, and are, on that account, more accessible, this extra care is particularly needed. The right time for feeding is in the autumn or spring. As stated at page 76, it is requisite to ascertain the condition of the hive at Michaelmas, and, if wanting, the deficiency can then be made up. [13] It is not wise to defer feeding until later in the season, because it is important that, when the food is placed b the cells, the bees should seal it up; and a tolerably warm temperature is required to enable them to secrete the wax for the delicately-formed lids of the cells. If the food remained unsealed, there is danger of its turning sour, and thereby causing disease among the bees. It is not well to feed in mid-winter or when the weather is very cold: bees at such times consume but little food, being in a state of torpor, from which it is better not to arouse them.
[13] A much greater quantity of food will have to be administered than the actual weight required to be furnished, because there is a very considerable decrease after it is taken by the bees
A little food in the spring stimulates the queen to lay more abundantly, for bees are provident and do not rear the young so rapidly when the supplies are short. In this particular, the intelligence of bees is very striking; they have needed no Malthus to teach them that the means of subsistence must regulate the increase of a prosperous population:—
"The prescient female rears the tender brood In strict proportion to the hoarded food."
E .
Judgment has, however, to be exercised by the apiarian in giving food, for it is quite possible to do mischief by over-feeding. The bees, when over-fed, will fill so many of the combs with honey, that the queen, in the early spring, cannot find empty cells in which to deposit her eggs, and, by this means, the progress of the hive is much retarded,—a result that should be guarded against. The following directions will show how the bottle-feeder is to be used:—Fill the bottle with liquid food; apply the net, affixed by an india-rubber band, over the mouth; place the block over the hole of the stock-hive, invert the bottle, the neck resting within the hole in the block; the bees will put their tongues through the perforations and imbibe the food, thus causing the bottle to act on the principle of a fountain. The bottle being glass, it is easy to see when the food is consumed. The piece of perforated zinc is for the purpose of preventing the bees from clinging to the net, or escaping from the hive when the bottle is taken away for the purpose of refilling. A very good syrup for bees may be made by boiling 6 lbs. of honey with 2 lbs. of water, for a few minutes; or loaf sugar, in the proportion of 3 lbs. to 2 lbs. of water, answers very well when honey is not to be obtained.
ROUND BEE-FEEDER.
Round bee-feeders are made of zinc and earthenware, eight inches across, three inches deep. The projection outside is a receptacle for pouring in the food; the bees gain access to the feeder through a round hole, which is placed either at the centre or nearer one side, whichever may best suit the openings on the top of the stock-hive. The feeder occupies a similar position to that of the glasses or cap hives in the gathering season. A circular piece of glass, cut so as to fit into a groove, prevents the bees escaping, and retains the warmth within the hive, whilst it affords opportunity for inspecting the bees when feeding.
The feeders were originally only made of zinc; but some beekeepers advised the use of earthenware, and a few have been made to meet the wishes of those who give the preference to that material.
When the bees are fed from above in this manner, the feeder is kept at a warm temperature by the heat of the hive. In common hives, cottagers feed the bees by pushing under the hive thin slips of wood scooped out, into which the food is poured. This plan of feeding can only be had recourse to at night, 'and the pieces of wood must be removed in the morning. By feeding at the top of the stockhive any interruption of the bees is avoided. For further instructions on this head, see the directions given for using the bottle-feeder.
ZINC FOUNTAIN BEE-FEEDER.
We have invented the fountain bee-feeder, in order that a larger supply of liquid food might be given to a hive than is practicable with the round feeder.
The liquid honey is poured in at, the opening, which unscrews; whilst being filled, the inside slide, closing the opening through which the food passes into the feeding-pan, should be shut down. When the reservoir is filled, the screw is made fast, and, the slide being withdrawn, a wooden float, pierced with small holes, through which the bees take the food, forms a false bottom, and rises and falls with the liquid. This feeder, being on the siphon principle, like a poultry or bird water-fountain, is supplied from the reservoir until that is empty. A piece of glass is fixed in the side of the reservoir, in order that the bee-keeper may see when it is emptied. A flat piece of glass on the top prevents the bees from escaping, and through it they may be inspected whilst feeding. The bees find access to the feeder on to the perforated float through the central round hole, which is placed over a corresponding hole in the stock-hive.
HONEY CUTTERS.
Honey cutters are used for removing comb from boxes and glasses without damaging it. The flat-bladed knife is for disconnecting the combs from the sides; the hook-shaped one is for the same purpose, to be applied to the top or horizontal part of the box or glass.
BOX FUMIGATOR.
This fumigator is a tin box, somewhat like a pepper-box upon a foot. It is a simple adaptation of the fumigating apparatus described by Mr. Nutt, and is used in the following manner:—Have a straw hive or other vessel ready that will match in circumference the hive intended to be fumigated. If the empty hive have a conical top, it will not remain crown downwards without a rest; in this case, it will be convenient to invert it on a pail. Having ascertained that the hive to be operated upon and the empty one in its reversed position nearly match in size, take half a packet of the prepared fungus, fire it well, and place it in the box or fumigator; place this in the centre of the empty hive, then bring the occupied hive directly over, so as to receive the fumes of smoke. To keep all close, put a wet cloth round the place where the two hives meet. In a minute or two, the bees may be heard dropping heavily into the lower empty hive, where they lie stupefied. After a little while, the old hive may be tapped upon to make the bees fall more quickly. On removing; the upper hive, the bees from it will be found lying quiet at the bottom of the lower one. Place a sheet on the ground, and spread the bees on it; then, with a feather, sort them over, in order to pick out the queen-bee. As soon as the queen is found, pour the rest of the lethargic swarm from off the sheet back into the inverted hive again. The stupefied bees must now be sprinkled freely with a syrup made of honey and water, or sugar and ale boiled together. Some' apiarians recommend a few drops 'of peppermint to be mixed with the syrup, in order to drown the peculiar odour which is special to each hive of bees,—this is more necessary when two-hives of bees are fumigated, and whilst under the influence of smoke are well mixed together. The hive containing the bees with which it is intended to unite the stupefied bees must now be placed on the top of that 'containing the latter, just as the hive was from which they have come. A wet cloth must be
fastened round the two hives, so as to prevent any of the bees from escaping. The hives in this position must be placed where they are not likely to be knocked down or meddled with. The fresh bees in the upper hive, attracted by the scent of the bees besmeared with honey, go down and commence licking off the sweets from the sleepy ones. The latter gradually revive, when all get mingled together and ascend in company to the upper hive, where they live as if they had not been separate families. The two hives should be left undisturbed for twenty-four hours, then the upper hive may be removed and placed immediately on the spot from whence it was brought.
The reason the queen is recommended to be taken is to prevent any fighting. She should be kept alive and fed as long as she will live, in case any harm should befall the sovereign of the other community.
TUBE FUMIGATOR.
The tube fumigator[14] is useful for several purposes. When a frame-hive has to be disturbed it is requisite to raise the lid and blow a little smoke into the hive, so as to check the angry passions of the bees. If it be desirable to stupefy the bees, ignited fungus must be placed in the box and the flattened end applied to the entrance of the hive; the smoke is then blown in, either with bellows or by applying the mouth of the operator, taking care to close all openings through which it can escape. The bees fall down stupefied, generally in about ten minutes; but the effect varies according to the populousness of the hive and the quantity of comb in it. The projected operations must now be performed speedily, as activity will soon be regained. See preceding directions.
[14] This fumigator will be found to possess many advantages over the box fumigator before mentioned
THE BEE DRESS OR PROTECTOR.
All operations connected with the removal or the hiving of bees should be conducted with calmness and circumspection. Bees, although the busiest of creatures, entertain a great dislike to fussiness in their masters, and become irritable at once if the apiarian allows them to see that he is in a hurry. Hence, there is great advantage in having the face and hands covered whilst at work amongst the bees; for when the operator knows he cannot possibly be stung, he can open his hives, take out the combs, gather in his swarms, or take the honey, with all the deliberation of a philosopher. Various kinds of bee-dresses have been contrived; one that we keep ready in stock is of a very simple construction. It is made of strong black net, in shape like an inverted bag, large enough to allow of a gentleman's wide-awake or a lady's hat being worn underneath. The projection of the hat or cap causes the dress to stand off from the face, and the meshes, of the net, though much too small for a bee to penetrate, are wide enough to allow of clear vision for the operator. An elastic band secures the dress round the waist; the sleeves also, made of durable black calico, are secured at the wrists by a similar method. The hands of the bee-master may be effectually protected with a pair of india-rubber gloves, which should be put on before the dress is fastened round the wrists. This kind of glove is regularly used by photographers, and allows of greater ease in manipulation than any other description.
Thus a very simple and inexpensive means of protection will enable even a novice in bee-keeping to make his observations and conduct his experiments under a sense of perfect security. Still, he need not be careless as to the feelings of his bees; his success and their comfort will be promoted by his "handling them gently, and as if he loved them." "Familiarity" between bees and their master "breeds" not "contempt," but affection.
Any sudden or clumsy movement, which jars the combs or frames, will excite the bees, and if but one should be crushed, the odour of their slaughtered comrade rouses the inhabitants of the hive to a pitch of exasperation. Their powers of smelling are very acute. The human breath is abomination to them; therefore, when operating upon bees, be careful to close the mouth and breathe only through the nostrils. The best time for most operations is in the middle of a fine day.
ENGRAVED
PRESSING ROLLER FOR THE GUIDANCE OF BEES IN THE CONSTRUCTION OF HONEY-COMB ON THE BARS.
This is an engraved metal roller, which, when applied to the coated underside of a comb-bar, leaves an impression as shown in the diagram. The wax having been spread on the flat bar, the roller, heated by being put into hot water, is heavily pressed over it. The roller has two wooden handles, so that considerable pressure may be given to it. The roller is a little less than two inches in diameter,
seven-eighths of an inch wide, and the length from handle to handle is six inches. The diagram shows the full size of the impressions as left on the wax, after passing the roller along the comb-bar, in the manner above described. It is a contrivance invented in Switzerland, and exhibited in the International Exhibition of 1862, when the pattern roller was purchased by ourselves.
The bars of a hive prepared with these markings in wax afford ready-made foundations for regular combs, which very much facilitate the operations of the bees.
IMPRESSED WAX SHEETS FOR ARTIFICIAL COMBS.
These artificial partition walls for combs are sheets of genuine wax, about the substance of thin cardboard. They receive rhomboidal impressions by being pressed between two metal plates, carefully and mathematically prepared and cast so that the impressions are exactly the same size as the base of the cells of a honey-comb. An inspection of a piece of comb will show that the division of the opposite cells is made by a thin partition wall, common to both. Now the substance of this is said to be only the one hundred and eightieth part of an inch, whilst the artificial ones we are recommending are between the thirtieth and fortieth part of an inch, more than four times the thickness of the handiwork of the bees themselves. It would, indeed, be vain to attempt to furnish sheets of wax at all approaching their own delicate fabric; the impressed sheets are quite as thin as they can be to bear the handling which is requisite for fixing them in the hives. We find, however, that the thickness is no disadvantage; the bees speedily excavate and pare the artificial sheet so as to suit their own notions of the substance required; then, with admirable economy, they use the surplus thus obtained for the construction of the cells. After a sheet has been partly worked at by the bees, it is interesting to hold it up to the light and observe the beautiful transparency of that part of it, contrasted with the opaqueness of the part not yet laboured upon.
When it is considered, as writers tell us, that more than 14 lbs. of honey are required for the secretion and elaboration of a single pound of comb, it will not be difficult to form a just estimate of the value of this invention, which thus furnishes cheap and excellent assistance to our industrious favourites. It also shows the beekeeper that all clean empty combs should be carefully preserved and considered as valuable stock. Another great advantage that it affords us is, that it renders us independent of guide-comb, which is not always obtainable. When a sheet or a strip of this impressed wax is properly fixed to the comb-bar, it is certain to be the guide and foundation of a straight comb. This invention has been derived from Germany, where it has been adopted many years with success. At the International Exhibition of 1862, we purchased the metal plates or castings, so as to manufacture the impressed sheets with which we are now able to supply our customers; and, after the careful trials we have made, we have great confidence in recommending them.
In the season of 1863 we furnished a Woodbury glass super, with the wax sheets fixed to the bars, in the manner hereafter to be explained, and it was truly astonishing to see the rapidity with which these sheets of wax were worked into comb. Receptacles were quickly made ready for the storing of honey, and the new combs soon became beautifully white; for, although the artificial wax has a yellow tinge, yet, after being worked at and made thinner, it is as good in colour as ordinary combs. For supers we cut the wax plates in half, making one serve for two bars.
We have received from Germany the following directions for the fastening of the artificial plates to the comb-bars. Hereafter will be described a plan which we have adopted, and to which preference is given.
(Translation.)—"The unstamped edge of the plate receives incisions half an inch distant from one another, made with a sharp knife, the plate having been a little warmed; then it is pinched between two equally strong ledges, which have been well moistened. The projecting edge of the plate which received the incisions is alternately bent to the right and to the left. The comb-bar is well besmeared with artificial sticking wax (a mixture of two parts
of wax and one part of American resin), and is well warmed at a fire. Afterwards the besmeared side is laid upon the bent end of the plate, and pressed to it as firmly as possible. A small wooden ledge, besmeared with sticking wax, and fastened by means of pressure to the lower edge of the plate, prevents it from bending, which sometimes happens when the bees work it."
To carry out the directions here given, it is necessary to warm the besmeared comb-bar at a fire; the wax plate has also to be warmed. Having tried this plan, and found inconvenience attending it, especially from the wax curling with the heat and the difficulty of making it stick firm, to say nothing of the uncomfortableness of performing the operation before a fire on a hot day in July, we began to consider if a little carpentering might not do the work better and more pleasantly, and adopted the following plan:—We split or cut the comb-bars of the Woodbury super in half, lengthways, and, taking the unstamped edge between the two strips, joined them together again by small screws at the side, confining the wax plate tightly in the centre, with no possibility of its falling down. Where frames are used, of course the bar could not be cut in two (except with the "compound bar and frame," where the bar being loose, it might be as easily managed). The plan we adopt with an ordinary frame is to saw out an opening, about an inch or an inch and a half from either end, where the sides are morticed in; this opening we make with a keyhole-saw. Through it the wax plate is easily put, and, with a heated iron passed over the upper side of the bar, is made sufficiently firm. If the wax plates are too large, a portion may be cut off; an opening of full eleven inches long can be made without materially weakening the bar and frame.
Another, and perhaps the simplest, plan is, to fix a strip of wood with brads to the underside of the top frame or bar: place the wax sheet against this, then wedge another strip close to it, and thus hold the wax sheet firmly in the centre of the frame, taking care also to make the second strip of wood fast with brads.
The wax plates must not extend to the bottom of the frame; a space of at least one inch should be left for expansion, because the bees, in working the plate, stretch it down lower. We also use a few
pins firmly pressed into the frames, and long enough to reach the edge of the plate; for by fixing three or four pins on either side, both at the sides and at the bottom, the plate may be held in an exactly central position within the frame. As before mentioned, when these directions are carried out, there is no fear of being troubled with crooked combs or bars.
The secretion of wax, and the method of its adaptation by the bees, is thus admirably described by Evans:—
"Thus filtered through your flutterer's folded mail Clings the cooled wax, and hardens to a scale. Swift at the well-known call, the ready train (For not a buzz boon Nature breathes in vain) Spring to each falling flake, and bear along Their glossy burdens to the builder throng. These, with sharp sickle, or with sharper tooth, Pare each excrescence and each angle smooth, Till now, in finish'd pride, two radiant rows Of snow-white cells one mutual base disclose; Six shining panels gird each polish'd round, The door's fine rim, with waxen fillet bound, While walls so thin, with sister walls combined, Weak in themselves, a sure dependance find.
Others in firm phalanx ply their twinkling feet, Stretch out the ductile mass, and form the street. With many a cross-way, path, and postern gate, That shorten to their range the spreading state."
MANIPULATION AND USES OF BAR AND FRAME HIVES.
AVING, at page 84, given a description of the mechanical arrangements of bar and frame hives, the next thing is, to describe the mode of introducing the bees, and of thus bringing the humane and scientific hives into operation. The swarm should be first hived into a common straw hive from the bough or shrub upon which they may have alighted; place this hive, into which we will suppose the bees have been shaken, on the ground, propped up on one side with a brick or a flower-pot, or anything of the sort that may be handy, in order that straggler-bees may join the swarm. The spot selected for this should be as shady an one as can be found, near to the place where the swarm settled; or it may be shaded from the rays of the sun by fixing matting on two poles, so as to prevent the heat falling on the hive; spread a sheet or cloth on the ground where an even surface can be obtained; stake this sheet down at the four corners, to prevent ruts and inequalities, which are great hindrances to the bees going into the bar and frame hive; place the latter upon the sheet, without its floor-board, having its front raised on blocks or sticks rather more than an inch,—not more, otherwise the bees will cluster, and attach themselves to the lower part of the frames, instead of going up between. These preparations will, perhaps, occupy ten minutes, by which time the swarm will have become settled and tolerably quiet. Then, with a sharp rap, precipitate the bees out of the straw hive on to the sheet immediately in front of the frame hive; give the straw hive another knock, so as to dislodge all the bees, and then take it quite away, otherwise they may, if it be left near, perversely choose to go into that, instead of the one desired. In some cases, as when the swarm has to be brought from a distance and procured from a cottager about whose skill in carrying out these directions there may be misgivings, it is best to give instructions that
the swarm be brought home after sunset, and then the foregoing directions for inducing the bees to tenant the frame hive may be better carried out. For ourselves, we much prefer the evening for the purpose. A little water sprinkled over them from a watering-pot is likely to induce the bees to quit the ground and go up into the hive more quickly.
Mr. Langstroth, in his admirable book, "The Hive and Honey Bee," writes:—"If they are too dilatory in entering the new hive, they may be gently separated with a spoon or leafy twig where they gather in bunches on the sheet, or they may be carefully 'spooned up' and shaken out close to the front of the hive. As these go in with fanning wings, they will raise a peculiar note, which communicates to their companions that they have found a home, and in a short time the whole swarm will enter, without injury to a single bee." In the Journal of Horticulture, Mr. Woodbury says:—"If combs be fixed in the frames, the crown-board may be removed and the cluster knocked out of the straw hive on to the top of the exposed frames. The bees will disappear between them with the utmost alacrity, delighted to have met with a ready-furnished dwelling, and the top, or crown-board, having been replaced, the hive should at once be removed to the position it is intended to permanently occupy."
No one should attempt these operations without being protected by a bee dress and a pair of india-rubber gloves, which are stingproof. Some persons also take the precaution of tying strings round the ancles of the trousers, lest some straggler should determine to attack the outposts of the enemy, which, to say the least, might perplex the operator in the midst of his task. Elastic india-rubber bands are good for this purpose, or a pair of "knickerbockers" would be useful. If Wellington boots are worn, the trousers may be tucked within the leather, in which case no bee can molest the operator, and no string or band will be needed. Practice makes perfect in beetending, as in other matters, and when a light hand is gained, there is little danger of the apiarian being stung.
If the weather be wet the next day or so after hiving, it will be well to give a little assistance to the new colony in the shape of food, for although, when a swarm leaves a hive, almost every bee composing
it fills itself with honey, we have known not a few instances, in case of very wet weather, in which the whole swarm has been starved for the want of this little timely help. Of course, the first work of the bees is to build themselves combs, and these combs being produced by the secretion of wax from honey, a great drain upon their resources immediately begins, and any little outlay at this juncture is abundantly compensated by its enabling these industrious emigrants the more quickly to push forward the furnishing of their new home.
Clean combs from hives that may have lost their bees are readily accepted, and cause a great saving in time and; material to the bees; these combs may easily be fixed by cutting them the proper size to fit within the frames, and making them firm by tying with tape or fixing them with pliable wire. In any case where the combs are too small to fit within the frame, a temporary bar may be fixed, and held firm by being sprung within the two upright sides of a frame, and thus pushed up until it presses the comb; then a piece of tape wound round, or a clip made of tin or zinc shaped to the top bar, prevents its falling out. All these supports may be removed[15] as soon as the bees have made the foundation secure; the comb will then be added to. In this way, every loose piece of comb may be economised.[16]
[15] They should be first dismembered from the comb by running a penknife between.
[16] Artificial comb may be advantageously used, especially if a little time (say a couple of days) be allowed to elapse before it is put into the hive; because, at first, so eager is a swarm to push forward the work of comb-building, that the sheets are liable to become mutilated. For guide-comb, cut the sheets in strips of rather more than an inch in depth, and fix them as mentioned at page 154.
These preparations must be made prior to 'the bees being hived, so that when a hive is so prepared, a swarm may begin to adapt whatever advantages they find ready for them; and it is truly marvellous what a swarm will do when thus furnished with combs in their new habitation. In these the queen can immediately begin to deposit her eggs, and the workers to store their honey, without
having to wait for the construction of combs, which is a laborious occupation for the bees.
In some cases, fine white combs of honey may be taken from the stock-hive; the end frames are always the most free from brood. Care must be exercised not to rob this part of the hive too much; one comb may, perhaps, be removed in the course of the season without impoverishing the bees, but it is not wise to take more.
PUTTING ON SUPER HIVE.
A colony established a year or more is called a "stock," by way of distinction from a swarm of the present year. Supposing the hive to be a stock, the super should be given them at the early part of the season, say, if fine and warm, at the latter end of April or beginning of May; if the weather be then unfavourable, it is better to delay doing so until a more genial temperature. If the colony be a swarm of the present year, two weeks should be allowed to elapse from the time of tenanting a hive, before putting on the super; this delay is necessary to give the bees the opportunity of building combs in their new domicile, and of getting a store of honey for themselves before working for their master.
When it is wished to use a super, the crown-board or roof of the stock-hive must be taken away, the thin adapting or honey-board taking its place. The two long slits at the sides are to give admission to the super. The bees will begin sooner, and work faster, if the eight bars are each furnished with artificial comb (as described at page 152). We have had depriving-hives very quickly filled when the bees were thus assisted. Combs that have been left unfilled may be fixed to the bars as before described; these must be white and clean, as dark comb should not be used for super hives. The combs, when filled, may be taken out singly, if desired for consumption, substituting an empty bar or comb; or, should the bee-keeper desire to see a handsome super, he must wait until the bees have filled and sealed up all the combs, and then he may proceed to disconnect the super by drawing a string or wire between the adapting-board and the stock-hive. After waiting a short time for the commotion to
subside, the operator must raise the super on its board and blow in a little smoke. The bees may be induced to quit by adopting either of the means described at pages 58 and 73. When the super has been removed, another may be put on; but if the honey-gathering be over, the crown-board should be replaced.
TAKING OUT FRAMES WITH COMBS.
It is well for a beginner to practise the directions for opening and shutting up hives, by using an empty hive until he becomes familiar with the handling of the frames.
The first thing to do is, to loosen the crown-board, or lid, with a knife, drawing a piece of string underneath it, to divide the wax or cement with which the bees make all secure. This string should be drawn through very slowly, so as not to irritate the bees. In hot weather, the crown-board may be loosened by a lateral movement; but sometimes, for want of care, this loosening of the lid disturbs the bees, and, as soon as it is removed, a number of them, enraged thereby, rush out and attack the operator. This and all other operations ought to be performed very carefully and gently. Especial care should be taken not to prise the lid upwards, by way of wrenching it off, for the frames and combs are generally secured thereto, and there is a liability of rending the combs with it; this will greatly irritate the bees, and be otherwise injurious. When a hive of bees is enraged, there is little chance of pacifying them; it is best, under such circumstances, to "give in," at once, and not attempt to perform any operation, but to shut the hive up and beat a retreat, benefiting by the experience, in order to do better a day or so afterwards. There are various devices for intimidating or conciliating the bees, and one of these already spoken of is—smoke. So next time the experimenter makes his attempt let him raise the lid an inch or so, and blow a few puffs of smoke into the hive, which will cause the bees to retreat This is best done by using our tube fumigator, with a little of the prepared fungus lighted. Pipes or cigars are not convenient to use for this purpose when the head is enveloped in the dress. As soon as the lid is removed, a few bees will fly out to learn
