Yeast Cell Surface Engineering

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Mitsuyoshi Ueda Editor

Yeast Cell
Surface
Engineering
Biological Mechanisms and Practical
Applications
Yeast Cell Surface Engineering
Mitsuyoshi Ueda
Editor

Yeast Cell Surface

Engineering
Biological Mechanisms and Practical
Applications
Editor
Mitsuyoshi Ueda
Graduate School of Agriculture
Kyoto University
Sakyo-ku, Kyoto, Japan

ISBN 978-981-13-5867-8    ISBN 978-981-13-5868-5 (eBook)

https://doi.org/10.1007/978-981-13-5868-5

Library of Congress Control Number: 2019932981

© Springer Nature Singapore Pte Ltd. 2019

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Preface

Cell surface engineering, in contrast to the conventional intracellular expression,

has many attractive features. This engineering is especially effective when yeasts
are used as a host, because eukaryotic modifications that are often required for func-
tional use can be added to the surface-displayed proteins/peptides. This engineer-
ing, leading to the generation of the so-called arming (molecular display) technology,
can be employed for basic and applied research purposes. In this book, various
strategies for the construction of surface-engineered yeasts and the diverse applica-
tions of this technology to industrial processes such as biofuel and chemical produc-
tions, pollutant removal, and health-related processes, including in vitro antibody
preparation and oral vaccines, are outlined. In addition, this engineering is suitable
for protein engineering and directed evolution through high-throughput screening
that is made possible by the feature that proteins/peptides displayed on cell surface
can be directly analyzed using intact cells without concentration and purification.
Actually, novel proteins/peptides with improved or developed functions have been
created.

Sakyo-ku, Kyoto, Japan Mitsuyoshi Ueda

v
Contents

Part I Principle
1 Principle of Cell Surface Engineering of Yeast...................................... 3
Mitsuyoshi Ueda

Part II Whole-Cell Biocatalyst

2 Energy Production: Biomass – Starch, Cellulose,
and Hemicellulose.................................................................................... 17
Kouichi Kuroda
3 Energy Production: Biomass – Marine.................................................. 29
Toshiyuki Takagi
4 Energy Production: Biodiesel................................................................. 43
Chiaki Ogino and Jerome Amoah
5 Cleanup of Pollution: Heavy Metal Ions
and Environmental Hormones................................................................ 63
Kouichi Kuroda
6 Recovery of Rare Metal Ions................................................................... 73
Kouichi Kuroda
7 Preparation of Functional Cells:
Improvement of Stress Tolerance........................................................... 85
Kouichi Kuroda
8 Bio-sensing Using Cell Surface Display:
Principles and Variations of a Cell Sensor............................................. 93
Seiji Shibasaki
9 Application of Cell Surface Engineering to Biosensing System........... 107
Shin-ichiro Suye

vii
viii Contents

Part III Medical Applications

10 Engineering Antibodies and Alternative Binders
for Therapeutic Uses................................................................................ 123
Wataru Aoki
11 Oral Vaccine Development Using Cell Surface
Display Technology.................................................................................. 149
Seiji Shibasaki

Part IV Protein Engineering

12 Combinatorial Engineering.................................................................... 161
Mitsuyoshi Ueda
13 Enzyme Evolution.................................................................................... 175
Natsuko Miura
 Part I
Principle

The cell surface engineering has also been established in gram-negative bacteria,
gram-positive bacteria, and phages. So far, many proteins/peptides have been suc-
cessfully displayed with maintenance of their functions. In contrast to bacteria and
phages, yeasts are equipped with the quality control and modification systems of
eukaryotic secretory pathways. Therefore, in the case of target proteins that have a
high molecular mass or require glycosylation modification, yeasts are suitable hosts
for cell surface display. In addition, simultaneous display of multiple kinds of pro-
teins/peptides on the same cell surface can be performed in yeasts by using different
auxotrophic markers, leading to the enhanced potential of surface-­ engineered
yeasts. The principles are described.
Chapter 1: Principle of Cell Surface Engineering of Yeast (Dr. Mitsuyoshi Ueda)
Chapter 1
Principle of Cell Surface Engineering
of Yeast

Mitsuyoshi Ueda

Abstract The cell surface is a functional interface between the interior and exterior
of cells. Surface proteins are responsible for most of the cell surface functions, serv-
ing as cell–cell adhesion molecules, specific receptors, enzymes, and transport pro-
teins. Cells have systems for anchoring and confining surface proteins to particular
domains on the cell surface. Fundamental analyses of how these molecules are tar-
geted and localized to the cell surface have been performed. For applications, the
cell surface should be exploited by making use of the known transport mechanisms
of proteins to the cell surface. The use of the abovementioned systems to display
heterologous proteins on the cell surface of microorganisms is expected to facilitate
the segregation of produced polypeptides and the production of microbial biocata-
lysts, whole-cell adsorbents, and live vaccines. The use of the cell surface of living
cells is also attractive for many applications in microbiology and molecular biology.

Keywords Cell surface engineering · Molecular display · GPI anchor · Arming

yeast · GPI-anchor attachment signal sequence

The cell surface is a functional interface between the interior and exterior of cells.
Surface proteins are responsible for most of the cell surface functions, serving as
cell–cell adhesion molecules, specific receptors, enzymes, and transport proteins.
Some surface proteins extend across the plasma membrane, whereas others are
bound by noncovalent or covalent interactions to cell surface components. Cells
have systems for anchoring and confining surface proteins to particular domains on
the cell surface. Fundamental analyses of how these molecules are targeted and
localized to the cell surface have been performed. For applications, the cell surface

M. Ueda (*)
Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan
e-mail: miueda@kais.kyoto-u.ac.jp

© Springer Nature Singapore Pte Ltd. 2019 3

M. Ueda (ed.), Yeast Cell Surface Engineering,
https://doi.org/10.1007/978-981-13-5868-5_1
4 M. Ueda

should be exploited by making use of the known transport mechanisms of proteins

to the cell surface. The use of the abovementioned systems to display heterologous
proteins on the cell surface of microorganisms is expected to facilitate the segrega-
tion of produced polypeptides and the production of microbial biocatalysts, whole-­
cell adsorbents, and live vaccines. The use of the cell surface of living cells is also
attractive for many applications in microbiology and molecular biology (Ueda and
Tanaka 2000; Kuroda and Ueda 2013; Ueda 2016).
Surface expression systems were first reported by showing that peptides could be
fused to the docking proteins (p111) of a filamentous phage without affecting its abil-
ity to infect Escherichia coli (Scott and Smith 1990). This led to the development of
phage display systems (Chiswell and McCaBerty 1992, 2018 Nobel Prize Chemistry).
These systems have already facilitated the isolation of specific ligands, antigens, and
antibodies from complex libraries (Hoogeoboom 1997). However, hybrids of larger
polypeptides with the major coat protein of a phage were not readily incorporated
into the phage particles. The bacterial surface may be more suitable for displaying
higher numbers of proteins. On the surface of gram-negative bacteria, which have an
outer membrane as their outermost cell surface, a number of heterologous proteins
have been displayed (Little et al. 1993; Georgiou et al. 1993, 1997), and these pro-
teins were fused to the surface-exposed termini of the outer membrane proteins
(Francisco et al. 1992, 1993). Lipoproteins (Harrison et al. 1990), fimbriae (Hedegaard
and Klemm 1989), and flagellar proteins (Newton et al. 1989) have also been display-
ing heterologous proteins on the cell surface. Gram-­positive bacteria have been used
for the purpose of displaying proteins on the bacterial surface, and the surface display
of heterologous proteins was achieved using proteins localized on the cell wall
(Gunneriusson et al. 1996; Samuelson et al. 1995; Schneewind et al. 1995). If the
system for cell surface display is intended to be applied to bioindustrial processes for
food, alcoholic beverages, medicines, and so on, it needs to be safe. For practical use,
the most suitable microorganism is the yeast Saccharomyces cerevisiae, which has a
“generally regarded as safe” status and can be used in the production of food and
pharmaceuticals. Thus, S. cerevisiae is a useful organism for the development of a
cell surface expression system. It is also useful as a host for genetic engineering,
since it enables the folding and glycosylation of expressed eukaryotic heterologous
proteins and can be subjected to many genetic manipulations. Moreover, the yeast
can be cultivated to a high cell density in a cheap medium.
S. cerevisiae has a thick and rigid cell wall (Fig. 1.1), of about 200 nm, which lies
outside the plasma membrane, which is in contrast to that of bacteria (Balloi 1982).
The cell wall of S. cerevisiae is mainly composed of mannoproteins and β-linked
glucans (Fleet 1991) and has a bilayered structure consisting of an internal skeletal
layer of glucans, composed of β-1,3- and β-l,6-linked glucose (Manners et al. 1973a,
b), and a fibrillar or brushlike outer layer, which is composed predominantly of
mannoproteins (Horisberger and Vonlanthen 1977). These proteins are linked to
glucans through covalent bonds. Two types of mannoproteins are present in the cell
wall of S. cerevisiae (Cid et al. 1995; Klis 1994). Mannoproteins loosely associated
with the cell wall through noncovalent bonds are extractable with sodium dodecyl
sulfate (SDS). When the isolated cell wall is solubilized with hot SDS, about 60
1 Principle of Cell Surface Engineering of Yeast 5

N, O-Glycosylated proteins (α-Agglutinin, Flo1p, Sed1p…..)

O-Glycosylated
proteins (Cwp1p,
Tip1p, Tir1p…..)
β-1,6-Glucan

β-1,3-Glucan
Chitin
N-Glycosidic chain
O-Glycosidic chain
Periplasmic proteins

Plasma membrane

Fig. 1.1 Structure of cell surface of S. cerevisiae

low-molecular-weight proteins are released (Valentin et al. 1984). Other types of

mannoproteins are extractable by glucanase and are released by β-1,3- or β-1,6-­
glucanase digestion of the glucan layer of the cell wall but not by SDS extraction
(Fleet and Manners 1977). Among these glucanase-extractable mannoproteins on
the cell surface of S. cerevisiae, the mating-type-specific agglutinins (Lipke and
Kurjan 1992), which mediate the direct cell–cell adhesion between cells of the
opposite mating type during mating and represent minor cell wall components, are
assumed to be located on the outermost surface. Mating-type a and α cells express
a-agglutinin and α-agglutinin, respectively (Terrance et al. 1987; Watzele et al.
1988). α-Agglutinin is encoded by the AGαl gene (Lipke et al. 1989) and interacts
with the binding subunit of the agglutinin complex of a-type cells (Cappellaro et al.
1991). a-Agglutinin consists of a core subunit encoded by the AGA1 gene (Roy
et al. 1991), which is linked through disulfide bridges to a small binding subunit
encoded by the AGA2 gene (Cappellaro et al. 1991). Both α-agglutinin and the core
subunit of a-agglutinin are composed of a secretion signal region, an active region,
a support region rich in serine and threonine, and a putative glycosylphosphati-
dylinositol (GPI) anchor attachment signal, which presumably exists in heavily
O-glycosylated forms (Cappellaro et al. 1991; Roy et al. 1991; Wojciechowicz et al.
1993). α-Agglutinin has a predicted length of 650 amino acids before processing.
GPI anchors have been found in many eukaryotic plasma membrane proteins
ranging from the coat proteins of protozoa to mammalian cell-adhesion molecules
(Cross 1990; Fredette et al. 1993; Homans et al. 1988; Dustin et al. 1987). The
structure of the GPI anchor (Fig. 1.2) is highly conserved among molecules from
various organisms (Ferguson and Williams 1988). The core structure of the yeast
GPI anchor is similar to that found in other eukaryotes (Lipke et al. 1989;
Conzelmarm et al. 1988; Leidicb et al. 1994), consisting of ethanolamine phosphate
6 M. Ueda

GPI anchor GPI anchor structure

Cell wall 3‘ Half of glycosylated
α-agglutinin
Plasma
membrane Protein Ethanolamine
phosphate
bridge
Inositol
Cleavage by PI-PLC
Exocytosis

Transglycosylation
Transport
vesicle Cleavage by PI-PLC
ER
C-terminal of 320
a.a. sequence of Signal sequence
Secretion signal sequence α-agglutinin
Promoter of GPI anchor

Gene encoding
displayed protein
Expression

Fig. 1.2 Transportation of GPI-anchor protein, GPI anchor structure, and protein display system
via GPI-anchor protein transportation

Table 1.1 GPI-anchor attachment signal

(6)-mannose(αl,2)-mannose(αl,6)-mannose(αl,4)-glucosamine(αl,6)-inositol phos-
pholipid. The lipid composition among yeast GPI anchors varies. Many cell surface
proteins in yeast, for example, Agnl (Lipke et al. 1989), Agal (Roy et al. 1991), Flol
(Watari et al. 1994), Sedl (Hardwick et al. 1992), Cwpl, Cwp2, Tipl, and Tirl/Srpl
(Table 1.1) (Van der Vaart et al. 1995), have GPI anchors that play important roles
in the surface expression of cell surface proteins and are essential for the viability of
yeasts. These glycophospholipid moieties are covalently attached to the C-termini
1 Principle of Cell Surface Engineering of Yeast 7

of proteins, and their primary function is to allow for the stable association of pro-
teins with the membrane. GPI-anchored proteins contain hydrophobic peptides at
their C-termini. After the completion of protein synthesis, the precursor protein
remains anchored in the endoplasmic reticulum (ER) membrane by the hydrophobic
carboxyl-terminal sequence, with the rest of the protein being located in the ER
lumen. In less than a minute, the hydrophobic carboxy-terminal sequence is cleaved
at the ω site (an arrow in Table 1.1) and concomitantly replaced with a GPI anchor,
presumably by the action of a transamidase. Because of the covalently linked lipid
anchor, the protein remains membrane-bound, exposed initially on the luminal side
of the ER and eventually on the cell exterior (Fig. 1.2).
The localization of both a-agglutinin and α-agglutinin to the cell surface occurs
through the secretory pathway (Tohoyama and Yanagishima 1985, 1987). Protein
secretion in S. cerevisiae comprises transfer through various membrane-enclosed
compartments constituting the secretory pathway. Secreted proteins are first trans-
located into the lumen of the ER and then transported from the ER to the Golgi
apparatus and finally from there to the plasma membrane in membrane-enclosed
vesicles (Schekman and Novick 1982; Schekman 1992). Fusion of the Golgi-­
derived secretory vesicles with the plasma membrane releases the secreted proteins
into the cell exterior. Posttranslational proteolytic modification of the precursors of
secretory peptides occurs late in the secretory pathway (in the trans cisternae of the
Golgi apparatus and secretory vesicles). The Kex2 endopeptidase is located in the
trans cisternae of the Golgi apparatus in S. cerevisiae to remove the proregion of
precursors, such as the α-factor pheromone (Wagner et al. 1987). It was proposed
that α-agglutinin is further transported to the outside of the plasma membrane
through the general secretory pathway in a GPI-anchored form, then released from
the plasma membrane by a phosphatidylinositol-specific phospholipase C, and
finally transferred to the outermost surface of the cell wall (Lu et al. 1994). Cell wall
anchorage of α-agglutinin was accomplished by the addition of a β-1,6-glucan to
the GPI anchor remnant of α-agglutinin, in a manner dependent on the prior addi-
tion of a GPI anchor to a-agglutinin (Lu et al. 1995; Kapteyn et al. 1996).
In order to target heterologous proteins to the outermost surface of the glycopro-
tein layer of the cell wall, information on α-agglutinin on a molecular scale was
utilized (Murai et al. 1997). The anchoring signal of α-agglutinin was combined
with the signal of the secreted enzymes using genetic engineering techniques.
Figure 1.2 shows the general structure of the gene necessary for the display of an
enzyme on the cell surface. The C-terminal half of α-agglutinin (320 amino acid
residues) contains a GPI anchor attachment signal at the C-terminal end, like other
cell surface proteins, and is used as an anchoring domain for heterologous proteins
since these proteins are covalently linked with glucans.
Schreuder et al. (1993, 1996) reported the successful targeting of α-galactosidase
from Cyamopsis tetragonoloba seeds, as a reporter, to the cell wall of S. cerevisiae.
Many proteins/peptides, including those with a relatively large molecular mass and
glycosylation requirements, have been successfully displayed on the cell surface of
yeast using the cell wall-anchoring domain of α-agglutinin. In the α-agglutinin-­
based display system, target proteins/peptides are fused to the secretion signal
sequence at the N-terminus and to the cell wall-anchoring domain (including the
8 M. Ueda

a-Agglutinin a-Agglutinin Flocculin

N
Target C
Protein Target
Protein
Aga 2 N
C-Terminal
half of
C a-agglutinin
GPI anchor
Aga 1
Cell wall Flocculation functional
domain of Flo1p

Plasma membrane

Yeast S. cerevisiae

Fig. 1.3 Cell surface display system in S. cerevisiae. a-Agglutinin-based display, α-agglutinin-­
based display, and Flo1p-based display are illustrated

GPI anchor attachment signal sequence) at the C-terminus. Several strategies have
been employed to improve the efficiency of cell surface display by S. cerevisiae.
Vector engineering using a high-copy-number plasmid and an improved host strain
enhances the efficiency of the α-agglutinin-based display system (Kuroda et al.
2009). On the other hand, a-agglutinin has been used in the C-terminus-free display
system. a-Agglutinin consists of Aga1p and Aga2p subunits. The secreted Aga2p
subunit is linked to the Aga1p subunit via two disulfide bonds, which are incorpo-
rated into the cell wall. Therefore, in the a-agglutinin-based display system, target
proteins/peptides are fused with the C-terminus of Aga2p. In addition, this system
has also been used in the N-terminal-free display by fusing target proteins/peptides
with the N-terminus of Aga2p (Boder and Wittrup 1997; Wang et al. 2005).
Additionally, screening from a cDNA library identified five genes whose overex-
pression improved the efficiency of the a-agglutinin-based display system (Fig. 1.3)
(Wentz and Shusta 2007).
Flo1p, which is a GPI-anchored cell wall protein involved in flocculation, is avail-
able for the N-terminal-free display in a similar manner (Sato et al. 2002). Although
other GPI-anchored cell wall proteins (Cwp1p, Cwp2p, Tip1p, Sed1p, YCR89w, and
Tir1p) have the potential to act as cell wall-anchoring domains, they are less fre-
quently used because of the previous successful performance of α- and a-agglutinin-
based display systems (Van der Vaart et al. 1997). Flo1p is also available for the
C-terminus-free display, in which truncated Flo1p without the GPI anchor attachment
signal sequence is used as an adhesive region. Pir proteins (Pir1–4p) have been used
as anchoring proteins in the C-terminus-free display, which permits the extraction of
displayed proteins/peptides from the cell wall by alkali treatment (Abe et al. 2004).
1 Principle of Cell Surface Engineering of Yeast 9

Cell wall

Random Linker
Human Peptide
FLAG Anchoring domain of Yps1p
GPCR library
GPI-anchor
Lipid raft Cellular membrane
Intracellular
Gα Gβ
Chimeric Ga Gγ MAPK
Cascade
Transcription factor STE12p

Fig. 1.4 Structure of cell membrane protein (the pheromone receptor Ste2p, which leads to activa-
tion of G proteins, the MAPK cascade, and the transcription factor Ste12p) of S. cerevisiae and its
application. Membrane-displayed alpha-factor (peptide agonist of the yeast pheromone response
pathway) activates the pheromone receptor Ste2p, which leads to activation of G proteins, the
MAPK cascade, and the transcription factor Ste12p

The methylotrophic strain Pichia pastoris can grow on an economical carbon

source and allows for high-density culture. Therefore, it is also a suitable host for
use in large-scale fermentation cultures of surface-engineered cells. In P. pastoris,
both N- and C-terminus-free display systems have been established using cell wall
proteins (α-agglutinin, a-agglutinin, Flo1p, Pir1p, Sed1p, and Tip1p) from S. cere-
visiae via the same strategy (Fig. 1.3) (Tanino et al. 2006; Wang et al. 2007, 2008).
The oleaginous yeast Yarrowia lipolytica is a heterothallic and dimorphic yeast
and has high potential for secreting heterologous proteins, which is a preferred fea-
ture in industrial uses. Several cell wall proteins in Y. lipolytica have been identified.
YlCWP1 and YlPIR1 from Y. lipolytica, which are GPI-anchored cell wall proteins,
allow N-terminus- and C-terminus-free display systems, respectively (Yuzbasheva
et al. 2011).
Almost all displayed proteins/peptides in yeast are localized to the cell wall, as
described above. However, the display of proteins/peptides on the plasma mem-
brane is desirable when there is a requirement for interaction with membrane pro-
teins such as receptors. Membrane display of proteins/peptides is performed using
the anchoring domain of Yps1p, a GPI-anchored plasma membrane protein. The
fusion of this domain with the C-terminus of the peptide ligand allows for display
on the plasma membrane and activation of either endogenous or heterologous
G protein-coupled receptors (GPCRs) in S. cerevisiae (Fig. 1.4) (Hara et al. 2012a, b).
The S. cerevisiae strains are the first examples of surface-engineered yeasts in
which active enzymes targeted to the cell surface endow the cells with new benefi-
cial properties. These surface-engineered yeast strains were termed “arming yeasts”
(Anonymous 1997). The displayed enzymes are also regarded as types of self-­
immobilized enzymes on the cell surface, with this feature being passed on to
daughter cells as long as the genes are retained by the cells. This display system
should further enhance the status of S. cerevisiae as a novel and attractive microor-
10 M. Ueda

Fig. 1.5 Display of GFP

on the yeast cell surface

Fig. 1.6 Illustration of arming yeast

ganism, a “charming yeast (Figs. 1.5 and 1.6),” owing to the fact that it can act as a
whole-cell biocatalyst as a result of the surface expression of various enzymes. This
is particularly important when the target substrates cannot be taken up by the cells,
and this system will make it possible to produce renewable self-immobilized bio-
catalysts. In studies of the surface display of proteins, previously reported systems
were used mainly to obtain possible single-chain antibodies, as in the phage display
system, or to explore the use of surface-displayed enzymes as catalysts. On the
1 Principle of Cell Surface Engineering of Yeast 11

Enzyme

Receptor
Antibody

Functional
protein Cell Surface Antigen
Engineering

Cell Application Immobilized

adhesion biocatalyst
Molecular Basic Practical
recognition Change of cell
function
Signal
transduction Biosensor
Protein Vaccine
engineering
Bioremediation
Cell physiology

Fig. 1.7 Various applications of cell surface engineering

other hand, cellular engineering studies have focused on changing or improving

intracellular metabolic capabilities by the addition or deletion of certain enzymes.
Here, cell surface engineering enabled combination of the cell surface display sys-
tem with the endowment of additional metabolic functions on yeast cells for the first
time. Thus, the cell surface can be regarded as a new target for providing additional
metabolic functions. This research heralds a new era for cellular or metabolic engi-
neering, not only in yeast but also in all living cells. We believe that cell surface
engineering will enable novel abilities to be conferred on all living cells and open
up a new field in biotechnology (Fig. 1.7).

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Anonymous (1997) Arming yeast with cell-surface catalysts. Chem Eng News 75:32
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 Part II
Whole-Cell Biocatalyst

The arming technology has been applied to yeasts for the construction of whole-cell
biocatalysts that can perform saccharification and fermentation. Starch and cellu-
lose are major components in grain and cellulosic biomass, respectively. Therefore,
the cell surface display of enzymes for hydrolytic degradation of these components
is attempted in order to achieve consolidated bioprocessing (CBP). Furthermore,
arming technology has been applied to bioadsorption of toxic metal ions and rare-­
metal ions, leading to bioremediation and resource recovery. To remove or degrade
environmental pollutants other than heavy metal ions, arming yeasts for bioreme-
diation and sensing of pesticides etc. have been constructed. Finally, arming tech-
nology has been also used in molecular breeding of stress-tolerant S. cerevisiae.
Chapter 2: Energy Production: Biomass – Starch, Cellulose, and Hemicellulose
(Dr. Kouichi Kuroda (Japan, Kyoto))
Chapter 3: Energy Production: Biomass – Marine (Dr. Toshiyuki Takagi (Japan,
Kashiwa))
Chapter 4: Energy Production: Biodiesel (Dr. Chiaki Ogino (Japan, Kobe) and
Dr. Jerome Amoah (Japan, Kobe))
Chapter 5: Cleanup of Pollution: Heavy Metal Ions and Environmental
Hormones (Dr. Kouichi Kuroda (Japan, Kyoto))
Chapter 6: Recovery of Rare Metal Ions (Dr. Kouichi Kuroda (Japan, Kyoto))
Chapter 7: Preparation of Functional Cells: Improvement of Stress Tolerance
(Dr. Kouichi Kuroda (Japan, Kyoto))
Chapter 8: Bio-sensing Using Cell Surface Display: Principles and Variations
of a Cell Sensor (Dr. Seiji Shibasaki (Japan, Kobe))
Chapter 9: Application of Cell Surface Engineering to Biosensing System
(Dr. Shin-ichiro Suye (Japan, Fukui))
Chapter 2
Energy Production: Biomass – Starch,
Cellulose, and Hemicellulose

Kouichi Kuroda

Abstract Starch, cellulose, and hemicellulose are promising renewable feedstock

from terrestrial plants for biofuel production. Cell surface engineering was applied
to the construction of whole-cell biocatalyst for the direct production of ethanol
from these polysaccharides by displaying enzymes on yeast cell surface. The envi-
ronmental polysaccharides can be efficiently degraded into monosaccharides by the
synergistic effects between the displayed enzymes. The generated monosaccharides
are quickly incorporated into the cells and assimilated into ethanol by intracellular
metabolic activities. In this chapter, ethanol production from starch, cellulose, and
hemicellulose by surface-engineered yeasts is introduced, and the advantages of cell
surface display of enzymes, such as their suitability for consolidated bioprocessing
(CBP), are described.

Keywords Cell surface engineering · Consolidated bioprocessing · Ethanol

production · Starch · Cellulose · Hemicellulose

1 Introduction

1.1 Situation of World Energy

As symbolized by the rapid fluctuations of energy price in recent years, economic

growth in developing countries triggers (i) shortage of resource and energy and (ii)
global warming, leading to a threat to the world in the near future. According to the
recent world energy outlook (WEO) 2017 (International Energy Agency 2017), the
global energy demand will rise more slowly than that in the past but is estimated to
increase by 30% in 2040 compared to that in the present. Most of the increase is
accounted for by emerging countries other than OECD (Organization for Economic
Co-operation and Development), especially China, India, and Middle East

K. Kuroda (*)
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto, Japan
e-mail: k_kuro@kais.kyoto-u.ac.jp

© Springer Nature Singapore Pte Ltd. 2019 17

M. Ueda (ed.), Yeast Cell Surface Engineering,
https://doi.org/10.1007/978-981-13-5868-5_2
18 K. Kuroda

countries. In the breakout of energy resources, it is estimated that coal consumption

will be about twice the current amount. Demand for crude oil is also expected to
increase greatly, and, in terms of production, even if newly developed oil fields and
LNG (liquefied natural gas) are added, concern about the shortage of crude oil
would still exist. Although shale gas and shale oil have been known for a long time
as potential natural gas and oil resources, escalation of crude oil prices stimulated
and prompted the development of mining technologies for shale gas and shale oil
(International Energy Agency 2017). Through further technical developments, the
production cost of shale oil has declined greatly but is still quite high compared to
the cost in large oil fields in the Middle East.
Another major global issue is the accumulation of carbon dioxide due to the
increased use of fossil fuels; and climate change due to global warming is a matter
of great concern. According to the WEO report, the amount of carbon dioxide emis-
sions related to global energy would increase slightly till 2040, which is far from
enough to avoid the serious impacts of climate change (International Energy Agency
2017). Along with the future international cooperations and efforts for reduction of
emission, if the concentration of atmospheric carbon dioxide can be maintained at
450 ppm in 2030, the temperature rise will be suppressed by 2 °C. However, this
goal cannot be achieved unless we make major policy changeovers and huge invest-
ments under the agreement of countries all over the world. Therefore, in addition to
reducing the absolute amount of energy use, it is necessary to drastically reduce the
consumption of fossil fuel and shift to alternative energy sources. To this end, the
use of renewable energy as well as the development of energy-saving equipment is
desirable.

1.2 Biomass Feedstock for Energy Production

Biomass energy is one of the promising renewable energies. Biomass is a generic

term for organic matter synthesized from water and carbon dioxide by photochemi-
cal action of sunlight. Carbon dioxide generated by the combustion of biomass is
absorbed by biomass again through photochemical action, so carbon neutrality
without increase in net carbon dioxide emission can be realized. Since biomass has
low energy density, it is intended not only to burn but also to improve the utilization
efficiency by converting it to biogas or liquid fuel by chemical/biochemical pro-
cesses. Biomass can be classified as follows: (i) biomass used in large quantities as
resources (resource crops), (ii) biomass whose existence is known but not suffi-
ciently used and whose utilization can be increased by development (unused bio-
mass), and (iii) valuable biomass that can be used but discarded without being used
(waste biomass). In the early stages of biomass energy development, attempts had
been made to produce ethanol from the starch of grain biomass such as corn and
sugarcane and replace some of the gasoline. However, there is a limitation to the
availability of this biomass because they are food crops, and there is competition
with food supply. Lignocellulosic biomass is a promising feedstock for ethanol
2 Energy Production: Biomass – Starch, Cellulose, and Hemicellulose 19

production by engineered yeasts for the following reasons: (i) cheap and readily
available because of more abundant renewable resource than grain biomass and (ii)
noncompetitive with food supply. Therefore, in the development of the second gen-
eration of biomass energy, lignocellulosic biomass such as waste wood, agricultural
waste (rice straw), and herbaceous biomass has been utilized as raw materials for
ethanol production.
The main polysaccharide components of lignocellulosic biomass are cellulose
(40–50%), hemicellulose (25–35%), and lignin (15–20%). Among them, cellulose
is most abundant on earth and is embedded in the cell wall matrix, together with
hemicellulose and lignin. In cellulose, a long straight chain of d-glucose is formed
by β-1,4 glucosidic linkage, converting the cellulose into the amorphous state due to
strong hydrogen bond between the microfibrils. Hemicellulose (glucomannan, man-
nan, xylan, and xyloglucan), nonhomogeneously distributed in the plant cell wall, is
bound to cellulose via hydrogen bond and to lignin via ester and glycoside bonds.
Lignin is a hydrophobic and complicated organic polymer, consisting of phenylpro-
pane monomers. The rigid structure of lignin strengthens the physical properties of
cell wall but makes cellulosic biomass difficult to degrade.

1.3  onsolidated Bioprocessing (CBP) by Cell Surface

C
Engineering

Ethanol production from cellulosic biomass includes the following four biopro-
cesses: (i) production of cellulases and hemicellulases, (ii) hydrolytic degradation
of cellulose and hemicellulose, (iii) C6 sugar fermentation, and (iv) C5 sugar fer-
mentation. Simultaneous saccharification and cofermentation (SSCF) is a major
process for ethanol production. Meanwhile, the process of performing these four
bioprocesses in a single fermenter is called “consolidated bioprocessing” (CBP)
(Fig. 2.1) (Lynd et al. 2005). In SSCF, the process of enzyme production is sepa-
rately required, but enzymes are produced in a single fermenter in CBP. Since CBP
can make ethanol production more efficiently and downsize the fermenter, it is
superior to the other ethanol production processes in terms of reduced cost of pro-
duction and equipment. To actually realize CBP, it is necessary to construct single
species or consortium of multiple species of microorganisms that can produce
enzymes, degrade polysaccharides, and perform fermentation. The yeast
Saccharomyces cerevisiae can perform ethanol fermentation and thus becomes an
ideal host for ethanol production in CBP by endowing its saccharification ability.
The technology of cell surface engineering that enables the display of functional
proteins on cell surface is suitable for strategy of constructing yeast for CBP system.
Using this technology, novel whole-cell biocatalysts that display enzymes have
been constructed (Kuroda and Ueda 2011, 2013, 2014). In Saccharomyces cerevi-
siae, 105–106 target proteins are displayed on the cell surface of a single cell by
using α-agglutinin as an anchoring protein. When displaying enzymes, yeast cells
20 K. Kuroda

Fig. 2.1 Consolidated bioprocessing (CBP) performing enzyme production, saccharification,

pentose fermentation, and hexose fermentation in a single fermenter

function not only as the host for enzyme production but also as the carrier for immo-
bilizing enzymes. Thus, only by a simple operation of cell cultivation, enzyme pro-
duction and immobilization on the cells can be simultaneously and automatically
achieved, and a large amount of whole-cell biocatalyst is easily prepared at low
cost. In addition, the displayed enzymes can be easily separated from the product by
cell recovery and are functional during the long durations of reaction due to the
improved stability of enzymes. Another advantage is that the concentration of
monosaccharides in the medium is kept almost zero, because monosaccharides gen-
erated by degradation of polysaccharides on yeast cell surface are rapidly imported
into the cells. This feature is advantageous in the prevention of bacterial contamina-
tion during ethanol production. Furthermore, several kinds of enzymes can be dis-
played simultaneously on the single yeast cell. The simultaneous display enables
multistep reactions on the cell surface and prompts a synergistic reaction between
the different kinds of enzymes due to the short distance between them (proximity
effect) (Bae et al. 2015).

2 Ethanol Production from Starch

Starch consists of amylose and amylopectin and is one of the most convenient mate-
rials in biomass. In the strategy for conversion of starch into ethanol, starch is first
degraded to glucose by Aspergillus, and then the generated glucose is assimilated
into ethanol by S. cerevisiae. Since yeast cannot directly utilize starch, it is neces-
sary to confer the ability to degrade starch into glucose, so that they can qualify for
CBP. Cell surface display of a starch-degrading enzyme allows yeast to utilize
starch directly as a sole carbon source. Glucoamylase from Rhizopus oryzae is an
exo-type amylolytic enzyme that cleaves α-1,4-linked and α-1,6-linked glucose
effectively from starch (Ashikari et al. 1986). A yeast strain displaying glucoamy-
lase from R. oryzae was constructed by cell surface engineering technology using
 2 Energy Production: Biomass – Starch, Cellulose, and Hemicellulose 21

α-agglutinin as an anchoring protein, which grew aerobically on starch, used as a

sole carbon source, to produce ethanol (Murai et al. 1997). Additional display of
α-amylase, an endo-type amylolytic enzyme from Bacillus stearothermophilus, on
glucoamylase-displaying yeast improved cell growth on starch-­containing medium,
compared to the yeast displaying only glucoamylase (Murai et al. 1999). By using
another anchoring protein, Flo1p, α-amylase from Streptococcus bovis was dis-
played along with display of glucoamylase from R. oryzae by using the anchoring
domain of α-agglutinin (Shigechi et al. 2004). The constructed yeast was able to
degrade raw corn starch and produce ethanol by assimilating the generated glucose
under an anaerobic condition. Therefore, co-­display of amylolytic enzymes on yeast
cell surface enables the integration of multiple conventional processes from two
different microorganisms into a single process.

3 Ethanol Production from Cellulose

Cellulose is a high-molecular-weight polysaccharide consisting of β-1,4 glycoside-­

linked linear chains of glucose and is a major component of lignocellulosic bio-
mass. There are crystalline and amorphous regions in cellulose, and multiple kinds
of enzymes are required for cellulose degradation. This feature makes the degrada-
tion of cellulose more difficult than that of starch. Three kinds of cellulolytic
enzymes, including endoglucanases (EGs), cellobiohydrolases (CBHs), and
β-glucosidases (BGLs), play an important role in cellulose degradation (Fig. 2.2).
Endoglucanases act on the amorphous region of the cellulose chain and randomly

β-Glucosidase
Cellobiohydrolase II Cellobiohydrolase I (BG)
(CBH II) (CBH I)

Endoglucanase Glucose
(EG)
Non-reducing ends

Reducing ends

Crystalline region Amorphous region Crystalline region

Fig. 2.2 Enzymatic hydrolysis of cellulose by the synergistic action of cellulases (cellobiohydro-
lases, endoglucanase, and β-glucosidase)
22 K. Kuroda

cleave internal bonds to create new reducing or nonreducing ends. Cellobiohydrolases

cleave cellobiose and cellooligosaccharide from the ends of the exposed cellulose
chain. By the endo-exo synergism of EGs and CBHs, cellulose chains are efficiently
degraded to soluble cellobiose and cellooligosaccharide (Medve et al. 1994).
β-Glucosidases hydrolyze the products generated by CBHs into glucose. Therefore,
the co-display of these enzymes on yeast cell surface can endow yeast cells with the
ability to degrade cellulose into glucose.
Carboxymethyl cellulase (CMCase) and β-glucosidase 1 (BGL1) from Aspergillus
aculeatus were co-displayed on yeast cell surface. The degradation and assimilation
of cellooligosaccharides were performed by this surface-engineered yeast in the
medium containing cellooligosaccharides as the sole carbon source (Murai et al.
1998). Then, the yeast strain co-displaying BGL1 from A. aculeatus and endogluca-
nase II (EG II) from Trichoderma reesei was constructed. This strain showed the
ability to grow in the medium containing barley β-glucan as the sole carbon source
and directly fermented 45 g/L β-glucan to produce 16.5 g/L ethanol (Fujita et al.
2002). As a further attempt, EG II and cellobiohydrolase II (CBH II) from T. reesei
and BGL1 from A. aculeatus were displayed on yeast cell surface (Fig. 2.3). The
cellulolytic enzyme-displaying yeast achieved simultaneous saccharification and
fermentation of 10 g/L phosphoric-acid-swollen cellulose (PASC) to produce 2.9 g/L
ethanol (Fujita et al. 2004). Although the displayed EG II showed slight activity
against PASC, the cellobiose production from PASC by EG II- and CBH II-co-
displaying yeast significantly exceeded that by EG II-displaying yeast. This result
indicates the importance of CBH II and the synergism between EG II and CBH II.
The display ratio of these cellulolytic enzymes is an important factor that deter-
mines the degradation efficiency of lignocellulosic biomass. However, the control
of the display ratio of multiple kinds of enzymes was a difficult challenge. A cock-

Fig. 2.3 Ethanol Cellooligosaccharides

production from cellulose
by surface-engineered
yeast co-displaying Glucose
cellulolytic enzymes.
Endoglucanase II (EG II), Cellulose
cellobiohydrolase II (CBH
II), and β-glucosidase 1
(BGL 1) were displayed by
fusion with cell wall-­
anchoring domain of
α-agglutinin Ethanol

Endoglucanase II (EG II) Cellobiohydrolase II (CBH II)

β-Glucosidase 1 (BGL1) C-terminal half of α-agglutinin

2 Energy Production: Biomass – Starch, Cellulose, and Hemicellulose 23

Mixture of yeast Yeast strain co-displaying

strains three kinds of enzymes

Distance between
each enzyme : Far Close

Endoglucanase II (EG II) Cellobiohydrolase II (CBH II) β-Glucosidase 1 (BGL1)

Fig. 2.4 Comparison of PASC-degrading activity between the yeast strain co-displaying cellulo-
lytic enzymes (EG II, CBH II, and BGL1) and the mixture of EG II-displaying yeast, CBH
II-displaying yeast, and BGL1-displaying yeast. Mixture of yeast strains was prepared by mixing
three kinds of surface-engineered yeasts in a ratio corresponding to the display ratio of enzymes
displayed on yeast co-displaying enzymes

tail δ-integration system and evolutionary engineering approach were employed to

optimize the display ratio of cellulolytic enzymes by controlling the copy number
of the integrated gene expression cassettes. As a result, the yeast strain with opti-
mized display ratio of EG II, CBH II, and BGL1 showed improved degradation
ability of PASC (Yamada et al. 2010). Proximity effect promotes synergistic effect
of cellulolytic enzymes when they are present within a short distance from each
other, which is also an important factor to be considered to improve the degradation
efficiency. Actually, Bae et al. (2015) evaluated the proximity effect between the
three kinds of cellulolytic enzymes (EG II and CBH II from T. reesei and BGL1
from A. aculeatus) displayed on yeast cell surface (Fig. 2.4). PASC-degrading activ-
ity of surface-engineered yeast co-displaying these enzymes was compared to that
of a mixture of EG II-displaying yeast, CBH II-displaying yeast, and BGL1-­
displaying yeast. The yeast co-displaying the enzymes showed 1.25- or 2.22-fold
higher activity than mixture of yeasts displaying single enzyme with an optical den-
sity at 600 nm (OD600) of 10 or 0.1, respectively (Fig. 2.5) (Bae et al. 2015). This
indicates that proximity effect between the displayed enzymes improves degrada-
tion efficiency and that this effect is more prominent at lower OD600.

4 Ethanol Production from Hemicellulose

Hemicellulose is also a major component of lignocellulosic biomass and mainly

consists of β-1,4-xylan. β-1,4-Xylan is a complex polysaccharide and is composed
of a backbone of β-1,4-linked xylopyranoside. Xylan is degraded into xylose
through the hydrolysis of xylan to xylooligosaccharides by endo-β-xylanase and the
24 K. Kuroda

Fig. 2.5 Proximity effect between cellulolytic enzymes co-displayed on yeast cell surface. PASC-­
degrading reactions were performed by using high (a) or low (b) concentrations of yeast cells. The
reducing ends generated by yeast cells were quantified using the 2,4-dinitrosalicylic acid (DNS)
assay

subsequent hydrolysis of xylooligosaccharides to xylose by β-xylosidase (Kulkarni

et al. 1999). In addition, the ability to metabolize xylose is required for ethanol
production. As the first step toward ethanol production from xylan, these enzymes
were displayed on yeast cell surface. Xylanase II from T. reesei (XYN II) and
β-xylosidase from Aspergillus oryzae (XylA) co-displayed on yeast cell surface
showed the activity, leading to the successful conversion of xylan to xylose (Katahira
et al. 2004). The second step is to convert the produced xylose to xylulose that can
be assimilated to ethanol by native metabolic pathway of yeast. There are two avail-
able pathways for achieving this step. One is the pathway including conversion of
xylose to xylitol by xylose reductase (XR) and conversion of xylitol to xylulose by
xylitol dehydrogenase (XDH). However, in this pathway, the difference in coen-
zyme specificity between XR and XDH causes intracellular redox imbalance and
the accumulation of by-products including xylitol and glycerol (Van Vleet and
Jeffries 2009; Matsushika et al. 2009). Even so, XR and XDH from Pichia stipites
and xylulokinase (XK) from S. cerevisiae were overproduced in the yeast cells co-­
displaying XYN II and XylA, resulting in direct production of 7.1 g/L ethanol from
birchwood xylan under anaerobic conditions (Katahira et al. 2004). Therefore, the
simultaneous saccharification and fermentation of xylan were achieved by a combi-
nation of cell surface engineering and metabolic engineering in yeast.
In the other pathway, xylose is directly converted to xylulose by xylose isomer-
ase (XI). The advantage of this pathway is that it does not need coenzymes for the
conversion reaction; thus the intracellular redox imbalance can be avoided. However,
there are only a few reports showing the successful intracellular production of active
XIs (Kuyper et al. 2003; Walfridsson et al. 1996; Brat et al. 2009), and it was a chal-
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 the future, philosophy, by discarding its abstractionism and its
(closely allied) solipsism, will do its share in making this “group
consciousness,” this consciousness of our being indeed
“fellow-workers” with all men, once again a property of our
minds and our thoughts.
278
One of Professor James’s last books is called A Pluralistic
Universe, and both he and Professor Dewey have always
written under the pressure of the sociological interest of
modern times. In short, it is obvious that the “reality” underlying
the entire pragmatist polemic against the hypothetical
character of the reading of the world afforded us by the
sciences, is the social and personal life that is the deepest
thing in our experience.
279
This idea of a “world of inter-subjective intercourse,” although
now a commonplace of sociology, was first expressed for the
writer in the first series of the Gifford Lectures of Professor
James Ward upon “Naturalism and Agnosticism,” in chapters
xv. and xvi. The first of these chapters deals with “Experience
and Life,” and the second with the “inter-subjective intercourse”
that is really presupposed in the so-called individual
experience of which the old psychology used to make so
much. The reader who wishes to follow out a development of
this idea of a “world of inter-subjective intercourse” cannot do
better than follow the argument of Professor Ward’s second
series of Gifford Lectures (“The Realm of Ends,” or “Pluralism
and Theism”), in which he will find a Humanism and Theism
that is at least akin to the theodicy, or the natural theology, of
which we might suppose Pragmatism to be enamoured. The
double series of these Lectures might well be referred to as an
instance of the kind of classical English work in philosophy of
which we have spoken as not falling into the extremes either of
Pragmatism or of Rationalism. The strong point of the “Realm
of Ends,” from the point of view of this book upon Pragmatism
and Idealism, is that it moves from first to last in the reality of
that world to which the science and the philosophy of the day
both seem to point the way. In opposition to “subjectivism” it
 teaches a Humanism and a Pluralism that we recognise as an
expression of the realities of the world of our common life and
our common efforts, and from this Humanism it proceeds to a
Theism which its author seeks to defend from many of the
familiar difficulties of Naturalism. Were the writer concerned
with the matter of the development and the elaboration of the
philosophy that seems to have precipitated itself into his mind
after some years of reflection on the issues between the
realists and the idealists, between the rationalists and the
pragmatists, he would have to begin by saying that its outlines
are at least represented for him in the theistic and pluralistic
philosophy of Professor Ward.
280
According to Professor Dawes Hicks in the Hibbert Journal for
April 1913, there is a great deal in the articles of Professor
Alexander on “Collective Willing and Truth” that supports some
of the positions I am here attempting to indicate, as part of the
outcome of the pragmatist-rationalist controversy. “Both
goodness and truth depend, in the first place, on the
recognition by one man of consciousness in others, and,
secondly, upon intersubjective intercourse” (p. 658).
281
I owe this reference (which I have attempted to verify) to a
suggestive and ingenious book (The New Word, by Mr. Allen
Upward) lent to me by a Montreal friend. Skeat, in his
Dictionary, gives as the meaning of truth, “firm, established,
certain, honest, faithful,” connecting it with A.S. tréou, tryw
(“preservation of a compact”), Teut. trewa, saying that the
“root” is “unknown.” I suppose that similar things might be said
about the Greek word ἐτεόν in its different forms, which Liddel
and Scott connect with “Sans., satyas (verus), O. Nor. Sannr,
A.S. sóth (sooth).” All this seems to justify the idea of the social
confirmation of truth for which I am inclined to stand, and the
connexion of intellectual truth with ethical truth, with the truth of
human life. I agree with Lotze that truths do not float above, or
over, or between, things, but that they exist only in the thought
of a thinker, in so far as he thinks, or in the action of a living
being in the moment of his action—the Microcosmos as quoted
 in Eisler, article “Wahrheit” in the Wörterbuch. The Truth for
man would be the coherence of his knowledge and his beliefs,
and there is no abstract truth, or truth in and for itself, no
impersonal “whole” of truth.
282
As in the Hegelian dialectic.
283
There is another important thing to think of in connexion with
this sociological character of Pragmatism. It is a characteristic
that may be used to overcome what we have elsewhere talked
of as its “subjectivism” and its “individualism,” and its
revolutionary tendencies. It is, we might urge, a social and a
collective standard of truth that Pragmatism has in view when it
thinks of “consequences” and of the test of truth. Lalande takes
up this idea in an article in the Revue Philosophique (1906) on
“Pragmatisme et Pragmaticisme,” pointing out that Dr. Peirce
would apparently tend to base his pragmatism on the
subordination of individual to collective thought. Dr. Schiller
too, I think, contemplates this social test of truth in his would-
be revival of the philosophy of Protagoras—that man is the
measure of reality—for man.
284
See below, p. 197, where we speak of this “mediation” as the
first fact for Professor Bosanquet as a prominent “Neo-
Hegelian” rationalist.
285
I have been asked by a friendly critic if I would include
“inference” in this “real thought.” I certainly would, because in
all real inference we are, or ought to be, concerned with a real
subject-matter, a set of relations among realities of one kind or
another. Possibly all students in all subjects (especially in
philosophy) have lost time in following out a set of inferences
in and for themselves. But such a procedure is justified by the
increased power that we get over the real subject-matter of our
thought. When thought cannot be thus checked by the idea of
such increased power, it is idle thought.
286
I am thinking, of course, of the entire revolutionary and radical
social philosophy that harks back (in theory at least) to the
“Social Contract” and to the State of Nature philosophy of
Rousseau and his associates and predecessors.
287
See p. 184 of Chapter VII., where I speak of the ability to do
this as the invariable possession of the successful American
teacher of philosophy.
288
An equivalent of it, of course, exists in many sayings, in many
countries, in the conception of the task of the metaphysician as
that of “a blind man in a dark room hunting for a black cat
which—is not there,” reproduced by Sir Ray Lankester in the
recent book of H. S. R. Eliot, Modern Science and the Illusions
of Bergson. There is generally an error or a fallacy in such
descriptions of philosophy—in this Lankester story the error
that the secret of the world is a kind of “thing in itself” out of all
relation to everything we know and experience—the very error
against which the pragmatists are protesting.
289
Mr. Bertrand Russell, for example, seems to me to have the
prejudice that philosophy is at its best only when occupied with
studies which (like the mathematics of his affections) are as
remote as possible from human life. “Real life is,” he says, “to
most men a long second-best, a perpetual compromise
between the ideal and the possible; but the world of pure
reason knows no compromise, no practical limitations, no
barrier to the creative activity embodying in splendid edifices
the passionate aspiration after the perfect form from which all
great work springs. Remote from human passions, remote
even from the pitiful facts of nature, the generations have
gradually created an ordered cosmos where pure thought can
dwell as in its natural home, and where one, at least, of our
nobler impulses can escape from the dreary exile of the actual
world.” I cannot—as I have indicated elsewhere in regard to
Mr. Russell—see for one moment how there is any justification
for looking upon this “ordered cosmos” of mathematical
physics as anything other than an abstraction from the real
 world with which we are acquainted. It is the creation of only
one of our many human interests. And I cannot see that the
thought that occupies itself with this world is any nobler than
the thought that occupies itself with the more complex worlds
of life, and of birth and death, and of knowledge and feeling
and conduct. Mr. Russell might remember, for one thing, that
there have been men (Spinoza among them) who have
attempted to treat of human passions under the light of
ascertainable laws, and that it is (to say the very least) as
legitimate for philosophy to seek for reason and law in human
life, and in the evolution of human history, as in the abstract
world of physical and mathematical science. Can, too, a
mathematical philosophy afford any final haven for the spirit of
man, without an examination of the mind of the mathematician
and of the nature of the concepts and symbols that he uses in
his researches? There is a whole world of dispute and
discussion about all these things.
290
I have in view in fact only (or mainly) such American
characteristics as may be thought of in connexion with the
newer intellectual and social atmosphere of the present time,
the atmosphere that impresses the visitor and the resident
from the old world, the atmosphere to the creation of which he
himself and his fellow-immigrants have contributed, as well as
the native-born American of two generations ago—to go no
further back. I mean that anything like a far-reaching analysis
or consideration of the great qualities that go to make up the
“soul” of the United States is, of course, altogether beyond the
sphere of my attention for the present. I fully subscribe, in
short, to the truth of the following words of Professor
Santayana, one of the most scholarly and competent American
students (both of philosophy and of life) of the passing
generation: “America is not simply a young country with an old
mentality; it is a country with two mentalities, one a survival of
the beliefs and standards of the fathers, the other an
expression of the instincts, practice, and discoveries of the
younger generation. In all the higher things of the mind—in
 religion, literature, in the moral emotions, it is the hereditary
spirit that still prevails, so much so, that Mr. Bernard Shaw
finds that America is a hundred years behind the times.”—“The
Genteel Tradition in American Philosophy,” in Winds of
Doctrine (p. 187).
291
A contemporary American authority, Professor Bliss Perry, in
his book upon The American Mind naturally singles out
radicalism as one of the well-marked characteristics of
Americans. Among the other characteristics of which he
speaks are those of the “love of exaggeration,” “idealism,”
“optimism,” “individualism,” “public spirit.” I refer, I think, to
nearly all these things in my pages, although of course I had
not the benefit of Professor Perry’s book in writing the present
chapter.
292
I am certainly one of those who insist that we must think of
America as (despite some appearances to the contrary—
appearances to be seen also, for example, in the West of
Canada) fundamentally a religious country. It was founded
upon certain great religious ideas that were a highly important
counterpart to some of the eighteenth-century fallacies about
liberty and equality that exercised their influence upon the
fathers of the republic.
293
He has recently published a volume dealing especially with the
contributions of Biology and Darwinism to philosophy.
294
See p. 252.
295
The crucial characteristics of the Presidential campaign of
1912 clearly showed this.
296
We can see this in the many valuable studies and addresses
of Professor Dewey upon educational and social problems.
297
It is this fact, or the body of fact and tendency upon which it
rests, that causes Americans and all who know them or
observe them, to think and speak of the apparently purely
 “economic” or “business-like” character of the greater part of
their activities. Let me quote Professor Bliss Perry here ... “the
overwhelming preponderance of the unmitigated business-man
face [italics mine], the consummate monotonous commonness
of the pushing male crowd” (p. 158). “There exists, in other
words, in all classes of American society to-day, just as there
existed during the Revolution, during the ‘transcendental’
movement, in the Civil War, an immense mass of
unspiritualised, unvitalised American manhood and
womanhood” (p. 160).
298
And this despite of what I have called elsewhere the
comparative failure of Pragmatism to give a rational, and
tenable account of “personality” and of the “self.”
299
At the moment of his death (scribens est mortuus) James was
undoubtedly throughout the world the most talked-about
English-speaking philosopher, and nowhere more so than in
Germany, the home of the transcendentalism that he so
doughtily and brilliantly attacked. Stein says, for example, in
his article upon “Pragmatism” (Archiv für Philosophie, 14,
1907, II. Ab.), that we “have had nothing like it since
Schopenhauer.” I have often thought that James and his work,
along with the life and work of other notable American thinkers
(and along with the “lead” that America now certainly has over
at least England in some departments of study, like political
and economic science, experimental psychology, and so on),
are part of the debt America owed, some decades ago, to the
Old World in the matter of the training of many of her best
professors—a debt she has long since cancelled and overpaid.
Readers, by the way, who desire more authentic information
about James and his work than the present writer is either
capable, or desirous, of giving in this book, may peruse either
the recent work of Professor Perry of Harvard upon Present
Philosophical Tendencies, or the work of M. Flournoy already
spoken of. Boutroux has a fine appreciation of the value of
James’s philosophical work in the work to which I have already
 referred. And there was naturally a crop of invaluable articles
upon James in the American reviews shortly after his death.
300
Think alone, for example, of what James says he learnt there
from a teacher like Agassiz: “The hours I spent with Agassiz so
taught me the difference between all possible abstractionists
and all livers in the light of the world’s concrete fulness that I
have never been able to forget it.”—From an article upon
James in the Journal of Philosophy, ix. p. 527.
301
While this book was passing through the press my eye fell
upon the following words of Professor Santayana in respect of
this very personality of James: “It was his personal
spontaneity, similar to that of Emerson, and his personal vitality
similar to that of nobody else. Conviction and ideas came to
him, so to speak, from the subsoil. He had a prophetic
sympathy with the dawning sentiments of the age, with the
moods of the dumb majority. His way of thinking and feeling
represents the true America, and represented in a measure the
whole ultra-modern radical world” (Winds of Doctrine, p. 205).
302
Including, say, the facilities of a completely indexed and
authenticated estimate of the work that has been done in
different countries upon his particular subject. It is easy to see
that the habit and the possibility of work in an environment
such as this [and again and again its system and its facilities
simply stagger the European] is a thing of the greatest value to
the American professor so far as the idea of his own best
possible contribution to his age is concerned. Should he
merely do over again what others have done? Or shall he try to
work in a really new field? Or shall he give himself to the work
of real teaching, to the training of competent men, or to the
“organization” of his subject with his public? It must be
admitted, I think, that the average American professor is a
better teacher and guide in his subject than his average
colleague in many places in Europe. Hence the justifiable
discontent of many American students with what they
 occasionally find abroad in the way of academic facilities for
investigation and advanced study.
303
The latter (it is perhaps needless to state) have long been
perfectly evident to all American teachers of the first rank in the
shape, say, of the worthless “research” that is often
represented in theses and studies handed in for the degree of
Doctor of Philosophy, or for other purposes. Anything that
seems to be “work done,” anything that has attained to some
“consequences” or other, has often been published as studies
and researches, and this despite the valuable things that are to
be associated with the idea of the pragmatist element in
American scholarship. The faults, too, of the undue
specialization that still obtains in many American institutions is
also, as I have indicated, becoming more and more apparent
to American authorities.
304
I cannot see why idealists should have been so slow to accord
to Pragmatism the element of truth in this idea, and to admit
that it connects the pragmatist philosophy of “consequences”
with the idealist “value-philosophy.”
305
The greater part, for example, of our British teaching and
writing about Kant and Hegel has taken little or no recognition
of the peculiar intellectual and social atmosphere under which
Criticism and Transcendentalism became intelligible and
influential in Germany and elsewhere, or of the equally
important matter of the very different ways in which the Kantian
and the Hegelian philosophies were interpreted by different
schools and different tendencies of thought. A similar thing
might, I think, too, be said of the unduly “intellectualistic”
manner in which the teachings of Plato and Aristotle have
often been presented to our British students—under the
influence partly of Hegelianism and partly of the doctrinairism
and the intellectualism of our academic Humanism since the
time of the Renaissance. Hence the great importance in Greek
philosophy of such a recent work as that of F. M. Cornford
upon the relation of Religion to Philosophy (From Religion to
 Philosophy, Arnold, 1912), or of Professor Burnet’s well-known
Early Greek Philosophy.
306
As suggestive of the scant respect for authorities felt by the
active-minded American student, I may refer to the boast of
Papini that Pragmatism appeals to the virile and the proud-
spirited who do not wish to accept their thought from the past.
307
I am thinking of such events as the “World’s Parliament of
Religions” (in Chicago in 1893), the recent international
conferences upon “ethical instruction in different countries,”
upon “racial problems,” upon “missions,” etc. It would be idle to
think that such attempts at the organisation of the knowledge
and the effort of the thinking people in the world are quite
devoid of philosophical importance. One has only to study, say,
von Hartmann, or modern social reform, to be convinced of the
contrary.
308
I trust I may be pardoned if I venture to suggest that in
opposition to the democratic attitude of Pragmatism to the
ordinary facts of life, and to the ordinary (but often heroic) life
of ordinary men, the view of man and the universe that is taken
in such an important idealistic book as Dr. Bosanquet’s
Individuality and Value is doubtless unduly aristocratic or
intellectualistic. It speaks rather of the Greek view of life than
of the modern democratic view. As an expression of the quasi
democratic attitude of James even in philosophy, we may cite
the following: “In this real world of sweat and dirt, it seems to
me that when a view of things is noble, that ought to count as a
presumption its truth, as a philosophical disqualification. The
Prince of Darkness may be a gentleman, as we are told he is,
but whatever the God of earth and heaven is, he can surely be
no gentleman. His menial services are needed in the dust of
our human trials.” [Having rewritten this quotation two or three
times, I have lost the reference to its place in James’s writings.
It is one of the three books upon Pragmatism and Pluralism.]
309
We may quote, I think, the following passage from Professor
Perry to show that the open-mindedness of James was not
merely a temperamental and an American characteristic in his
case, but a quality or attitude that rested upon an intellectual
conviction in respect of the function of ideas. “Since it is their
office [i.e. the office of ideas] to pave the way for direct
knowledge, or to be temporarily substituted for it, then
efficiency is conditioned by their unobtrusiveness, by the
readiness with which they subordinate themselves. The
commonest case of an idea in James’s sense is the word, and
the most notable example of his pragmatic or empirical method
is his own scrupulous avoidance of verbalism. It follows that
since ideas are in and of themselves of no cognitive value,
since they are essentially instrumental, they are always on
trial, and ‘liable to modification in the course of future
experience.’”—Present Philosophical Tendencies, p. 364
(italics mine).
310
It is known to all students that some of the more important
writings of this prince of thinkers cannot be intelligibly
approached without a long preliminary study of the peculiar
“dressing up,” or transformation, to which he subjects the
various facts of life and existence. And the same thing is true
(to a more modified extent) of the writings of Kant.
311
See the wise remark, in this very connexion, of the possible
service of philosophy to-day, of Dr. Bosanquet, reproduced
upon p. 226. And then, again, we must remember that an
unduly pragmatist view of life would tend to make people
impervious to ideas that transcend the range and the level of
their ordinary interests and activities.
312
Cf. the following from Professor Pace’s Preface to Introduction
to Philosophy, by Charles A. Dubray. “In Catholic colleges,
importance has always been attached to the study of
philosophy both as a means of culture and as a source of
information regarding the great truths which are influential in
supporting Christian belief and in shaping character.” Of
 course these same words might be used as descriptive of what
Professor Santayana calls the “older tradition” in all American
colleges. It is interesting, by the way, to note also the
pragmatist touch in the same Preface to this Catholic manual.
“But if this training is to be successful, philosophy must be
presented, not as a complex of abstruse speculations on far-off
inaccessible topics, but as a system of truths that enter with
vital consequence into our ordinary thinking and our everyday
conduct.”
313
See p. 136.
314
See above, p. 34 and p. 165.
315
It is not, however, “rest” that the pragmatists want, even in
heaven, but renewed opportunities for achievement. “‘There
shall be news,’ W. James was fond of saying with rapture,
quoting from the unpublished poem of a new friend, ‘there shall
be news in heaven.’”—Professor Santayana in Winds of
Doctrine, p. 209.
316
In using this expression I am acutely conscious of its
limitations and of its misleading character. There is nothing in
which Americans so thoroughly believe as knowledge and
instruction and information. A belief in education is in fact the
one prevailing religion of the country—the one thing in which
all classes, without any exception, unfeignedly believe, and for
which the entire country makes enormous sacrifices.
317
In using this expression I am not blind to such outstanding
characteristics of American life as (1) the enormous amount of
preventive philanthropy that exists in the United States; (2) the
well-known system of checks in the governmental machinery
of the country; (3) the readiness with which Americans fly to
legislation for the cure of evils; (4) the American sensitiveness
to pain and their hesitation about the infliction of suffering or
punishment, etc. Nor do I forget the sacrifice of life entailed by
modern necessities and modern inventions in countries other
 than America. I simply mean that owing to the constant stream
of immigration, and to the spirit of youthfulness that pervades
the country, the willingness of people to make experiments
with themselves and their lives is one of the many remarkable
things about the United States.
318
See p. 117.
319
And this despite the enormous amount of work that has been
done by American biologists upon the “factors” of evolution,
and upon a true interpretation of Darwinism and of
Weismannism and of the evolutionary theory generally.
320
Even Professor James, for example, dismissed (far too readily,
in my opinion) as a “sociological romance” a well-known book
(published both in French and in English) by Professor Schinz
entitled Anti-Pragmatism. Although in some respects a
superficial and exaggerated piece of work, this book did
discover certain important things about Pragmatism and about
its relation to American life.
321
It is probably a perception of this truth that has led Dr.
Bosanquet to express the opinion that the whole pragmatist
issue may be settled by an examination of the notion of
“satisfaction.” He must mean, I think, that satisfaction is
impossible to man without a recognition of many of the ideal
factors that are almost entirely neglected by the pragmatists—
except by Bergson, if it be fair to call him a pragmatist.
322
Bourdeau, for example, has suggested that its God is not really
God, but merely an old domestic servant destined to do us
personal services—-help us to carry our trunk and our cross in
the midst of sweat and dirt. He is not a gentleman even. “No
wonder,” he adds, “it was condemned at Rome.” See his
Pragmatisme et Modernisme, p. 82.
323
I am thinking here of the words in the Constitution of the State
of California (they are printed in Mr. Bryce’s American
Commonwealth—at least in the earlier editions) to the effect
 that it is the natural right of all men to seek and to “obtain [!]”
happiness.
324
“Epigramme,” Venice, 1790. [“I could never abide any of those
freedom-gospellers. All that they ever wanted was to get things
running so as to suit themselves. If you are anxious to set
people free, just make a beginning by trying to serve them.
The simplest attempt will teach you how dangerous this effort
may be.”]
325
See Chapter IX.
326
On what grounds does Professor Bosanquet think of
“compensating justice” as a naïve idea? It is on the contrary
one of the highest and deepest, and one of the most
comprehensive to which the human mind has ever attained—
giving rise to the various theogonies and theodicies and
religious systems of mankind. It is at the bottom, for example,
of the theodicy and the philosophy of Leibniz, the founder of
the Rationalism of the seventeenth and eighteenth centuries in
Europe.
327
Could any system of ethics which took such an impossible and
such a belated conception of the individual be regarded as
ethics at all?
328
I do not think that this is a fair preliminary description of the
problem of teleology. A person who believes in the realization
of purpose in some experiences with which he thinks himself to
be acquainted does not plead for the guidance of the universe
by finite minds, but simply for a view of it that shall include the
truth of human purposes. And of course there may be in the
universe beings other than ourselves who also realize
purposes.
329
Italics and exclamation mine.
330
Italics mine. There is a large element of truth in this great idea
of Professor Bosanquet’s, connecting [for our purposes] his
 philosophy with the theism and the personalism for which we
are contending as the only true and real basis for Humanism.
331
Readers who remember Green’s Prolegomena to Ethics will
remember that it is one of the difficulties of that remarkable,
but one-sided, production (exposed, I think, with many other
defects in Professor Taylor’s brilliant, but unduly intellectualistic
Problem of Conduct) that it also seems to teach a kind of
“Determinism” in ethics, in what our nature is unduly
communicated to us by the Absolute, or the “Eternal
Consciousness.” This whole way of looking at things must
largely be abandoned to-day.
332
See below, p. 226.
333
It is, I am inclined to think, the existence of this contradiction in
Dr. Bosanquet’s Lectures that will cause the average intelligent
person to turn away from them as not affording an adequate
account of the reality of the world of persons and things with
which he knows himself to be directly and indirectly
acquainted. Another way of stating the same thing would be to
say that Absolutism fails to take any adequate recognition of
that most serious contradiction (or “defect”) in our experience
of which we have already spoken as the great dualism of
modern times, the opposition between reason and faith an
opposition that is not relieved either by the greatest of the
continually increasing discoveries of science, or by any, or all,
of the systems of all the thinkers. Hegelianism in general
assures us that from the point of view of a “higher synthesis”
this opposition does not exist or that it is somehow
“transcended.” And its method of effecting this synthesis is to
convert the opposition between faith and reason into the
opposition between what it calls “Understanding” and what it
calls “Reason” [an opposition that is to some extent a fictitious
one, “reason” being, to begin with, but another name for our
power of framing general conceptions or notions, and not
therefore different from “understanding”]. It removes, that is to
say, the opposition between two different phases or aspects of
 our experience by denying the existence of one of them
altogether. It changes the opposition between knowledge and
faith into an opposition between an alleged lower and an
alleged higher way of knowing. This alleged higher way of
knowing, however, is, when we look into it, but the old ideal of
the perfect demonstration of all the supposed contents of our
knowledge (principles and facts alike) that has haunted
modern philosophy from the time of Descartes. It is an
unattainable ideal because no philosophy in the world can
begin without some assumption (either of “fact” or of principle),
and because our knowledge of the world comes to us in a
piecemeal fashion—under the conditions of time and space. A
fact prior to all the issues of the demand of Rationalism for a
supposedly perfect demonstration is the existence of the
conscious beings (Dr. Bosanquet himself, for example) who
seek this supposed certainty in order that they may act better
—in ignorance of the fact that complete initial certainty on our
part as to all the issues and aspects of our actions would tend
to destroy the personal character of our choice as moral
agents, as beings who may occasionally act beyond the given
and the calculable, and set up precedents and ideals for
ourselves and for others—for humanity. It is this underlying
faith then in the reality of our moral and spiritual nature that we
would alone oppose (and only in a relative sense) to the
supposed certainties of a completely rational, or a priori,
demonstration, the whole contention of humanism being that it
is in the interests of the former reality that the latter certainties
exist. The apparent opposition between faith and reason would
be surmounted by a philosophy that should make
consciousness of ourselves as persons the primal certainty,
and all other forms of consciousness or of knowledge
secondary and tributary, as it were, to this.
334
I am aware that there is a difference between the “universal” of
ordinary formal logic and Dr. Bosanquet’s (or Hegel’s)
“concrete universal,” but it is needless for me to think of it here.
Dr. Bosanquet uses in his Lectures the phrase “logical
 universal” for his “concrete universal” or his principle of positive
coherence. It is always logical coherence that he has in view.
335
“For everywhere it is creative Logic, the nature of the whole
working in the detail, which constitutes experience, and is
appreciable in so far as experience has value.” Now Logic of
itself does not thus “work” or “do” anything. It is men or
persons who do things by the help of logic and reasoning and
other things—realities and forces, etc.
336
Cf. p. 31. “We are minds,” he says, “i.e. living microcosms, not
with hard and fast limits, but determined by our range and
powers which fluctuate very greatly.” My point simply is that
this is too intellectualistic a conception of man’s personality.
We have minds, but we are not minds.
337
See p. 192. “But as the self is essentially a world of content
engaged in certain transformations”; and p. 193, “a conscious
being ... is a world ... in which the Absolute begins to reveal its
proper nature.” How can a “world of content” [that is to say, the
“sphere of discourse” of what some person is thinking for some
purpose or other] be “engaged” in certain transformations? It is
the person, or the thinker, who is transforming the various data
of his experience for his purposes as a man among men. It is
time that philosophy ceased to make itself ridiculous by calmly
writing down such abstractions as if they were facts.
338
Cf. “Mind as the significance and interpretation of reality,” p.
27.
339
“Mind has nothing of its own but the active form of totality,
everything positive it draws from Nature.”
340
This again is an abstraction, and how on earth can it be said
that “mind” and conscious life “reflect” merely certain
abstractions (or creations) of their own? They have invented
such terms as “content” for certain purposes, and their own
being and nature is therefore more than these terms. Mind is
not a “content”; it makes all other things “contents” for itself.
341
It has even there, according to Dr. Bosanquet, only its purely
theoretic function of working after its own perfection in the way
of attaining to a logical “universal.” “The peculiarity of mind for
us, is to be a world of experience working itself out towards
harmony and completeness.” This is simply not true.
342
“Finite consciousness, whether animal or human, did not make
its body.”
343
“Thus there is nothing in mind which the physical counterpart
cannot represent.” (Italics mine.)
344
“What we call the individual, then, is not a fixed essence, but a
living world of content representing a certain range of
externality.” P. 289.
345
“The system of the universe, as was said in an earlier Lecture,
might be described as a representative system. Nature, or
externality[!] lives in the lives of conscious beings. (Italics
mine.)
346
“Spirit is a light, a focus, a significance[!] which can only be by
contact with a ‘nature’ an external world.”
347
“For, on the other hand, it has been urged and we feel, that it is
thought which constructs and sustains the fabric of experience,
and that it is thought-determinations which invest even sense-
experience with its value and its meaning. . . . The ultimate
tendency of thought, we have seen, is not to generalise, but to
constitute a world,” p. 55. Again, “the true office of thought, we
begin to see, is to build up, to inspire with meaning, to
intensify, to vivify. The object which thought, in the true sense,
has worked upon, is not a relic of decaying sense, but is a
living world, analogous to a perception of the beautiful, in
which every thought-determination adds fresh point and
deeper bearing to every element of the whole,” p. 58. And on
p. 178 he says that he sees no objection to an idealist
recognising the “use made of” “laws” and “dispositions” in
 recent psychology. [How one wishes that Dr. Bosanquet had
really worked into his philosophy the idea that every mental
“element” is in a sense a “disposition” to activity!] Some of
these statements of Dr. Bosanquet’s have almost a pragmatist
ring about them, a suggestion of a living and dynamic (rather
than a merely intellectualistic) conception of thought. They may
therefore be associated by the reader with the concessions to
Pragmatism by other rationalists of which we spoke in an early
chapter (see p. 74).
348
See Chapter III. p. 90.
349
I must say that apart from any questions in detail about this
rejection of teleology by Dr. Bosanquet, there is something
inexplicable about it to me. He cannot retain his own great
notion of “wholeness” without the idea of “end,” because
“wholeness” is a demand of thought that is guided by some
idea of purpose or end.
350
See p. 90.
351
Italics mine.
352
P. 225.
353
See p. 90.
354
Having already given instances of this abstractionism in the
case of such things as the “self” and the “universal” and “spirit,”
it will suffice to point out here in addition (1) its tendency to talk
of “experience” and “experiences” as if there could be such
things apart from the prior real existence of the experients or
the experiencing persons with whom we are acquainted in our
daily life, and (2) its tendency to talk of getting at “the heart of
actual life and love” in a “system” which leaves no place for the
real existence of either gods or men who live and love. And
then I trust that it may not be regarded as an impertinence to
allege as another puzzling piece of abstractionism on the part
of Dr. Bosanquet, that he has allowed himself to speak and
 think in his book as if his theory of the “concrete universal”
were practically a new thing in the thought of our time—apart
altogether, that is to say, from the important work in this same
direction of other Neo-Hegelian writers, and apart, too, from
the unique work of Hegel in the same connexion.
355
See below, p. 230.
356
This is revealed in the main in its exposition of the world as the
logical system of a single complete individual experience—a
tendency that students of philosophy know to exist in Neo-
Hegelianism generally from Green to Bradley. I admit that this
tendency is literally a different thing from solipsism in the
ordinary sense, as the inability of a particular finite person to
prove to himself that any person or thing exists except himself.
It is still, however, it seems to me, possible to regard as
solipsistic the tendency to set forth the universe as the
experience, or the thought, of a single experient or a single
thinker, even although the impersonalism of Dr. Bosanquet’s
logical “whole” conflicts somewhat with the individuality of his
Absolute.
357
Cf. p. 160.
358
The well-known inability of Mr. Bradley, for example, to be
content with the reality of any portion or any phase of reality
that falls short of what he regards as absolute reality, and with
the merely relative meaning that he attaches to any category of
the “finite.” Also the well-known Neo-Hegelian tendency to
make an opponent forge the weapon by which he is to be
dislodged from any particular point of view. In the case of Dr.
Bosanquet this tendency takes the form of making out any one
who holds to a belief in the real existence of finite conscious
persons to hold the absurd position of believing in “an
impervious and isolated self,” a thing, of course, that no one
who knows anything about biology or ethics, or social
psychology, really does.