Microbial Biotechnology: Progress and Trends

Microbial

Production

Hideharu Anazawa

Sakayu Shimizu Editors

From Genome Design to Cell Engineering

Microbial Production

Hideharu Anazawa • Sakayu Shimizu

Editors

Microbial Production

From Genome Design to Cell Engineering

ISBN 978-4-431-54606-1 ISBN 978-4-431-54607-8 (eBook)

DOI 10.1007/978-4-431-54607-8

Springer Tokyo Heidelberg New York Dordrecht London

Library of Congress Control Number: 2013956366

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Editors

Hideharu Anazawa, Ph.D.

Director

Japan Bioindustry Association

Grande Bldg. 8F, 2-26-9 Hatchobori, Chuo-ku

Tokyo 104-0032 , Japan

[email protected]

Sakayu Shimizu, Ph.D.

Professor

Department of Bioscience and Biotechnology

Graduate School of Enviromental Science

Kyoto Gakuen University

Nanjo-Ohtani, Sogabe, Kameoka

Kyoto 621-8555 , Japan

[email protected]

v

Pref ace

It has long been considered essential to introduce energy-saving and environmentally

friendly bioprocesses to incorporate resource-saving concepts in production sys￾tems. Production of useful substances using microbial and enzymatic reactions, for

example, is a truly environmentally friendly process and should be actively explored

if there is even a small possibility for the process to replace a chemical- industrial

one based on conventional petrochemical reactions.

Historically speaking, the microbial production of useful substances has shown

expansion of its fundamental and technological platforms and evolved in a unique

manner, mainly through fermentative or enzymatic transformation of bioactive

compounds such as antibiotics, amino acids, nucleic acid-related compounds, and

vitamins. There have been some relatively recent developments in technologically

and industrially new areas, such as the production of chiral chemicals using chemo￾enzymatic methods, the production of commodity chemicals (e.g., acrylamide,

ethanol, isopropanol, and n -butanol), and single-cell oil production.

Many of the technologies originated in Japan and have made prominent contribu￾tions to mankind. One of the bases of these developments has been established

through extensive screening using the rich and diverse microbial resources of Japan,

a country that has been one of the major players in the establishment and develop￾ment of scientifi c and technological platforms.

As already mentioned, a bioprocess, especially a microbial one, is essentially

environmentally friendly. However, there are many unresolved issues related to

energy savings and resource depletion. Is CO 2 reduction really possible by introduc￾ing biosystems in place of petrochemical systems? Are biosystems really clean? At

this time, unfortunately, we still do not have enough data, concrete evidence, and

in-depth discussions about these issues. What are always referred to are the cases of

the nitrile hydratase process for acrylamide production and the lactonase process for

pantothenate production. In each instance, it is evident that the overall process is

simple and rapid, and requires less energy (30 % CO 2 reduction compared with

conventional chemical processes). Undoubtedly, this tendency can be found in

many of the processes already in use, but to our regret, no relevant data have been

presented to society.

vi

According to a report from the Department of Trade and Industries in the UK,

Japan’s strength in this area of biotechnology lies in the fact that chemical industries

have been actively promoting the industrialization of bioprocesses with the use of

their rich microbial resources and have incorporated their technologies into their

industrial structures. However, I believe that these facts may not, in themselves, be

obvious in Japan, or may already be self-evident and allow no room for further

debate, which could be why there are not many active discussions about these mat￾ters now. Am I the only person who has the impression that all relevant political

actions at the national level supporting this biotechnology are also inadequate?

Many of the chapters collected here are based on the results of the work for the

decade-long METI/NEDO project, the so-called Minimum Genome Factory, in

which I was involved as a project leader.

Kyoto , Japan Sakayu Shimizu

Preface

vii

Contents

Part I Minimum Genome Factory

1 Creation of Novel Technologies for Extracellular

Protein Production Toward the Development

of Bacillus subtilis Genome Factories .................................................... 3

Katsutoshi Ara, Kenji Manabe, Shenghao Liu,

Yasushi Kageyama, Tadahiro Ozawa, Masatoshi Tohata,

Keiji Endo, Kazuhisa Sawada, Nozomu Shibata,

Akihito Kawahara, Kazuhiro Saito, Hiroshi Kodama,

Yoshiharu Kimura, Katsuya Ozaki, Yoshinori Takema,

Hiroshi Kakeshita, Kouji Nakamura, Kunio Yamane,

Takeko Kodama, Junichi Sekiguchi, Takuya Morimoto,

Ryosuke Kadoya, Shigehiko Kanaya, Yasutaro Fujita,

Fujio Kawamura, and Naotake Ogasawara

2 Minimum Genome Factories in Schizosaccharomyces pombe............. 17

Hiromichi Kumagai, Mayumi Sasaki, Alimjan Idiris,

and Hideki Tohda

3 The Concept of the Escherichia coli Minimum Genome Factory ....... 25

Hideharu Anazawa

Part II Whole Genome Manipulation for Genome Design

4 Effi cient and Accurate Production

of De Novo Designed Large-Size Gene Clusters

by a Novel Bacillus subtilis-Based System ............................................ 35

Mitsuhiro Itaya, Shinya Kaneko, and Kenji Tsuge

5 Development and Application of Novel Genome

Engineering Technologies in Saccharomyces cerevisiae ....................... 53

Yu Sasano, Minetaka Sugiyama, and Satoshi Harashima

viii

6 Genome Design of Actinomycetes for Secondary Metabolism............ 63

Kiyoko T. Miyamoto and Haruo Ikeda

Part III Application of Omics Information and Construction

of Mutant Libraries

7 Application Methodology of Whole Omics Information ..................... 75

Myco Umemura and Masayuki Machida

8 Application of Genomics in Molecular Breeding

of the koji Molds Aspergillus oryzae and Aspergillus sojae .................. 87

Tadashi Takahashi

9 Comprehensive Libraries of Escherichia coli K-12

and Their Application ............................................................................. 97

Hirotada Mori, Rikiya Takeuchi, Yuta Otsuka, Yong Han Tek,

Wataru Nomura, and Barry L. Wanner

10 Insights into Metabolism and the Galactose Recognition

System from Microarray Analysis in the Fission Yeast

Schizosaccharomyces pombe ................................................................... 109

Kaoru Takegawa and Tomohiko Matsuzawa

Part IV Applications of Advanced Technologies for Production

11 Multi-enzymatic Systems for the Production

of Chiral Compounds ............................................................................. 121

Akira Iwasaki, Noriyuki Ito, and Yoshihiko Yasohara

12 Use of Organic Solvent-Tolerant Microorganisms

in Bioconversion ...................................................................................... 131

Akinobu Matsuyama

13 Approaches for Improving Protein Production

by Cell Surface Engineering .................................................................. 141

Takeko Kodama, Kenji Manabe, Katsutoshi Ara,

and Junichi Sekiguchi

14 Strategies for Increasing the Production Level

of Heterologous Proteins in Aspergillus oryzae ..................................... 149

Mizuki Tanaka and Katsuya Gomi

15 Overproduction of L-Glutamate in Corynebacterium glutamicum ...... 165

Hisashi Yasueda

Contents

ix

Part V Pharmaceuticals

16 Microbial Hormones as a Master Switch for Secondary

Metabolism in Streptomyces ................................................................... 179

Takeaki Tezuka and Yasuo Ohnishi

17 Enzymatic Production of Designed Peptide.......................................... 191

Kuniki Kino

Part VI Functional Foods

18 Microbial Production of Functional Polyunsaturated

Fatty Acids and Their Derivatives ......................................................... 207

Jun Ogawa, Eiji Sakuradani, Shigenobu Kishino, Akinori Ando,

Kenzo Yokozeki, and Sakayu Shimizu

19 Enzymatic Production of Oligosaccharides .......................................... 219

Takashi Kuroiwa

Part VII Cosmetics

20 Cosmetic Ingredients Fermented by Lactic Acid Bacteria.................. 233

Naoki Izawa and Toshiro Sone

21 Structure of Tyrosinase and Its Inhibitor from Sake Lees .................. 243

Yasuyuki Matoba and Masanori Sugiyama

Part VIII Energy and Chemicals

22 Toward Realization of New Biorefi nery Industries Using

Corynebacterium glutamicum ................................................................. 253

Haruhiko Teramoto, Masayuki Inui, and Hideaki Yukawa

23 Hydrogen Production Using Photosynthetic Bacteria ......................... 263

Jun Miyake

24 Production of Biofuels and Useful Materials by Anaerobic

Organisms in Ecosystem of Methane Fermentation ............................ 283

Yutaka Nakashimada and Naomichi Nishio

Index ................................................................................................................. 301

Contents

Part I

Minimum Genome Factory

H. Anazawa and S. Shimizu (eds.), Microbial Production: From Genome Design 3

to Cell Engineering, DOI 10.1007/978-4-431-54607-8_1, © Springer Japan 2014

Chapter 1

Creation of Novel Technologies for Extracellular

Protein Production Toward the Development

of Bacillus subtilis Genome Factories

Katsutoshi Ara , Kenji Manabe , Shenghao Liu , Yasushi Kageyama ,

Tadahiro Ozawa , Masatoshi Tohata , Keiji Endo , Kazuhisa Sawada ,

Nozomu Shibata , Akihito Kawahara , Kazuhiro Saito , Hiroshi Kodama ,

Yoshiharu Kimura , Katsuya Ozaki , Yoshinori Takema , Hiroshi Kakeshita ,

Kouji Nakamura , Kunio Yamane , Takeko Kodama , Junichi Sekiguchi ,

Takuya Morimoto , Ryosuke Kadoya , Shigehiko Kanaya , Yasutaro Fujita ,

Fujio Kawamura , and Naotake Ogasawara

K. Ara (*)

Integrated Medical Research Laboratories , Kao (China) Research &

Development Center Co., Ltd , Shanghai , China

Biological Science Laboratories , Kao Corp , Tochigi , Japan

e-mail: [email protected]

K. Manabe • S. Liu • Y. Kageyama • T. Ozawa • M. Tohata • K. Endo • K. Sawada

N. Shibata • A. Kawahara • K. Saito • H. Kodama • Y. Kimura • K. Ozaki • Y. Takema

Biological Science Laboratories , Kao Corp , Tochigi , Japan

H. Kakeshita

Biological Science Laboratories , Kao Corp , Tochigi , Japan

Graduate School of Life and Environmental Sciences, University of Tsukuba , Tsukuba , Japan

K. Nakamura • K. Yamane

Graduate School of Life and Environmental Sciences, University of Tsukuba , Tsukuba , Japan

Abstract Bacillus subtilis has been widely used for the industrial production of

useful proteins because of its high protein secretion ability and safety. We focused on

genome reduction as a new concept for enhancing production of recombinant

enzymes in B. subtilis cells based on detailed analysis of the genome mechanism.

First, we reported that a novel B. subtilis strain, MGB874, depleted 20.7 % of the

genomic sequence of the wild type by rationally designed deletions to create simpli￾fi ed cells for protein production. When compared with wild-type cells, the productiv￾ity of cellulase and protease from transformed plasmids harboring the corresponding

genes was markedly enhanced. These results indicate that a bacterial factory special￾izing in the production of substances can be constructed by deleting the genomic

regions unimportant for growth and substance production from B. subtilis . Second,

deletion of the rocDEF-rocR region, which is involved in arginine degradation, was

found to contribute to the improvement of enzyme production in strain MGB874.

4

The present study indicated that our results demonstrated the effectiveness of a

synthetic genomic approach with reduction of genome size to generate novel and

useful bacteria for industrial uses. Furthermore, the design of the changes in the

transcriptional regulatory network of the nitrogen metabolic pathway in B. subtilis

cells could facilitate the generation of improved industrial protein production.

Keywords Bacillus subtilis • Recombinant protein productivity • Refi ned genome

factory

1.1 Introduction

Bacillus subtilis ( B. subtilis ), a gram-positive soporiferous bacillus, has been widely

used for the industrial production of useful proteins because of its high protein

secretion ability and safety (Simonen and Palva 1993 ). Beginning about 1990,

mainly European and Japanese research groups implemented a project to sequence

the entire B. subtilis strain 168 genome, and reported the sequence of the entire

4,215-kbp genome in 1997 (Kunst et al. 1997 ). At that time, they reported 4,101

genes in the entire genome and identifi ed or inferred the functions of 58 % of these

genes. A subsequent project to analyze the functions of the unknown genes identi￾fi ed the functions of about half of the functionally unknown genes. In addition, the

individual disruption of 4,101 genes of B. subtilis showed that 271 genes (essential

genes) were absolutely essential for growth, and the majority was involved in DNA

replication, gene transcription and translation, cell structure formation, and cell

division (Schumann et al. 2000 ; Kobayashi et al. 2003 ).

Deleting genes unnecessary for the production and secretion of useful proteins

from the B. subtilis 168 strain and introducing necessary genes to improve the B. sub￾tilis genome, we aimed to create a host microorganism cell (MGF) that can effi ciently

T. Kodama

Biological Science Laboratories , Kao Corp , Tochigi , Japan

Faculty of Textile Science and Technology, University of Shinshu , Matsumoto , Japan

J. Sekiguchi

Faculty of Textile Science and Technology, University of Shinshu , Matsumoto , Japan

T. Morimoto • R. Kadoya

Graduate School of Information Science, Nara Institute of Science and Technology , Ikoma , Japan

Biological Science Laboratories , Kao Corp , Tochigi , Japan

S. Kanaya • N. Ogasawara

Graduate School of Information Science, Nara Institute of Science and Technology , Ikoma , Japan

Y. Fujita

Faculty of Life Science and Biotechnology, Fukuyama University , Fukuyama , Japan

F. Kawamura

Laboratory of Molecular Genetics , Rikkyo University , Tokyo , Japan

K. Ara et al.

5

produce commercial enzymes promising for application in a wide range of production

processes. The number of essential genes required for the growth of eukaryotic bacte￾ria is expected to be about 300–500, regardless of the genus and species, and the

majority of genomes consist of nonessential genes, which can probably be deleted

from the chromosome without infl uencing growth. Westers et al. deleted two pro￾phage regions (SP* and PBSX), three prophage-like regions (prophage 1, prophage 3,

and skin), and the largest operon pks from the B. subtilis genome, thereby constructing

a sextuple-deletion strain lacking 332 genes, accounting for 7.7 % of the entire

genome (Westers et al. 2003 ). In this sextuple-deletion strain, the deletion of regions

was found not to infl uence the growth of B. subtilis , protein secretion, transformation

competence, or sporulation ability, indicating that the genome of B. subtilis can be

artifi cially reduced by deleting nonessential gene regions.

We expected that it would be possible to construct a database for determination

of the minimum set of genes in B. subtilis by effi ciently deleting these regions. In

addition, it is interesting to know whether the deletion of a large genomic region

infl uences the production of useful enzymes and cell growth. We previously

attempted to delete regions of more than 10 kbp present in essential genes, and suc￾cessfully constructed a strain (MGF874) with a reduced genome, with deletions of

866 genes accounting for 20.7 % (about 874 kbp) of the B. subtilis genome, which

was similar in morphology and growth to the wild strain. Moreover, a genome￾reducing strain ( B. subtilis strain RGB1334) was constructed from B. subtilis

MGB874 by deletion of 27 gene regions, which resulted in a 20 % increase of cel￾lulose productivity in the later growth phase when compared to B. subtilis MGB874

(Ara et al. 2007 ; Morimoto et al. 2008 ; Kageyama et al. 2009 ). These results indi￾cate that a bacterial factory specializing in the production of substances can be

constructed by deleting the genomic regions unimportant for growth and substance

production from B. subtilis . Furthermore, based on detailed analysis of the genome

mechanism (Kobayashi et al. 2012 ), the design of the amino-acid metabolism of a

B. subtilis (Manabe et al. 2011 , 2012 ), improvement of secretion equipment

(Kakeshita et al. 2010 , 2011a , b , c ), a high translation system of a target gene

( Tagami et al. 2012 ), and cell surface engineering (see Chap. 15; Kodama et al.

2007a ; Kodama et al. 2007b ; Kodama et al. 2011 ), etc., were implemented.

1.2 Genome Reduction in B. subtilis

Strategies for genome reduction, which represents a relatively new fi eld in synthetic

genomics, have been used with Escherichia coli and B. subtilis to investigate micro￾bial genomic architecture and improve their characteristics (Westers et al. 2003 ;

Posfai et al. 2006 ; Mizoguchi et al. 2007 , 2008 ; Fabret et al. 2002 ). For effective

production of enzymes, we have been performing a study aiming at the creation of

B. subtilis MGF, by deleting genes that are not essential under normal cultivating

conditions, and enhancing essential genes. Single deletion of each of a number of

genes and multiple deletions of dozens of genomic regions were carried out effi -

ciently by using different deletion techniques.

1 Creation of Novel Technologies for Extracellular…

6

1.2.1 Genome Deletion Technology

A genetic tool to introduce marker-free deletions is essential for multiple manipula￾tions of genomes (Liu et al. 2007 ). We reduced the B. subtilis genome by step-by￾step deletion, using the upp (encoding uracil-phosphoribosyltransferase) cassette

and 5-fl uorouracil (5-FU) selection (Fabret et al. 2002 ), to select cells that had lost

a drug-resistant cassette used to introduce primary deletions through intramolecular

homologous recombination at repeated sequences fl anking the cassette (Morimoto

et al. 2008 ) (Fig. 1.1a ). We also developed a system using the AraR repressor to

improve the effi ciency at which marker-free mutants can be obtained (Liu et al.

2008 ). In this method, we replaced the native araR gene with a promoter-less neo￾mycin resistance gene ( neo ) fused to the arabinose operon ( ara ) promoter, and the

selection marker cassette containing a chloramphenicol resistance gene and the

araR gene encoding the repressor for the ara operon was then integrated into the

target site. Transformants became neomycin sensitive after integration of the marker

cassette because of the repression of neo expression by AraR, and marker cassette￾free cells were obtained by selection for neomycin resistance. However, the meth￾ods just described require the use of specifi c genetic backgrounds, such as

inactivation of the native upp gene for 5-FU selection, or replacement of the araR

gene with the Para - neo construct in the latter system (Liu et al. 2008 ). We devel￾oped a simple and effi cient method to create marker-free deletion mutants of B.

subtilis through transformation with recombinant PCR products, using the E. coli

mazF gene encoding an endoribonuclease that cleaves free mRNAs as a counter￾selection tool (Morimoto et al. 2009 , 2011a , b ).

The mazF -encoding cassette is fused with the fl anking sequences of the target

region using splicing by overlap extension-polymerase chain reaction (SOE-PCR).

Upstream and downstream sequences (fragments A and B) of the fl anking region to

be deleted are amplifi ed from the genomic DNA of the B. subtilis strain to be manip￾ulated. The mazF cassette is amplifi ed from the genomic DNA of B. subtilis strains

that contain a drug resistance gene and the mazF gene under the control of an iso￾propyl β-D-1-thiogzalactopyranoside (IPTG)-inducible spac promoter (Fig. 1.1b ).

An internal sequence (fragment C) in the target region is also amplifi ed. These PCR

products are fused by recombinant PCR in the order A–B– mazF- cassette–C (as

illustrated in Fig. 1.1) and integrated into the target region through homologous

recombination between fragment A and C loci. The resulting recombinants are

selected for drug resistance in the absence of IPTG. Thereafter, the primary trans￾formant is cultivated in the presence of IPTG (i.e., mazF toxin-inducing conditions),

and clones in which the mazF cassette has been excised by intramolecular homolo￾gous recombination at region B are selected (Fig. 1.1b ).

1.2.2 Multiple Deletion Design for MGF

To construct the multiple deletion series mutants, we rationally designed to maintain

cellular function for recombinant protein production. Among 4,106 genes in the

K. Ara et al.