Microorganisms in Biorefineries

Microbiology Monographs

Series Editor: Alexander Steinbüchel

Birgit Kamm Editor

Microorganisms

in Biorefineries

Microbiology Monographs

Volume 26

Series Editor: Alexander Steinbu¨chel

Mu¨nster, Germany

More information about this series at

http://www.springer.com/series/7171

Birgit Kamm

Editor

Microorganisms in

Biorefineries

Editor

Birgit Kamm

FI Biopos e.V. and BTU Cottbus Research Center

Teltow-Seehof

Teltow

Germany

ISSN 1862-5576 ISSN 1862-5584 (electronic)

ISBN 978-3-662-45208-0 ISBN 978-3-662-45209-7 (eBook)

DOI 10.1007/978-3-662-45209-7

Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2014957319

© Springer-Verlag Berlin Heidelberg 2015

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part

of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,

recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or

information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar

methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts

in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being

entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication

of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the

Publisher’s location, in its current version, and permission for use must always be obtained from

Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center.

Violations are liable to prosecution under the respective Copyright Law.

The use of general descriptive names, registered names, trademarks, service marks, etc. in this

publication does not imply, even in the absence of a specific statement, that such names are exempt

from the relevant protective laws and regulations and therefore free for general use.

While the advice and information in this book are believed to be true and accurate at the date of

publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for

any errors or omissions that may be made. The publisher makes no warranty, express or implied, with

respect to the material contained herein.

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Series Editor

Alexander Steinbu¨chel

Institut fu¨r Molekulare Mikrobiologie und Biotechnologie

Westfa¨lische Wilhelms-Universita¨t

Mu¨nster

Germany

Dedicated to Michael Kamm, founder of

biorefinery.de GmbH

ThiS is a FM Blank Page

Preface

Although the chemical industry today still works with fossil raw materials such as

petrol and natural gas, even this sector will have a stronger focus on the use of

renewable feedstock: biomass from plants. A particular advantage of biorefineries

will be effective in this development for exploiting biomass perfectly: the genera￾tion of a high number of products and material for further processing in the

chemical industry. The development of microbial processes both for the digestion

of biomass and for the synthesis of platform chemicals and secondary products is an

important object of research in this context.

This monograph delivers a selective outlook on developments regarding micro￾organisms and their use in several product lines of the biorefinery. Microorganisms

in lignocellulosic feedstock biorefineries (chapters by Arkady P. Sinitsyn and

Alexandra M. Rozhkova; Alessandro Luis Venega Coradini et al.; M. Teresa

F. Cesa´rio and M. Catarina M. Dias de Almeida; and Dzˇenan Hozic´), particularly

concerning the production of polyhydroxyalkanoates and lipids, alcohol fuels, and

hydrocarbons, microorganisms in the green biorefinery focused on organic acids

(chapter by Petra Scho¨nicke et al.; Mette Hedegaard Thomsen et al.); and micro￾organisms for the synthesis of defined platform chemicals and specialty chemicals

containing heteroatoms (chapters by Qiang LI and Jianmin Xing; Nick Wierckx

et al.; Christine Idler, Joachim Venus, and Birgit Kamm; Robert Kourist and Lutz

Hilterhaus). Furthermore, microorganisms for the generation of isoprenoids and

methane from biomass are part of the biorefining observations (chapters by Claudia

E. Vickers et al.; Vladimir V. Zverlov, Daniela E. Ko¨ck, and Wolfgang

H. Schwarz).”

Teltow, Germany Birgit Kamm

vii

ThiS is a FM Blank Page

Contents

Penicillium canescens Host as the Platform for Development

of a New Recombinant Strain Producers of Carbohydrases .......... 1

Arkady P. Sinitsyn and Alexandra M. Rozhkova

Microbial Life on Green Biomass and Their Use for Production

of Platform Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Petra Scho¨nicke, Robert Shahab, Rebekka Hamann, and Birgit Kamm

Microorganism for Bioconversion of Sugar Hydrolysates

into Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Alessandro Luis Venega Coradini, Andre´ia Anschau, Annamaria D
oria Souza

Vidotti, E´rika Marques Reis, Michelle da Cunha Abreu Xavier,

Renato Sano Coelho, and Telma Teixeira Franco

Lignocellulosic Hydrolysates for the Production

of Polyhydroxyalkanoates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

M. Teresa F. Cesa´rio and M. Catarina M. Dias de Almeida

Microbial Research in High-Value Biofuels . . . . . . . . . . . . . . . . . . . . . . 105

Dzˇenan Hozic´

Microorganisms for Biorefining of Green Biomass . . . . . . . . . . . . . . . . . 157

Mette Hedegaard Thomsen, Ayah Alassali, Iwona Cybulska,

Ahmed F. Yousef, Jonathan Jed Brown, Margrethe Andersen,

Alexander Ratkov, and Pauli Kiel

Microbial Succinic Acid Production Using Different Bacteria

Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

Qiang Li and Jianmin Xing

ix

Whole-Cell Biocatalytic Production of 2,5-Furandicarboxylic

Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

Nick Wierckx, Tom D. Elink Schuurman, Lars M. Blank,

and Harald J. Ruijssenaars

Microorganisms for the Production of Lactic Acid and Organic

Lactates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Christine Idler, Joachim Venus, and Birgit Kamm

Microbial Lactone Synthesis Based on Renewable Resources . . . . . . . . 275

Robert Kourist and Lutz Hilterhaus

Production of Industrially Relevant Isoprenoid Compounds

in Engineered Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Claudia E. Vickers, James B.Y.H. Behrendorff, Mareike Bongers,

Timothy C.R. Brennan, Michele Bruschi, and Lars K Nielsen

The Role of Cellulose-Hydrolyzing Bacteria in the Production

of Biogas from Plant Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Vladimir V. Zverlov, Daniela E. Ko¨ck, and Wolfgang H. Schwarz

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

x Contents

Penicillium canescens Host as the Platform

for Development of a New Recombinant

Strain Producers of Carbohydrases

Arkady P. Sinitsyn and Alexandra M. Rozhkova

Contents

1 Introduction ................................................................................... 2

2 Development of Penicillium canescens Genetic Tools ..................................... 4

2.1 Penicillium canescens Selection Marker Based on Auxotrophic or Nutritionally

Deficient Penicillium canescens Strains ............................................... 4

2.2 Identification and Isolation of Strong Promoters for Gene Expression .............. 6

2.3 Construction of Expression Vectors and Cloning of Target Genes .................. 7

3 Penicillium canescens as a Producer of Cellulases ......................................... 9

4 Penicillium canescens as a Producer of Other Carbohydrases .. .. .. .. .. .. .. .. .. .. .. .. ... .. 13

5 Penicillium canescens as a Producer of Inulinases ....... ....... ....... ....... ....... ...... 14

Conclusions ....................................................................................... 17

References ........................................................................................ 18

Abstract The filamentous fungi strain Penicillium canescens has been developed

as a host for the production of heterologous proteins and enzymes for biorefinery.

There are several features of this strain which make it an attractive option as a host

expression system. First, P. canescens has a high growth rate and the developed

system of biosynthesis of extracellular enzymes; second, strain needs inexpensive

fermentation medium using sugar beet pulp as a main substrate; third, the fermen￾tation process can be easily scaled up; and fourth, there is auxotrophic strain

A.P. Sinitsyn

M.V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia

Russian Academy of Sciences, A.N. Bach Institute of Biochemistry, Leninsky Prospect, 33-2,

Moscow 119071, Russia

e-mail: [email protected]

A.M. Rozhkova (*)

Russian Academy of Sciences, A.N. Bach Institute of Biochemistry, Leninsky Prospect, 33-2,

Moscow 119071, Russia

e-mail: [email protected]

© Springer-Verlag Berlin Heidelberg 2015

B. Kamm (ed.), Microorganisms in Biorefineries, Microbiology Monographs 26,

DOI 10.1007/978-3-662-45209-7_1

1

P. canescens which can be transformed by plasmid DNA with exogenous genes. All

these factors make possible to create new efficient recombinant strains and enzyme

preparations (different endo-glucanases and cellobiohydrolases, β-glucosidase,

pectin lyase, inulinases) that are in demand by various sectors of industry and

biorefinery.

1 Introduction

Many enzymes used in biorefinery are fungal; in their natural habitat fungi secrete

cellulases, hemicellulases, pectinases, amylases, chitinases, other carbohydrases, as

well as esterases, ligninases, and related enzymes taking places in renewable

biomass degradation. Filamentous fungi also can be efficient in protein secretion

and production; besides that, fungi can be relatively easily cultured on the relatively

cheap substrates. These circumstances make fungi as an important tool for produc￾tion of enzymes for the needs of biorefinery. At the same time the secretion level of

many fungal enzymes is not high enough, and a number of fungal hosts for fungal

gene expression and methods of transformation have been disclosed for improve￾ment of secretion level of target enzymes and enzymatic mixtures. Aspergillus

(Lubertoz and Keasling 2009; Punt et al. 2002) and Trichoderma (Nevalainen

et al. 2005; Keranen and Pentilla 1995) are currently the main fungal genera applied

as expression system to produce enzymes for biorefinery. Recently Myceliophthora

thermophila (former Chrysosporium lucknowense) was suggested to use as a host

system for expression of biomass hydrolyzing enzymes (Visser et al. 2011). But the

search for efficient fungal host system is still continued to fulfill the demand of

biorefinery area for the source of cheap and efficient enzymes.

The general demands to the host are the following: the host must be readily

fermented using inexpensive medium and easy to scale up, should be capable of

efficient secretion of the protein, must process the desired protein such that it is

produced in an active form not requiring additional activation or modification steps,

should be readily transformed, should allow a wide range of expression regulatory

elements to be used thus ensuring ease of application and versatility, should allow

use of easily selectable markers that are cheap to use, and should produce stable

transformants.

We have developed the filamentous fungi strain Penicillium canescens as a host

for the production of heterologous proteins (enzymes) with many demands to the

host listed above: first, P. canescens has a high growth rate and the developed

system of biosynthesis of extracellular enzymes; second, strain needs inexpensive

fermentation medium using sugar beet pulp as a main substrate; third, the fermen￾tation process can be easily scaled up; and fourth, there is auxotrophic strain

P. canescens which can be transformed by plasmid DNA with exogenous genes.

All these factors make possible to create new efficient recombinant strains and

enzyme preparations (different endo-glucanases and cellobiohydrolases,

2 A.P. Sinitsyn and A.M. Rozhkova

β-glucosidase, pectin lyase, inulinases) that are in demand by various sectors of

industry and biorefinery.

The general scheme of enzyme preparation obtained in filamentous fungi hosts

applying genetic engineering approaches is presented in Fig. 1. Briefly, the first step

is amplification and cloning of the target gene into suitable expression vectors.

Obtained shuttle expression plasmid transforms into E. coli cells to determine

sequence of cloned gene (to exclude mismatch, deletions, mutations, and inser￾tions). Then, large-scale DNA isolation is carried out, because large DNA amount

(around 10γ) is necessary for fungal transformation. Then expression plasmid

together with transformation plasmid containing selective gene to separate recom￾binant strains is transformed to fungal protoplasts. The next step is primary screen￾ing of recombinant fungal clones by PCR to find chromosomal integration of target

genes. Then small-scale fermentation of new recombinant strains in shaking flasks

is carried out to determine basal and target enzyme activities and level of new

recombinant strain productivity. And final step is concluded in a large-scale fer￾mentation for production of enzyme preparation for testing in application trials.

Fig. 1 Scheme of enzyme preparation obtaining in filamentous fungi hosts

Penicillium canescens Host as the Platform for Development of a New... 3

2 Development of Penicillium canescens Genetic Tools

It is difficult to imagine modern biotechnology and, in particular, modified strains

that produce commercially important enzymes, without the use of genetic engi￾neering methods. Advantages of genetic engineering approaches consist of the

(1) possibility of multienzymatic complexes obtained with specified ratio of con￾stituent carbohydrases, (2) reproducible low time for creation of new recombinant

strains, (3) possibility to obtain (mono)producers of individual commercially

important enzymes, and (4) stable integration of gene(s) of interest to the fungal

chromosome.

In the early 1980s, numerous fungal isolates were screened for their natural

ability to produce new hemicellulases. This screening resulted in the isolation of a

fungal strain from soil capable of secreting xylanases, β-galactosidases, and

arabinofuranosidases, and this strain was characterized as a haploid filamentous

fungus (USSR Patent 1982, 1984). The fungus showed broad pH (4.5–6.0) and

temperature (25–35
C) ranges for growth. Based on morphological characteristics,

the isolate was classified as P. canescens (deposited at the Russian Collection of

Microorganisms (VKM) of the Russian Academy of Sciences, Accession No. VKM

F-175). The P. canescens strain was developed by the State Research Institute of

Genetics and Selection of Industrial Microorganisms (“Genetika”) as a platform for

recombinant strain producers of biotechnologically relevant multienzymatic

complexes.

In 1994 it was found that the arabinose is the main inductor for biosynthesis of

β-galactosidase (Nikolaev and Vinetski 1998).

In 1995 plasmid transformation was developed for P. canescens (Aleksenko

et al. 1995).

During 1994–1997 multicopy producers of β-galactosidase were obtained. The

level of β-galactosidase expression was 200 and 600 U/ml in fermentation broth

(Patent RU 1997, 1999).

As a result of application of genetic engineering approaches, the productivity of

β-galactosidase was increased 12 times. The specific activity and other properties of

the enzyme obtained by the multicopy strain did not change compared to those of

the native enzyme.

2.1 Penicillium canescens Selection Marker Based

on Auxotrophic or Nutritionally Deficient Penicillium

canescens Strains

Random mutagenesis procedures using UV light or the mutagenic agent N-methyl￾N0

-nitro-N-nitrosoguanidine (NTG) resulted in a primary strain lineage (Fig. 2).

The selection marker was developed for P. canescens strain F178 based on com￾plementation of niaD mutants lacking nitrate reductase activity, using the

4 A.P. Sinitsyn and A.M. Rozhkova