Corynebacterium glutamicum: From Systems Biology to Biotechnological Applications
"Without question a valuable book" (BIOSpektrum)
Caister Academic Press
Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany
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Corynebacterium glutamicum is most widely known for its role in the industrial production of L-glutamate and L-lysine and as a platform organism for the production of a variety of fine chemicals, biofuels and polymers. The organism's accessibility to genetic manipulation has resulted in a wealth of data on its metabolism and regulatory networks; this in turn makes C. glutamicum the model organism of choice in white biotechnology. A key development in recent years has been the engineering of C. glutamicum to utilize a broader spectrum of carbon sources (e.g. glycerol, galactose and pentose sugars) in addition to the traditional hexoses. Given its unique ability to co-utilize mixed carbon sources, C. glutamicum could be used to clean-up of wastes from agricultural or other industries, simultaneously producing useful compounds such as L-lysine or putrescine.
This book provides a comprehensive overview of current knowledge and research on C. glutamicum systems biology and biotechnological applications. Written by a team of prominent scientists under the expert editorship of Andreas Burkovski, the topics covered include: proteomics; flux analysis technology for metabolic analysis; metabolic engineering for alternative carbon source utilization; manipulation of nitrogen metabolism; transport, degradation and assimilation of aromatic compounds and their regulation; engineering for production of organic acids and alcohols; microbial factory for the production of polyesters; biotechnological application under oxygen deprivation; the secretory production of heterologous proteins; genetically-encoded biosensors.
Packed with practical information and state-of-the-art science this concise volume is an essential handbook for everyone working with Corynebacterium and related organisms in academia, biotechnology companies, and the pharmaceutical industry and is a recommended volume for all microbiology libraries.
"Without question a valuable book with information that is not available elsewhere in this concise form" from Biospektrum (2015) 21: 459.
Table of contents
1. Trends in Corynebacterium glutamicum Research and Application
Industrial production of amino acid, a key sector of white biotechnology, is the classical field of application of Corynebacterium glutamicum. With a current annual production of more than 2.9 million tons of L-glutamate and more than 1.9 million tons of L-lysine (Ajinomoto, 2013a, b) C. glutamicum already represents a key organism for industrial biotechnology. Taking current megatrends in society, such as increasing population and demands for meat in account, it is likely that amino acid production will increase continuously as already estimated previously (Takors et al., 2007). Furthermore, within the last decade, C. glutamicum was applied in various production processes for fine chemicals, fuels and polymers. Based on these studies, it becomes increasingly obvious that C. glutamicum has an enormous potential for the biotechnological synthesis of many compounds and materials, making C. glutamicum a versatile platform organism and biotechnology workhorse.
2. Proteomics of Corynebacterium glutamicum
Andreas Harst and Ansgar Poetsch
Proteomics is an important technique to study the biology of Corynebacterium glutamicum with focus on processes related to the biotechnological production of amino acids and other chemicals. Within this review, a comprehensive overview is given about the methods established for C. glutamicum. The methods developed in C. glutamicum have been and can be applied to other organisms. Technologies for quantification, membrane enrichment and MS analysis tested in C. glutamicum might lead to higher coverage for membrane proteins or improve the accuracy and reliability of quantification based on isotope labeling. In summary, proteomics data are important for systems biology approaches in C. glutamicum.
3. Developing Interpretation of Intracellular Metabolism of Corynebacterium glutamicum by Using Flux Analysis Technology
Tomokazu Shirai and Hiroshi Shimizu
Corynebacterium glutamicum is often used as a model organism because of its ability to produce various useful substances. For achievement of high productivity, metabolic flux analysis (MFA) is a powerful technique, which is used to determine the state of intracellular metabolism in microorganisms. In order to develop the precise MFA, it is necessary to obtain 13C labeling information of intracellular metabolites, in addition to the data of extracellular measurements such as substrate consumption rates, cell growth rates, and production rates. Metabolic flux can be estimated accurately through a mathematical approach by using computer calculations to analyze 13C labeling information of intracellular metabolites by performing nuclear magnetic resonance and/or gas chromatography-mass spectrometry. The accurate determination of intracellular metabolisms by using MFA permits a deeper understanding of C. glutamicum physiology and can produce information for improving the production of useful compounds by using this bacterium.
4. Growth and Production Capabilities of Corynebacterium glutamicum: Interrogating a Genome-scale Metabolic Network Model
Elisabeth Zelle, Katharina Nööh and Wolfgang Wiechert
In recent years, the assembly of genome-scale metabolic networks has been established as a powerful tool for obtaining a comprehensive understanding of microbial production hosts and for exploring their industrial potential. Network modeling provides a versatile framework for analyzing, predicting and, eventually, optimizing cellular processes. This book chapter reviews the current state of stoichiometric modeling for Corynebacterium glutamicum and presents a new curated network containing 475 reaction steps and 408 metabolites. This network is interrogated to obtain answers for several fundamental questions on quantitative growth and production capabilities of the organism. Using flux balance analysis the model is validated with experimental data, maximal yields for all 20 amino acids are exemplarily computed and the impact of biomass composition data on model prediction is systematically investigated. As an extension of stoichiometry, the structural prerequisites for metabolic flux analysis are discussed. For the purpose of isotope-based flux analysis, the central metabolic network is extended by carbon transition information required for tracer studies. The genome-scale metabolic network presented in this chapter is supplied via a web server.
5. Metabolic Engineering of Corynebacterium glutamicum for Alternative Carbon Source Utilization
Lennart Leβmeier, Christian Matano, Ahmed Zahoor, Steffen N. Lindner and Volker F. Wendisch
Corynebacterium glutamicum is well known for the multi-million-ton-scale production of L-glutamate and L-lysine in biotechnological industry, which is currently based on hexoses derived from starch and molasses. Only recently, metabolic engineering approaches were used to broaden the substrate spectrum of C. glutamicum, allowing access to alternative carbon sources such as glycerol, galactose and pentose sugars present in lignocellulosic hydrolysates. Unlike most microorganisms, C. glutamicum is able to co-utilize mixed carbon sources, making it a preferential host for utilization of complex carbon sources such as wastes from agricultural industry. Several industrial byproducts have been successfully valorized by engineered C. glutamicum strains. Fermentative production of amino acids has been shown based on crude glycerol from biodiesel production, silage juice and rice straw hydrolysates. In this review, we will present a glimpse into the state of the art of metabolic engineering of C. glutamicum toward utilization of alternative carbon sources, such as wastes and unprocessed sources.
6. Manipulation of Nitrogen Metabolism and Alternative Nitrogen Sources for Corynebacterium glutamicum
Nadine Rehm, Julia Bürger and Andreas Burkovski
Nitrogen is an essential component of nearly all macromolecules in a bacterial cell, e.g. proteins, nucleic acids and cell wall components and their corresponding building blocks. To satisfy the nitrogen demand and to ensure a sufficient nitrogen supply even in situations of nitrogen limitation, microorganisms have evolved sophisticated uptake and assimilation mechanisms for different nitrogen sources. This review focuses on nitrogen metabolism and its control in the biotechnology workhorse Corynebacterium glutamicum, which is used for the industrial production of almost four million tons of L-amino acids each year. Ammonium assimilation and connected control mechanisms on activity and transcription level are summarized and the influence of mutations on amino acid pools and production of L-glutamate, L-glutamine and L-lysine is discussed.
7. Transport, Degradation and Assimilation of Aromatic Compounds and their Regulation in Corynebacterium glutamicum
Xi-Hui Shen, Tang Li, Ying Xu, Ning-Yi Zhou and Shuang-Jiang Liu
The closing of the last gap of the genome sequence of Corynebacterium glutamicum opened a new door to insight in physiology and genetics. For example, longtime overlooked pathways for aromatic degradation and assimilation by C. glutamicum were discovered by the access to genome data, and broad knowledge on the physiology and genetics of aromatic degradation and assimilation by C. glutamicum has been accumulated in the last ten years. This chapter summarizes the current understanding of transport and metabolism of aromatic compounds by C. glutamicum as well as their regulation.
8. Engineering Corynebacterium glutamicum for Production of Organic Acids and Alcohols
Bernhard J. Eikmanns and Michael Bott
Due its success in the large-scale production of L-glutamate and L-lysine and the tremendous increase in knowledge on its metabolism and regulatory networks as well as the establishment of reliable tools for genetic engineering, Corynebacterium glutamicum has become a favorite model organism in white biotechnology. Within the past decade, its potential for the production of other amino acids, but also of a variety of other metabolites of commercial interest has been explored extensively. A major driving force of these studies is the aim to establish a bio-based economy based on renewable carbon sources, which enables to reduce our dependency on fossil carbon sources and to establish sustainable production processes. Important targets in this direction are organic acids, which are employed e.g. as precursors for bulk chemicals and bio-based polymers, and alcohols, which serve e.g. as biofuels. In this review, we will summarize the current knowledge on C. glutamicum strains developed for the production of the organic acids succinate, L- and D-lactate, pyruvate, the 2-keto acids ketoisovalerate, ketoisocaproate and ketoglutarate and of the alcohols ethanol and isobutanol. From the data presented it becomes obvious that C. glutamicum has an enormous potential for the biotechnological synthesis of many of these compounds, which is comparable and often even better than that described for alternative production hosts.
9. Microbial Factory for the Production of Polyesters: A New Platform of Corynebacterium glutamicum
Yuyang Song, John Masani Nduko, Ken'ichiro Matsumoto and Seiichi Taguchi
Corynebacterium glutamicum has been extensively employed for the industrial production of many value-added compounds such as food grade amino acids. Recently, many promising studies have focused on the engineering of this bacterial platform beyond the scope of amino acid production such as; for the production of microbial polyesters called polyhydroxyalkanoates (PHAs), which have the potential to replace petroleum-derived plastics. Thus far, C. glutamicum has been engineered by expressing PHA synthase and related monomer supplying enzymes to produce a variety of PHAs including poly(3-hydroxybutyrate) [P(3HB)] homopolymer and copolymers like poly[3HB-co-3-hydroxyvalerate(3HV)]. In a current hot topic, the biosynthesis of poly(lactic acid)-like polyesters has been achieved in C. glutamicum by using an engineered PHA synthase, (lactate-polymerizing enzyme: LPE). Furthermore, the arming C. glutamicum displaying a-amylase on its cell surface has provided an attractive platform for the production of the microbial polyesters from starch as a less expensive feedstock. These studies demonstrate the potential of this organism as a platform for microbial polymer factory.
10. Biotechnological Application of Corynebacterium glutamicum Under Oxygen Deprivation
Toru Jojima, Masayuki Inui and Hideaki Yukawa
Traditional bioprocesses using Corynebacterium glutamicum as biocatalysts, including amino acid fermentation process, are operated under aerobic conditions. An emerging bioprocess in which the microorganism is utilized under oxygen deprivation, so-called growth-arrested bioprocess, is from these traditional fermentation processes. This novel bioprocess is expected to play a major role in the production of biofuels, commodity and fine chemicals, besides the aforementioned amino acids. In this chapter, we describe the physiology of C. glutamicum under oxygen deprived conditions with respect to recent advances in development of the microorganism as a mainstay biocatalysts used in growth-arrested bioprocess, with special emphasis on potential applications relevant to future lignocellulosic biorefineries.
11. Corynebacterium glutamicum as a Platform Organism for the Secretory Production of Heterologous Proteins
Corynebacterium glutamicum is widely used as an industrial workhorse for the production of amino acids and other low molecular weight compounds. However, an increasing amount of evidence indicates that this microorganism likewise might possess a great potential as an alternative platform organism for the secretory production of heterologous proteins. Besides giving an overview on the major protein secretion routes present in C. glutamicum, i.e. the general secretion (Sec) system and the twin-arginine translocation (Tat) system, this review summarizes the attempts that have been made so far to use C. glutamicum as a host system for the expression and subsequent secretion of foreign proteins. In addition, this review also addresses recent progress with respect to producer strain improvement and process optimization.
12. Genetically-encoded Biosensors for Strain Development and Single Cell Analysis of Corynebacterium glutamicum
Nurije Mustafi, Michael Bott and Julia Frunzke
Genetically-encoded single cell biosensors have long been used for the detection of pollutants and stress stimuli in the field of microengineering and bioremediation. In industrial microbiology their potential for the detection of various small molecules in single cells and their implementation in strain development and high-throughput (HT) screenings has rarely been exploited so far. However, several recent studies revealed suchlike sensor systems, which transform the production of inconspicuous small molecules into an optical readout (e.g. fluorescence), as a powerful tool for single cell quantification of metabolites. Recent studies reported on the construction of transcription factor-based biosensors for the intracellular detection of amino acid production in Corynebacterium glutamicum. In this chapter we will introduce the general design and characterization of sensor performance features. An overview of all currently available C. glutamicum biosensors is provided, including several sensors for the detection of amino acids and stress stimuli in single cells. Finally, we introduce the emerging field of applications for metabolite biosensors in flow cytometry-based HT screening approaches and live cell imaging of metabolite production using microfluidic lab-on-a-chip devices. First results, outlined in this chapter, already highlight the great potential of biosensor-driven strain development and single cell analysis and promise the revolution of traditional approaches towards a "bright" future of industrial microbiology.
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(EAN: 9781910190050 9781910190067 Subjects: [microbiology] [bacteriology] [molecular microbiology] [genomics] [environmental microbiology] )