ISOLATION, CHARACTERIZATION AND ENGINEERING OF BACILLUS SMITHII A NOVEL THERMOPHILIC PLATFORM ORGANISM FOR GREEN CHEMICAL PRODUCTION ELLEKE F. BOSMA Thesis committee Promotors Prof. Dr Willem M. de Vos Professor of Microbiology Wageningen University Prof. Dr John van der Oost Personal Chair at the Laboratory of Microbiology Wageningen University Co-promotor Dr Ir. Richard van Kranenburg Corporate Scientist Cell Factories and Team Leader Strain Development Corbion, Gorinchem Other members Prof. Dr Gerrit Eggink, Wageningen University Prof. Dr Oscar P. Kuipers, University of Groningen Dr Eric Johansen, Chr. Hansen A/S, Hørsholm, Denmark Dr Filipe Branco dos Santos, University of Amsterdam This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). ISOLATION, CHARACTERIZATION AND ENGINEERING OF BACILLUS SMITHII A NOVEL THERMOPHILIC PLATFORM ORGANISM FOR GREEN CHEMICAL PRODUCTION ELLEKE F. BOSMA Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 6 November 2015 at 4 p.m. in the Aula. Elleke F. Bosma Isolation, characterization and engineering of Bacillus smithii - A novel thermophilic platform organism for green chemical production. 220 pages. PhD thesis, Wageningen University, Wageningen, NL (2015) With references, with summary in English ISBN 978-94-6257-507-3 Voor Oma Sietske TABLE OF CONTENTS Chapter 1 General introduction and thesis outline 9 Chapter 2 Sustainable production of bio-based chemicals by extremophiles 21 Chapter 3 Isolation and screening of thermophilic bacilli from compost 57 for electrotransformation and fermentation: Characterization of Bacillus smithii ET 138 as a new biocatalyst Chapter 4 Complete genome sequence of thermophilic Bacillus smithii type 85 strain DSM 4216T Chapter 5 Establishment of markerless gene deletion tools in thermophilic 103 Bacillus smithii and construction of multiple mutant strains Chapter 6 Metabolic characterization of thermophilic Bacillus smithii and 127 mutant strains Chapter 7 Thesis summary and general discussion 159 References 181 Co-author affiliations 205 Acknowledgements / Dankwoord 209 About the author 216 List of publications 217 Overview of completed training activities 218 CHAPTER 1 GENERAL INTRODUCTION AND THESIS OUTLINE GENERAL INTRODUCTION 10 A SHORT HISTORY OF MICROORGANISMS AS CELL FACTORIES In the late 18th century, Antonie van Leeuwenhoek made his famous microscopic ob- servations of “animalcules” (“little animals”), which we now know as bacteria. In the century after that, Louis Pasteur made his ground-breaking discoveries on the spoilage of wine and milk, revealing that bacterial activity was the cause of this phenomenon. He also postulated that if bacteria could spoil wine and milk from the air, they also might be the cause of human disease. This theory was confirmed in the late 19th century by Robert Koch, who showed that Bacillus anthracis was the direct causative agent of anthrax. He was also the first one to establish methods to grow pure bacterial cultures in the laboratory as we do nowadays. Although micro-organisms such as bacteria and yeast had been used already for centuries in basic biotechnological processes such as those for making bread, wine, beer and sake, it was only after the discovery of bacteria as organisms and fermentation as a process that people consciously started to exploit bacteria (as well as other microbes such as yeast and fungi) as cell factories. In the late 19th century, beer brewers started to use pure yeast cul- tures in order to make their process more reliable and the taste of their beer more constant. During World War I, also other fermentation products started to be used as replacements for products for which a shortage occurred, such as acetone as solvent for the explosive cor- dite and lactic acid as hydraulic fluid – both products were made by bacterial fermentation and it was around this time that the word ‘biotechnology’ was coined for these processes, in which raw material was converted to valuable products by biological systems. From this time onwards, the field of biotechnology in general, and microbial biotechnology in particu- lar, has been ever-expanding and more and more useful products made by microbes were discovered. Biotechnology took an even larger flight after the discovery of the structure of DNA by Watson and Crick in 1953 (Watson & Crick, 1953) and the subsequent discovery of a technique to transfer DNA from one bacterium to another and from eukaryotes to bacteria in the 1970’s (Cohen & Chang, 1973; Morrow et al., 1974). The ability to modify the genome and with that the production capacities of the host organism, created a whole new range of possibilities to develop microorganisms into efficient cell factories for the production of valuable compounds such as medicines, food, chemicals and fuels. THE BIOREFINERY Whereas in a traditional oil refinery valuable products are made from raw fossil materi- als (oil), in a biorefinery valuable compounds are produced from raw renewable materials (biomass) by microbial fermentation (Figure 1). Like in a traditional refinery, in a biorefinery a wide range of products can be made in different, integrated streams of the refinery, in which waste products of one production stream can be used as substrate or input of another stream. Also comparable to a traditional refinery, the products in a biorefinery can range 11 from fuels that can be directly used, to so-called building block chemicals that can be used in further processes such as polymerization to create for example bio-plastics. Examples OUTLINE of such building blocks are lactic acid and succinic acid, which can be used as a basis for generating polymers such as plastics, nylons, solvents, pharmaceuticals, cosmetics and AND food ingredients such as preservatives (Bozell & Petersen, 2010). A major advantage of using microbial conversion of biomass instead of chemical conversion is the optical purity that can generally be achieved via microbial metabolism. Examples of products that can only be produced as racemic mixtures via chemical synthesis, but rather in their pure opti- INTRODUCTION . 1 cal forms by microbes are 2,3-butanediol (2,3-BDO), malate and lactate. To create uniform polymers of these molecules, optically pure building blocks are required (Nair & Laurencin, CH 2007). Whereas this requires an extra purification step when produced chemically, they can be purified relatively easily when produced by microbes containing only one of the stereo-selective enzymes. With the growing global demand for fuels and chemicals and because of the environ- mental impact of the use of fossil resources, the production of fuels and chemicals from renewable resources in a biorefinery is generally considered to be an attractive green alter- native. The initial focus of the green industry was mainly on bio-fuels (mainly bio-ethanol), but green chemicals are gaining attention. In 2013, the global market size for alcohols was 110 billion USD and is expected to grow with 4.4% until 2020, whereas the market for orga- nic acids and polymers was 3.5 billion USD and 0.6 billion USD, respectively, with expected respective growths of 8.8% and 13.5% until 2020 (Deloitte, 2014). One of the major con- cerns with the biological processes is that often food resources such as corn or sugar beet are used as raw material, potentially creating competition between fuels and chemicals and the human food chain. Therefore, current research focuses on the development of ‘second generation’ bio-fuels and chemicals, which are derived from non-food biomass such as non-edible parts of crops, agricultural, forestry and household waste. This is challenging as the pure sugars used in first generation biorefineries can be easily utilized by the microorga- nisms that are currently used as production hosts, whereas the second generation biomass consists of lignocellulose. Lignocellulose consists of tightly packed sugar chains containing different types of sugars, which is harder to utilize and requires both additional pre-treat- ment to make these substrates accessible to enzymatic hydrolysis as well as the addition of hydrolytic enzymes to generate fermentable sugars. ‘Third generation’ alternatives such as syngas fermentation and photosynthetic processes are also considered but these are very recently emerged strategies that require time to be developed (Sheldon, 2011). In order to make the second generation large scale biorefineries (Figure 1) economically and ethically feasible, the production costs need to be reduced and non-food substrates need to be used. There are ample possibilities to achieve cost reduction in each step of 12 the biorefinery such as using cheaper substrates; optimizing pre-treatment; reducing en- zyme load; reducing operational costs such as required tanks, cooling and contamination costs; optimizing the microorganisms to produce more of the compound of interest and less by-products; and optimizing down-stream processing. For example, different operation mo- des using separated or simultaneous saccharification of the biomass by hydrolytic enzymes and fermentation by the microbes are possible (Figure 1). This will be further explained in Chapter 2. To shift from food to non-food resources, either the current