Downloaded from orbit.dtu.dk on: Oct 07, 2021 Lactic Acid Bacteria as a new platform for sustainable production of fuels and chemicals Boguta, Anna Monika Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Boguta, A. M. (2016). Lactic Acid Bacteria as a new platform for sustainable production of fuels and chemicals. Department of Systems Biology, Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Department of Systems Biology Technical University of Denmark Lactic Acid Bacteria as a new platform for sustainable production of fuels and chemicals PhD thesis Anna Monika Boguta Supervisors: Associate Professor Jan Martinussen Professor Peter Ruhdal Jensen Submitted: March 2016 Preface and acknowledgements The work described in this PhD thesis has been performed at the Department of Systems Biology, Technical University of Denmark, from January 2012 to March 2016. The project was supervised by Associate Professor Jan Martinussen and Professor Peter Ruhdal Jensen. The research was initially carried out at the Center for Systems Microbiology, which has then split into two smaller groups: Center for Systems Biotechnology with Peter Ruhdal Jensen as a group leader and Metabolic Signaling and Regulation Group, formed by Jan Martinussen and Mogens Kilstrup. First and foremost, I would like to thank my supervisors, Jan Martinussen and Peter Ruhdal Jensen, for their support, guidance, and valuable suggestions throughout my entire PhD project. Thank you for being inspiring and encouraging, and for always having the time for scientific discussions. I would also like to thank Associate Professor Mogens Kilstrup for his positive attitude and for his valuable scientific advices. Special thanks to Christopher Workman from the Center for Biological Sequence Analysis for his support and kind assistance during the microarray data analysis. Many thanks to Marzanna Pulka- Amin and Regina Åris Schürmann for their technical assistance, and for always being helpful and supportive. Thanks to all my colleagues for creating a friendly atmosphere and making it a good experience to come to work; it has been a great pleasure working with you. Additionally, I would like to thank all the colleagues that I had the privilege and pleasure to share office with: Malene Mejer Pedersen, Zhihao Wang, Anne-Mette Meisner Hviid and Karen Imbæk Starlit; thank you for very inspiring both scientific and non-scientific discussions we had. Finally, I would like to thank my husband, Tomasz, and my lovely daughter, Zuzanna, for their love, patience and unconditional support. I couldn’t have done it without you. Anna Monika Boguta Kgs. Lyngby, March 2016 iii iv Summary The diminishing natural resources and environmental issues lead us to consider other ways of producing materials, chemicals and energy to satisfy the ever-increasing needs of our society. Lignocellulosic biomass is the most abundant type of substrate in the world; it is also cheap and renewable which makes it a perfect candidate substrate for production of value added products. The second generation biorefineries, employing microorganisms for conversion of lignocellulosic feedstocks into value added products, are not yet employed commercially in a large scale. To increase the economic feasibility of the process, robust microbial catalysts are necessary, both having a broad substrate utilization range and being tolerant to the common inhibitors generated during the lignocellulose pretreatment. Even though many microorganisms are already well characterized and commercially employed in 1st generation biorefineries, the conversion of lignocellulose is a more complex process; thus, the pursue for a suitable microbe continues. In this PhD study, a wide collection of Lactic Acid Bacteria was systematically screened for the strains’ tolerance levels towards various inhibitors coming from the pretreatment of lignocellulosic biomass, as well as for their capabilities to utilize various sugar substrates, including both pentoses and hexoses. Almost 300 strains were tested, including 141 different isolates of Lactobacillus plantarum, L. paraplantarum, L. pentosus, L. brevis, L. buchneri and L. paracasei, and all available Lactobacillus and Pediococcus type strains. Five most promising strains were subjected to further studies; these included L. pentosus LMG 17672, L. pentosus LMG 17673, L. pentosus 10-16, P. pentosaceous ATCC 25745 and P. acidilactici DSM 20284. The strains were tested in growth experiments with increased concentrations of the key inhibitors, such as furfural and HMF, as well as with the presence of the most common combinations of inhibitors, mimicking real-life lignocellulosic feedstocks: sugarcane bagasse, wheat straw and soft wood. The two most promising strains were selected; these were L. pentosus LMG 17673 and P. acidilactici DSM 20284. They were not only found highly resistant to the key inhibitors, but they were also demonstrated to utilize pentoses, xylose and arabinose. For one of the selected most promising strains, P. acidilactici DSM 20284, a chemically defined medium was developed and optimized. The resulting Pediococcus Defined Medium (PDM) proved to support the growth of a variety of other species as well, including all Pediococcus species and several fastidious Lactobacilli. Thus, the PDM medium appears to be superior to the previously published defined media, and can therefore be suitable for physiological, biochemical or nutritional investigations in other LAB species. An efficient transformation procedure is necessary for strain’s rational genetic engineering. To ease strategies for further strain improvement, a transformation procedure was developed and optimized for P. acidilactici DSM 20284, increasing the transformation efficiency by 2 log units. An optimized method allows for the transformation with an efficiency of 2.8·103 transformants per µg DNA, permitting the genetic modification of this strain. In order to even further enhance the P. acidilactici DSM 20284 tolerance to furfural, an adaptation experiment was performed by continuous serial-transfer method. After 408 generations, an adapted strain A28 was isolated and showed an increased growth rate on the rich MRS medium with addition of furfural; yet, it also demonstrated a 27% better growth in MRS medium alone. A whole genome resequencing analysis revealed 62 mutations in the genome of the adapted strain compared to the wild-type. The mutations were mainly single nucleotide polymorphisms, but there were also 12 single insertions v identified. More than half of the mutations were non-synonymous substitutions, leading to an amino acid change. Two transcriptional regulators, HrcA and CtsR, were affected by non-synonymous substitutions within the protein or the Shine-Dalgarno sequence, respectively. Several membrane proteins as well as proteins involved in the cell redox homeostasis were also mutated. Purine biosynthesis, salvage and transport related genes were also affected by mutations, likely having an influence on the intracellular nucleotide pool sizes, thereby allowing for an increased growth rate. The analysis of the transcriptomic profiles of the wild-type P. acidilactici DSM 20284 and the adapted strain A28 revealed that the applied furfural concentration did not induce the stress response neither in the wild- type nor in the adapted strain. This finding indicates that both strains are already well adapted to furfural; thereby during the adaptive laboratory evolution experiment the strain adapted towards a faster and more efficient growth on the medium rather than towards furfural resistance. However, several genes related to exopolysaccharide biosynthesis or encoding membrane proteins were induced in the adapted strain, indicating that the cell wall structure might be important for the cell’s protection against furfural. The higher growth rate on the other hand, occurred to be enabled by an optimization of the purine and pyrimidine biosynthesis and salvage pathways, up-regulation of the folic acid biosynthesis as well as several enzymes involved in glycolysis. Finally, the study confirmed the remarkable potential of LAB for their use as microbial cell factories for conversion of lignocellulosic substrates into value-added products. vi Dansk Resumé De faldende ressourcer kombineret med et stadigt stigende globalt forbrug, gør det tvingende nødvendigt at overveje andre måder at producere blandt andet kemikalier på. Biomasse der indeholder lignocellulose er det der er mest af i verden; det er derfor tvingende nødvendigt at udnytte det til produktion af forædlede produkter fremadrettet. Anden generations bioraffinaderier der bruger mikroorganismer til at omdanne lignocellulose-holdige råmaterialer til