Microbial production of Poly(3-hydroxybutyrate-3- hydroxyvalerate)-copolymers from starch-containing byproducts I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Mateusz Biernacki geboren am 28. September 1989 in Glogow, Polen Greifswald, 2018 i Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Hans-Joachim Schüller 2. Gutachter: Prof. Dr. Volkmar Passoth Tag der Promotion: 21.03.2019 ii Table of content Zusammenfassung ...................................................................................................................... 1 Summary .................................................................................................................................... 6 1 Introduction ....................................................................................................................... 10 1.1 Biopolymers ............................................................................................................... 10 1.2 Poly(hydroxyalkanoates) ........................................................................................... 10 1.3 Physical properties of PHAs ...................................................................................... 11 1.3.1 Poly(hydroxybutyrate) (PHB) ............................................................................ 11 1.3.2 Poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-V) ....................................... 12 1.3.3 Other PHAs ........................................................................................................ 12 1.4 Synthesis pathway of PHAs ...................................................................................... 13 1.5 Phasins ....................................................................................................................... 15 1.6 Microbial production of PHAs .................................................................................. 16 1.6.1 Bacteria ............................................................................................................... 16 1.6.2 Yeasts ................................................................................................................. 18 1.6.3 Plants .................................................................................................................. 19 1.6.4 Other organisms ................................................................................................. 20 1.7 Polymer extraction ..................................................................................................... 20 1.8 (R)-3-hydroxybutyric acid and (R)-3-hydroxyvaleric acid ....................................... 22 1.9 Applications ............................................................................................................... 24 1.9.1 Commercial products ......................................................................................... 25 1.10 Non-conventional yeast Arxula adeninivorans ......................................................... 27 2 Aim of the study and research strategy ............................................................................. 30 3 Materials and methods ...................................................................................................... 32 3.1 Oligonucleotide primers ............................................................................................ 32 3.2 Cultivation media ...................................................................................................... 33 3.2.1 Bacterial medium ............................................................................................... 33 3.2.2 A. adeninivorans medium .................................................................................. 34 3.2.3 A. nidulans medium ............................................................................................ 35 3.3 Cultivation conditions ................................................................................................ 36 3.3.1 Shaking flasks .................................................................................................... 36 3.3.2 Fed-batch cultivation .......................................................................................... 36 iii 3.3.3 Storage of DNA and bacterial and yeast strains. ................................................ 37 3.4 Molecular genetic techniques .................................................................................... 37 3.4.1 Small-scale plasmid DNA isolations from E. coli ............................................. 37 3.4.2 RNA isolation from yeast and fungi .................................................................. 37 3.4.3 Quantitative analysis of nucleic acids ................................................................ 39 3.4.4 Preparative and analytics agarose gel electrophoresis ....................................... 39 3.4.5 DNA sequencing ................................................................................................ 40 3.4.6 Polymerase Chain Reaction (PCR) .................................................................... 40 3.4.7 DNA restriction .................................................................................................. 42 3.4.8 Extraction of DNA from agarose gels ................................................................ 43 3.4.9 DNA ligation ...................................................................................................... 43 3.5 Yeast and bacterial transformation ............................................................................ 44 3.5.1 Bacterial transformation ..................................................................................... 44 3.5.2 Preparation of yeast competent cells .................................................................. 44 3.5.3 Yeast transformation .......................................................................................... 45 3.5.4 Stabilization of potential transformants. ............................................................ 45 3.6 Yeast protoplast fusion .............................................................................................. 45 3.7 Enzyme activity assays .............................................................................................. 46 3.7.1 β-ketothiolase ..................................................................................................... 47 3.7.2 Acetoacetyl-CoA reductase ................................................................................ 47 3.7.3 Thioesterase ........................................................................................................ 48 3.7.4 ATP citrate lyase ................................................................................................ 48 3.7.5 Statistics ............................................................................................................. 49 3.8 PHA and (R)-3-HB analysis ...................................................................................... 49 3.8.1 GC/MS qualitative analysis ................................................................................ 49 3.8.2 (R)-3-HB enantiopurity ...................................................................................... 49 3.8.3 Extraction and polymer analysis ........................................................................ 50 3.9 Polymer imaging ........................................................................................................ 50 3.9.1 Confocal light microscopy ................................................................................. 50 3.9.2 Transmission Electron Microscopy (TEM) ........................................................ 50 3.10 Metabolites determination ......................................................................................... 51 3.10.1 Fatty acids .......................................................................................................... 51 3.10.2 Glucose ............................................................................................................... 51 4 Results ............................................................................................................................... 52 iv 4.1 Primary research for polyhydroxyalkanoates production. ......................................... 52 4.1.1 Construction of expression plasmids for primary research ................................ 52 4.1.2 PCR analysis for introduction of genetic material into A. adeninivorans genome 53 4.1.3 GC/MS analysis of accumulated polyhydroxyalkanoates. ................................. 55 4.2 (R)-3-hydroxybutyric acid synthesis ......................................................................... 56 4.2.1 Searching for suitable β-ketothiolase and acetoacetyl-CoA reductase genes .... 57 4.2.2 Construction of expression plasmids .................................................................. 57 4.2.3 GC/MS (R)-3-HB standard curve ...................................................................... 59 4.2.4 Screening for transformants secreting 3-HB ...................................................... 59 4.2.5 Time course experiment. .................................................................................... 60 4.2.6 Relationship between (R)-3-HB production and enzymatic activity