Design, Construction, and Application of Synthetic Microbial Consortia by Alissa R. Kerner A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biomedical Engineering) in the University of Michigan 2013 Doctoral Committee: Assistant Professor Xiaoxia Nina Lin, Chair Professor Erdogan Gulari Professor Jennifer J. Linderman Professor Shuichi Takayama © Alissa R. Kerner 2013 All Rights Reserved Table of Contents List of Figures .................................................................................................................... v List of Tables ................................................................................................................... vii Abstract .................................................................................................................... viii Chapter 1 Background and Motivation ...................................................................... 1 1.1 Microbial Consortia ......................................................................................................... 1 1.2 Consolidated bioprocessing for biofuel production ...................................................... 5 1.3 Dissertation Overview ...................................................................................................... 7 Chapter 2 Design and construction of a programmable, synthetic E. coli consortium via tunable symbiosis .................................................................................... 9 2.1 Summary ........................................................................................................................... 9 2.2 Introduction .................................................................................................................... 10 2.2.1 General scheme and basic model for two cross-feeding auxotrophs ........................ 10 2.2.2 Tryptophan and tyrosine over-production ................................................................. 13 2.2.3 Choosing suitable inducible promoters ..................................................................... 17 2.2.4 Basic model implementation with E. coli strains W3 and Y3 .................................. 18 2.3 Methods ........................................................................................................................... 19 2.3.1 Auxotroph construction and YFP addition ............................................................... 19 2.3.2 Plasmid construction ................................................................................................. 19 2.3.3 Co-culture composition determination using YFP .................................................... 20 2.3.4 Measurement of Trp and Tyr concentrations in mono- and co-culture supernatants 22 2.3.5 Measurement of Trp and Tyr Affinities .................................................................... 23 2.3.6 3D surface and design space plots ............................................................................ 23 2.4 Results ............................................................................................................................. 24 2.4.1 Effect of inducers on metabolite secretion and growth properties of engineered strains 24 ii 2.4.2 Changes in the consortium growth and composition ................................................ 27 2.4.3 Incorporation of metabolic burden in mathematical modeling ................................. 32 2.4.4 Programming the synthetic consortium with the Design Space ................................ 35 2.5 Discussion and conclusion ............................................................................................. 38 Chapter 3 Engineering a two-member E. coli consortium for conversion of five- and six-carbon sugars to isobutanol .............................................................................. 41 3.1 Summary ......................................................................................................................... 41 3.2 Introduction .................................................................................................................... 42 3.2.1 Pretreatment methods and next-generation biofuels ................................................. 43 3.2.2 Isobutanol production in E. coli ................................................................................ 45 3.2.3 Consortia vs. “Superbug” for fuel production and sugar co-utilization .................... 48 3.2.4 C5 specialist construction and xylodextrin utilization .............................................. 51 3.3 Methods ........................................................................................................................... 53 3.3.1 Strain construction .................................................................................................... 53 3.3.2 pAK6 (pSA55-xynTB) plasmid construction ............................................................ 54 3.3.3 Microplate and flask cultivations using M9IPGX+YE media .................................. 54 3.3.4 Determination of isobutanol and sugar concentrations via HPLC ............................ 55 3.3.5 Corn stover hydrolysate preparation ......................................................................... 55 3.3.6 Growth experiments with CS hydrolysate ................................................................ 56 3.4 Results ............................................................................................................................. 56 3.4.1 Two bacterial strains engineered to co-convert hexose and pentose sugars to isobutanol ............................................................................................................................... 56 3.4.2 Co-culture performance on mixed-sugar defined media ........................................... 58 3.4.3 Sugar variation in defined media .............................................................................. 64 3.4.4 Isobutanol production on AFEX-pretreated corn stover hydrolysate ....................... 67 3.4.5 Xylodextrin utilization by NC5 pLOI3708 ............................................................... 71 3.5 Discussion and conclusion ............................................................................................. 73 Chapter 4 Use of microbial consortia in high-throughput screening for over- production of L-valine and isobutanol .......................................................................... 78 4.1 Summary ......................................................................................................................... 78 4.2 Introduction .................................................................................................................... 79 4.2.1 High-throughput microbial screening techniques ..................................................... 79 4.2.2 Detection of L-valine and isobutanol production using a secretor-sensor pair ......... 83 iii 4.3 Methods ........................................................................................................................... 87 4.3.1 Microplate growth studies for auxotroph selection and leucine titration .................. 87 4.3.2 Secretor and sensor strain construction ..................................................................... 87 4.3.3 Microplate growth cultivation with the secretor/sensor pair and ratio determination via plate counting ................................................................................................................... 88 4.4 Results and Discussion ................................................................................................... 89 4.4.1 Selection of a secretor base strain and microplate studies ........................................ 89 4.4.2 Identification of target mutations for L-valine over-production ............................... 93 4.4.3 Design of secretor and sensor strains ........................................................................ 96 4.4.4 Preliminary growth studies with the proof-of-concept strains .................................. 97 4.5 Future Work ................................................................................................................... 99 Chapter 5 Concluding Remarks and Future Directions ........................................ 101 5.1 Concluding Remarks ................................................................................................... 101 5.1.1 Design and construction of a programmable, synthetic E. coli consortium via tunable symbiosis ................................................................................................................. 101 5.1.2 Engineering a two-member E. coli consortium for conversion of five- and six- carbon sugars to isobutanol .................................................................................................. 102 5.1.3 Use of microbial consortia in high-throughput strain development for over- production of L-valine and isobutanol ................................................................................. 103 5.2 Future directions .......................................................................................................... 103 5.2.1 Remove metabolic burden of the tunable circuit and application of the circuit in isobutanol production .......................................................................................................... 103 5.2.2 Optimization of the isobutanol