Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate Shiqin Yu Master of Science A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Chemical Engineering I Abstract Bio-based chemicals have drawn research interests due to the need for sustainable development. Aromatics and the derived compounds are an important class of chemicals that are mostly derived from fossil resources. Microbial bio-production can produce many compounds from renewable feedstocks to reduce the current heavy dependency on fossil resources. The aromatic compound para-hydroxybenzoate (PHBA) is used to make parabens and high-value polymers (liquid crystal polymer). Biologically, this chemical can be derived from the shikimate pathway, the central pathway for the biosynthesis of aromatic amino acids in bacteria, plants and fungi and some protozoa. In recent years, the gram-negative soil bacterium Pseudomonas putida is becoming an interesting chassis for industrial biotechnology. The production of PHBA in recombinant P. putida KT2440 was engineered by overexpressing the chorismate lyase Ubic from Escherichia coli and a feedback resistant 3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroGD146N). Additionally, the pathways competing for the substrate chorismate (trpE and pheA) and the PHBA degradation pathway (pobA) were eliminated. Finally, deletion of the glucose metabolism repressor hexR led to an increase in erythrose-4-phosphate and NADPH supply. This resulted in a maximum titre of 1.73 g L-1 and a carbon yield of 18.1 % (C-mol C-mol-1) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase route. Large-scale aerobic fermentations are more expensive due to the limiting gas transfer and lead to substrate loss in the form of CO2, whereas anaerobic processes are easier to scale up and achieve high carbon yield. In the case of aromatics, anaerobic production could be beneficial. Microbial electrosynthesis is an emerging technology for biosynthesis of chemicals and fuels by microorganisms in an anaerobic bio-electrochemical system (BES). These systems can provide electrons to electroactive microbes for reductive production processes in the cathode or drain excessive electrons in oxidative production processes in the anode. P. putida was tested for the first time for its ability to perform anoxic metabolism in BES with ferricyanide as the mediator and anode as the electron sink. A dual-phase fermentation was employed, where the cells grew aerobically in the first phase and then performed an anaerobic oxidation process in the second phase. In this way, P. putida KT2440 could electrochemically produce 2-ketogluconate, a sugar acid used for biosynthesis of antioxidant E315 for the food industry under anaerobic conditions from glucose. This would have been impossible without the use of BES. The parental strain II obtained a carbon yield of around 65% (C-mol C-mol-1) and a productivity of 0.56 ± 0.01 -1 -1 mmolproduct gCDW day . Two genetic engineering targets were identified as potential targets for improvement of the productivity, pointing towards the key enzymes in the electron transport chain coupled catalytic reactions: the glucose dehydrogenase (mGCD) and gluconate dehydrogenase (GADH, enzyme complex). Strains were constructed to evaluate their performances in BES. Surprisingly, the double overexpression dehydrogenases strain did not lead to best performance in BES. Real-time RT-qPCR revealed the intrinsic regulation system of mGCD and GADH, in which mGCD was regulated by glucose and gluconate while GADH only regulated by gluconate. By utilizing the self-regulation, the single overexpression -1 mutant (mGCD) achieved a 2-ketogluconate productivity of 1.59 ± 0.01 mmolproduct gCDW day-1, a 300 % increase compared to the wildtype. This work demonstrated that expression of dehydrogenases coupled with the electron transfer chain can significantly improve productivity, and controlled expression level is necessary for the well-being of BES biocatalyst as well as productivity improvement. Different media and carbon sources were investigated for the production of the biomass prior to the BES experiments. Cells grown in terrific broth (TB) or minimal medium with glycerol as sole carbon source showed outstanding performance, and glucose-grown cells gave the worst performance in terms of carbon yield, electron transfer rate or productivity. The TB-grown cells achieved a carbon yield of 91 % (C-mol C-mol-1) with a productivity of -1 -1 2.53 ± 0.09 mmolproduct gCDW day , while the glycerol-grown cells achieved highest carbon yield of 98 % (C-mol C-mol-1) with a 9 % lower productivity than that in TB-grown cells. The proteomics analysis suggested the GADH enzyme complex (PP_3382, PP_3383, and PP_3384) overexpression was limited by the subunit PP_3382 biosynthesis. The slight upregulation of PP_3382 in glycerol-grown cells may contribute to its better performance in this condition, suggesting that the aerobic growth condition can be optimized for a better performance in bio-electrochemical production. In summary, using a chorismate lyase route in P. putida is a suitable strategy to produce aromatics. The use of a bio-electrochemical system in combination with metabolic engineering can be used to enable and accelerate anaerobic production with P. putida. In a first attempt, the two approaches showed promising results, paving the way for future application. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Peer-reviewed papers: 1. Metabolic Engineering of Pseudomonas putida KT2440 for the Production of para- Hydroxy benzoate. Yu, S., Plan, M. R., Winter, G., Krömer, J. O., 2016. Front Bioeng Biotechnol. 4, 90. 2. Anodic respiration of Pseudomonas putida KT2440 in a stirred-tank bioreactor. Hintermayer, S., Yu, S., Krömer, J. O., Weuster-Botz, D., 2016. Biochem Eng J. 115, 1-13. 3. Anoxic metabolism and biochemical production in Pseudomonas putida F1 driven by a bioelectrochemical system. Lai, B., Yu, S., Bernhardt, P. V., Rabaey, K., Virdis, B., Krömer, J. O., 2016. Biotechnol Biofuels. 9, 39. 4. Quantitative analysis of aromatics for synthetic biology using liquid chromatography. Lai, B., Plan, M. R., Averesch, N. J. H., Yu, S., Kracke, F., Lekieffre, N., Bydder, S., Hodson, M. P., Winter, G., Krömer, J. O., 2016. Quantitative analysis of aromatics for synthetic biology using liquid chromatography. J. Biotechnol. 5. Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase. Biotechnol Bioeng. Yu, S., Bin, L., Plan, M. P., Hodson, M.P., Lestari E.A., Song H., Krömer. J.O. Submitted to Biotechnol. Bioeng (accepted) Other papers or manuscripts under reviewed or revision are listed in Appendicx I Conference abstracts: Production of Aromatics in Microbes. Yu, S., Averesch N.J.H., Williams,T.C., Winter G., Vickers,C.E., Nielsen, L.K., and Krömer,J.O. ME-11,Kobe, 2016 Electrofermentation As a Tool to Balance Redox in Engineering Pseudomonas Putida. Yu S., Bin L., Krömer,J.O. ME-11, Kobe, 2016 V Publications included in this thesis Metabolic Engineering of Pseudomonas putida KT2440 for the Production of para-Hydroxy benzoate. Yu, S., Plan, M. R., Winter, G., Krömer, J. O., 2016. Front Bioeng Biotechnol. 4, 90.3. – incorporated as Chapter 2. Contributor Statement of contribution Shiqin Yu (Candidate) Designed experiments (90%) Conducted the experiments (95%) Drafted the paper (100%) Analysed the data (80%) Gal Winter Reviewed and edited paper (40%) Jens Kroemer Designed experiments (10%) Analysed the data (20%) Reviewed and edited paper (60%) Manuel R. Plan Conducted the experiments (5%). Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase. Biotechnol Bioeng.