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Fit-For-Purpose Yeast and Bacteria Via Directed Evolution

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Fit-For-Purpose Yeast and Bacteria Via Directed Evolution Fit-for-Purpose Yeast and Bacteria via Directed Evolution FINAL REPORT to AUSTRALIAN GRAPE AND WINE AUTHORITY Project Number: UA1302 Principal Investigator: Professor Vladimir Jiranek Research Organisation: University of Adelaide th Date: 30 December 2017 1 UA1302 Project Title: ‘Fit-for-purpose’ yeast and bacteria via directed evolution Authors: Michelle Walker, Krista Sumby, Jennie Gardner, Joanna Sundstrom, Tommaso Watson, Paul Grbin, Vladimir Jiranek Date: 30 December 2017 Publisher: University of Adelaide Copyright and Disclaimer: © The University of Adelaide 2017 This publication is copyright, no part may be reproduced by any process except in accordance with the provisions of the Copyright Act 1968, and with the written permission of The University of Adelaide. This publication may be of assistance, but The University of Adelaide and its employees do not guarantee that the publication is without flaw of any kind or is wholly appropriate for your particular purposes and therefore disclaims all liability for any error, loss or other consequence which may arise from you relying on any information in this publication. 2 Table of contents Abbreviations 7 Abstract 8 Executive Summary 9 Background 13 Project Aims and Performance Targets 15 1.0 Generation of microbes with improved fermentation characteristics by Directed Evolution or classical methods 20 Background 20 Methods 22 M 1.1 Microbial Strains 22 M 1.2 Yeast strain identification using Delta PCR 24 M 1.3 Generation of F2 hybrid progeny from a genetic cross between EC1118 and FM16 C7H 24 M 1.4 En masse sporulation and mating of yeast 24 M 1.5 Micro-fermentation screens for tolerance to single stress conditions: Yeast 25 M 1.6 Micro-fermentation evaluation of F2 hybrids originating from H3-13 (EC118 x FM16 C7H) 26 M 1.7 10 mL screens for tolerance to single and multiple stress conditions: Bacteria 26 M 1.8 Micro-fermentation screen for malic acid utilisation 27 M 1.9 Evaluation of fermentation performance in 100 mL scale using automated robotic platform (T-bot) 28 M 1.10 Sequential batch DE of microbes 28 M 1.11 Continuous DE of microbes in a bioreactor 29 M 1.12 Mutagenesis of Lb. plantarum 30 M 1.13 Screening LAB for improved MLF performance. 32 M 1.14 Evaluation of un-inoculated ferment sample for MLF performance 33 Summary of Outcomes 33 Results and Discussion 34 Output 1A 1.0 Preliminary screening trials for selection of yeast strains and key stressors to achieve sought-after outcomes 34 Output 1A 2.0 Preliminary screening trials for selection of bacterial strains and key stressors to achieve sought-after outcomes 38 Output 1D 1.0 Stage 1 DE experiments and intermittent evaluation of candidate strains with the aim of generating improved yeast strains 44 Output 1D 1.1 Sequential batch DE (in flasks) targeting DE strategy (i) tolerance to multiple stresses 46 Output 1D 1.2 Sequential batch DE (in bioreactor) targeting DE strategy (ii) improved fructose utilisation and ethanol tolerance 46 Output 1D 1.3 Directed Evolution of the mixed C7H BIO population by continuous culture 48 Output 1D 1.4 Increased genetic heterogeneity through chemical mutagenesis 48 Output 1D 1.5 Increased genetic heterogeneity through rare spore mating 48 Output 1D 1.6 Increased genetic heterogeneity through en masse sporulation and rare mating (RM) 49 Output 1D 1.7 Evaluation of existing collection of monosporic and hybrid strains for fermentation performance 49 Output 1D 1.8 Stage 2 DE strategy: batch flask fermentations targeting (i) tolerance to multiple stresses and ethanol tolerance 50 Output 1D 2.0 Lactic acid bacteria, Stage 1 DE and evaluation of candidate strains with the aim of generating candidate improved strains 54 Output 1D 2.1 Sequential batch DE of Lb. plantarum in Schott bottles, targeting tolerance to 3 multiple stresses. 54 Output 1D 2.2 Batch fermentations of of Lb. plantarum UV and EMS mutants. 55 Output 1D 2.3 Directed Evolution of LAB by continuous culture 55 Output 1D 2.4 Directed Evolution of O. oeni A90 by continuous culture 58 Output 2B 2.5 Directed Evolution of additional LAB 59 Output 2B 2.6 Screening for improved MLF performance during DE. 59 Output 1D 2.7 Evaluation of un-inoculated ferment samples for MLF performance 62 2.0 Fermentation performance of promising candidate microbes with improved fermentation attributes in laboratory scale fermentations 63 Background 63 Methods 64 M 2.1 Evaluation of yeast fermentation performance in microscale (0.2 mL) 64 M 2.2 Evaluation of yeast fermentation performance in 100 mL cultures 64 M 2.3 Evaluation of LAB MLF performance in microscale (0.2 mL) 64 M 2.4 Micro-plate screen of LAB bioreactor clones 64 M 2.5 Micro-plate screen of Lb. plantarum EMS mutants 64 M 2.6 Evaluation of MLF performance in 15 mL and 50 mL cultures 65 Summary of Outcomes 67 Results and Discussion 68 2.0 Periodic evaluation of evolved yeast populations from sequential batch DE 68 2.1 Periodic evaluation of evolved LAB populations from sequential batch DE and continuous culture 73 2.2 Micro-plate screen for MLF performance of Lb. plantarum EMS mutants 74 2.3 Evaluation of MLF by Lb. plantarum isolates grown in 15 mL and 50 mL cultures 75 2.4 Periodic evaluation of O. oeni A90 DE 79 2.6 Evaluation of O. oeni isolates from an un-inoculated fermentation (15 mL cultures) 81 3.0 Industrial scale winemaking trials of evolved microbes 84 Background 84 Methods 85 M3.1 Yeast strains 85 M 3.2 Winery scale yeast fermentations - Mataro 86 M 3.3 Winery scale yeast fermentations - Chardonnay 86 M 3.4 Winery scale yeast fermentations - Cabernet Sauvignon 86 M 3.5 Winery scale malolactic fermentation 87 M 3.6 Bottle (5L) scale MLF – Shiraz and Shiraz-Grenache blend 87 M 3.7 Winery scale bacteria MLF - Mataro 88 M 3.8 Winery scale bacteria MLF - Shiraz 89 M 3.9 Sensory analysis 89 M 3.9.1 Sensory analysis DE yeast 89 M 3.9.2 Sensory analysis LAB 5 L MLF 89 M 3.10 HPLC and GCMS analysis of key metabolites 90 Summary of Outcomes 90 Results and Discussion 90 Outputs 2E and 3E Lead strains evaluated (kinetic and sensory) for practical suitability 90 Outputs 2E and 3E LAB lead strains evaluated (kinetic and sensory) for practical suitability 97 Bacteria: 5 L scale MLF 97 Bacteria: Winery scale MLF 97 4.0 Extension of the Fermentome to include genes of protrophic lab and wine yeast. The effect of overexpression of key genes on fermentation progression and analysis of yeast deletion mutant effects on wine colour. 100 Background 100 4 Methods 101 M 4.1 Yeast strains and culture 101 M 4.2 Screen of prototrophic laboratory yeast deletion library (LYDL) and wine yeast deletion library (WYDL) in CDGJM under high and low nitrogen conditions 102 M 4.3 Evaluation of identified yeast deletants in laboratory scale (100 mL) fermentations in CDGJM under high and low nitrogen conditions 102 M 4.4 Confirmation of clonal identity of gene deletions in laboratory yeast strains 103 Summary of Outcomes 103 Results and Discussion 104 Output 1B. 4.1: Preliminary micro-fermentation screen of prototrophic yeast deletion libraries 104 Output 2A. 4.2: Comparison of the Fermentome between data sets 105 Output 1F. 4.3. Laboratory scale (100 mL) evaluation of selected candidate wine yeast deletants 105 Output 1F. 4.4. Effect of overexpressed genes on fermentation duration 107 5.0 Identification of SNPs unique to evolved microbes via genome sequencing and comparison to the Fermentome 109 Background 109 Methods 110 M 5.1 Genome sequencing 110 M 5.1.1 Whole genome sequencing - 454 technology 110 M 5.2 Yeast genome assembly 111 M 5.3 Bacterial genome assembly 111 M 5.3.1 Bacterial genome assembly with Geneious 111 M 5.3.2 Bacterial genome assembly with Galaxy 112 M 5.3.3 Re-assembly of O. oeni A90 DE strains and assembly of Lb. plantarum DE strains with customised pipeline 112 Summary of Outcomes 113 Results and Discussion 114 Outputs 3A and 3E. 5.1. Genome sequencing and assembly of evolved LAB and yeast isolates 114 Output 3C. 5.6 Comparison of Fermentome with SNPs of key evolved strains 124 6.0 Construction of recombinant strains to confirm importance of highlighted gene deletion mutants. 125 Background 125 Methods 125 Summary outcomes 125 Results and Discussion 126 7.0 Upgrade of the automated fermentation platform (T-bot) to allow for 384 fermentations. 129 8.0 Annual review of UA1302 and AWRI project funded by Wine Australia 130 9.0 Communication with yeast and bacteria manufacturers regarding commercialisation of evolved strains 132 10.0 Dissemination of Project findings to industry and academia 133 Conference, workshop and seminar presentations 133 Refereed journal articles 136 PhD Thesis 137 Refereed journal articles (under review) 138 Referred Journal Articles (In preparation) 138 11.0 Outcomes and Conclusion 139 5 12.0 Recommendations 141 Appendix 1: Communication 142 Appendix 2: Intellectual Property 143 Appendix 3: References 144 Appendix 4: Staff 149 Appendix 5: Supplementary Data 150 6 Abbreviations CDGJM Chemically Defined Grape Juice Medium DAP Diammonium Phosphate (Ammonium Dibasic Phosphate) DE Directed Evolution FAN Free Amino acid Nitrogen FCDGJM Fermented Chemically Defined Grape Juice Medium FEG Fermentation Essential Genes FSO2 Free sulfur dioxide TSO2 Total sulfur dioxide LYDL Lab Yeast Deletion Library LAB Lactic acid bacteria MFCA Medium Chain Fatty Acids MLF Malolactic fermentation MRS De Man, Rogosa and Sharpe medium MRSAJ MRS with Apple Juice NGS Next Generation Sequencing ORF Open Reading Frame PMS Potassium Metabisulfite RCDGJM Red CDGJM RFCDGJM Red Fermented CDGJM SD Standard deviation SNP Single Nucleotide Polymorphism WYDL Wine Yeast Deletion Library WGS Whole Genome Sequencing 7 Abstract This project sought to generate ‘fit-for-purpose’ yeast and lactic acid bacterial (LAB) strains better suited to problematic wine and juice conditions via Directed Evolution (DE).
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