Molecular
Genetics and
Biotechnology of
Microorganisms
,H0: Overview of the course
Learning outcomes – 6 credits
• This course aims to provide insight into the latest developments in microbial genetics and
biotechnology, as well as in the recent progress in fundamental and applied research on
bacteria and yeast.
• In addition, it aims to make the students familiar with the many industrial applications
(medical, industrial, ecological) of microorganisms and their derived products.
Course content
• First part: discuss design and use of engineered bacteria as live therapeutic agents,
biosensors and reporters, and as production platforms for industrially important natural
products and valuable enzymes.
• Second part: bacterial pathogens and communities and their potential for biotechnological
applications and exploring the intricate internal organization of bacterial cells
• Last part: pioneering role of yeast Saccharomyces cerevisiae in the elucidation of various
eukaryotic cellular processes
The exam
Type: partial assessment with exam during the examination period
→ written exam (2 questions for each professor, covering different lectures)
→ project during the year
Final grade:
• Written exam: 50%
• Project: 50%
,H1: MICROBIAL BIOSENSORS AND REPORTERS ........................................................................................................................ 1
1.1 GENERAL INTRODUCTION .............................................................................................................................................................. 1
1.1.1 What are biosensors?..................................................................................................................................................... 1
1.1.2 Why are microbial biosensors necessary? ...................................................................................................................... 1
1.1.3 Advantages of whole-cell based microbial biosensors ................................................................................................... 1
1.1.4 Disadvantages of whole-cell based microbial biosensors .............................................................................................. 2
1.2 THE DESIGN OF WHOLE-CELL MICROBIAL BIOSENSORS .......................................................................................................................... 2
1.2.1 Reporter module............................................................................................................................................................. 3
1.2.2 Receptor module ............................................................................................................................................................ 8
1.2.3 Signal processing module ............................................................................................................................................... 8
1.2.4 Ways to stabilize microbial biosensors ......................................................................................................................... 12
1.2.5 Overview: designing a microbial biosensor involves several key steps: ........................................................................ 13
1.3 APPLICATIONS OF MICROBIAL BIOSENSORS....................................................................................................................................... 13
1.3.1 Diagnostic and therapeutic applications ...................................................................................................................... 14
H2: ENGINEERING MICROBES FOR THERAPEUTIC APPLICATIONS............................................................................................ 16
2.1 INTRODUCTION ......................................................................................................................................................................... 16
2.1.1 Microbial therapeutics? ............................................................................................................................................... 16
2.1.2 Advantages of microbiota-based cell therapy compared to traditional drug .............................................................. 16
2.2 DESIGN PRINCIPLES .................................................................................................................................................................... 17
2.2.1 Chassis development .................................................................................................................................................... 17
2.2.2 Environmental sensing module .................................................................................................................................... 18
2.2.3 Designing the intracellular signal integration and transduction .................................................................................. 20
2.2.4 Production and delivery of therapeutics ...................................................................................................................... 21
2.2.5 Kill switches .................................................................................................................................................................. 23
2.2.6 Evaluating the design principles ................................................................................................................................... 26
2.3 RECENT (PRE)CLINICAL EXAMPLES AND NEW TRENDS ......................................................................................................................... 27
2.3.1 Multiple MOs have served as chassis for engineered microbial therapies ................................................................... 27
2.3.2 Factors governing the choice of bacterial species for clinical applications .................................................................. 29
2.3.3 Programming a non-model human commensal bacterium.......................................................................................... 29
2.3.4 Challenges .................................................................................................................................................................... 30
H3: MICROBIAL SPECIALIZED METABOLITES ........................................................................................................................... 31
3.1 INTRODUCTION TO MICROBIAL SPECIALIZED METABOLITES ................................................................................................................... 31
3.1.1 What are microbial specialized metabolites? ............................................................................................................... 31
3.1.2 Ecological roles of specialized metabolites ................................................................................................................... 31
3.1.3 Four major classes of specialized metabolites .............................................................................................................. 32
3.1.4 Why interesting for humans? ....................................................................................................................................... 33
3.1.5 Natural product biosynthesis engineering ................................................................................................................... 33
3.2 ENGINEERING NATURE’S DRUG FACTORIES: POLYKETIDE SYNTHASES (PKSS) .......................................................................................... 34
3.2.1 Erythromycin polyketide synthase – biosynthesis ......................................................................................................... 34
3.2.2 Strategies for engineering the biosynthesis of polyketides .......................................................................................... 38
3.3 ENGINEERING NATURE’S DRUG FACTORIES: NON-RIBOSOMAL PEPTIDE SYNTHASES (NRPSS) ..................................................................... 40
3.3.1 Biosynthesis .................................................................................................................................................................. 40
3.3.2 Strategies for engineering the biosynthesis of non-ribosomal peptide synthases ........................................................ 41
3.4 ENGINEERING NATURE’S DRUG FACTORIES: HYBRID ASSEMBLY LINES .................................................................................................... 42
3.4.1 Hybrid polyketide-non-ribosomal peptides biosynthesis .............................................................................................. 42
3.4.2 Strategies for engineering the biosynthesis of hybrid pathways .................................................................................. 42
3.5 GENOME MINING FOR NOVEL MEDICINES ........................................................................................................................................ 43
3.5.1 “Grind and find” methodology ..................................................................................................................................... 43
3.5.2 Genome mining tools ................................................................................................................................................... 43
H4: BACTERIAL CELL BIOLOGY ................................................................................................................................................ 44
4.1 RESEARCH IN MOLECULAR GENETICS AND BIOTECHNOLOGY OF MOS ..................................................................................................... 44
4.1.1 Fundamental vs applied research ................................................................................................................................. 44
4.1.2 False dichotomy: a spectrum, not separate entities ..................................................................................................... 44
, 4.1.3 The power of fundamental research: basic discovery .................................................................................................. 44
4.2 INTRODUCTION TO BACTERIAL CELL BIOLOGY .................................................................................................................................... 45
4.2.1 What and why: (bacterial) cell biology ......................................................................................................................... 45
4.2.2 Eukaryotic spatial organization and the historic view of bacteria ................................................................................ 45
4.2.3 The three workhorses of bacterial cell biology ............................................................................................................. 46
4.2.4 Spatial organization in bacteria ................................................................................................................................... 47
4.2.5 Bacterial diversity and morphological variability ......................................................................................................... 49
4.3 METHODS FOR STUDYING BACTERIAL CELL BIOLOGY ........................................................................................................................... 50
4.3.1 Different methodologies............................................................................................................................................... 50
4.3.2 What can we extract from it and what information does it give us ............................................................................. 51
4.3.3 Other types of microscopy ............................................................................................................................................ 51
4.4 COMMON STEPS IN THE BACTERIAL CELL CYCLE ................................................................................................................................. 52
4.4.1 General introduction to the bacterial cell cycle ............................................................................................................ 52
4.4.2 Cell wall synthesis ......................................................................................................................................................... 52
4.4.3 Chromosome replication and segregation ................................................................................................................... 54
4.4.4 Cell division................................................................................................................................................................... 61
4.5 RESEARCH STORY: NUCLEOID SIZE SCALING AND INTRACELLULAR ORGANIZATION OF TRANSLATION IN BACTERIA ............................................... 63
4.5.1 Class notes.................................................................................................................................................................... 63
4.5.2 The research paper....................................................................................................................................................... 65
H5: SUGAR AND AMINO ACID SENSING.................................................................................................................................. 66
5.1 GLUCOSE ACTIVATION OF THE PKA PATHWAY IN S. CEREVISIAE: REQUIREMENT OF A DUAL INPUT SYSTEM ...................................................... 66
5.1.1 Introduction .................................................................................................................................................................. 66
5.1.2 How does yeast sense glucose? .................................................................................................................................... 67
5.1.3 Glucose activation of Ras-cAMP-PKA pathway ............................................................................................................ 69
5.2 METHIONINE ACTIVATION OF THE PKA PATHWAY IN C. ALBICANS: REQUIREMENT OF A DUAL INPUT SYSTEM ................................................... 73
5.2.1 Gpr1 is involved in morphogenesis ............................................................................................................................... 73
5.2.2 The cAMP-PKA and MAPK pathways in C. albicans ...................................................................................................... 73
5.2.3 What does activate Gpr1? ............................................................................................................................................ 75
H6: YEAST: FACTORY FOR TERPENES & TERPENOIDS ............................................................................................................... 77
6.1 ESSENTIAL OILS AND NEW ANTIFUNGAL COMPOUNDS ........................................................................................................................ 77
6.1.1 What are essential oils (EOs) and their components (EOCs)? ....................................................................................... 77
6.1.2 Screening and identifying active compounds ............................................................................................................... 78
6.2 YEAST AS A PLATFORM FOR PRODUCTION OF PLANT EOS AND EOCS ..................................................................................................... 80
6.2.1 Design-Build-Test-Learn (DBTL) approach .................................................................................................................... 80
6.2.2 Key steps in terpene biosynthesis ................................................................................................................................. 82
6.2.3 Further improvement of cytoplasmic production of acetyl-CoA in S. cerevisiae........................................................... 84
6.2.4 Construction of an orthogonal pathway for monoterpene production ........................................................................ 84
6.2.5 Expanding the chemical variety of terpenes/terpenoids .............................................................................................. 84
6.3 OVERVIEW OF DIFFERENT APPROACHES........................................................................................................................................... 85
H7: BACTERIAL PATHOGENS & COMMUNITIES ....................................................................................................................... 86
7.1 GENERAL INTRODUCTION TO BACTERIAL PATHOGENS.......................................................................................................................... 86
7.1.1 Yersinia pestis ............................................................................................................................................................... 86
7.1.2 Borrelia burgdorferi ...................................................................................................................................................... 87
7.1.3 Staphylococcus aureus ................................................................................................................................................. 91
7.2 HOW TO FIGHT PATHOGENS: ANTIBIOTICS? ...................................................................................................................................... 92
7.2.1 Antibiotic resistance and ways bacteria bypass or inactivate antibiotics ..................................................................... 92
7.2.2 Antibiotic persistence is an additional problem ........................................................................................................... 94
7.3 BACTERIAL COMMUNITIES ........................................................................................................................................................... 96
7.3.1 Bacteria rarely live in isolation ..................................................................................................................................... 96
7.3.2 Drivers behind community assembly ............................................................................................................................ 96
7.3.3 Uncovering the spatial organization of microbial communities ................................................................................... 97
7.3.4 Bacterial biofilms........................................................................................................................................................ 100
7.3.5 Research into bacterial communities ......................................................................................................................... 100
Genetics and
Biotechnology of
Microorganisms
,H0: Overview of the course
Learning outcomes – 6 credits
• This course aims to provide insight into the latest developments in microbial genetics and
biotechnology, as well as in the recent progress in fundamental and applied research on
bacteria and yeast.
• In addition, it aims to make the students familiar with the many industrial applications
(medical, industrial, ecological) of microorganisms and their derived products.
Course content
• First part: discuss design and use of engineered bacteria as live therapeutic agents,
biosensors and reporters, and as production platforms for industrially important natural
products and valuable enzymes.
• Second part: bacterial pathogens and communities and their potential for biotechnological
applications and exploring the intricate internal organization of bacterial cells
• Last part: pioneering role of yeast Saccharomyces cerevisiae in the elucidation of various
eukaryotic cellular processes
The exam
Type: partial assessment with exam during the examination period
→ written exam (2 questions for each professor, covering different lectures)
→ project during the year
Final grade:
• Written exam: 50%
• Project: 50%
,H1: MICROBIAL BIOSENSORS AND REPORTERS ........................................................................................................................ 1
1.1 GENERAL INTRODUCTION .............................................................................................................................................................. 1
1.1.1 What are biosensors?..................................................................................................................................................... 1
1.1.2 Why are microbial biosensors necessary? ...................................................................................................................... 1
1.1.3 Advantages of whole-cell based microbial biosensors ................................................................................................... 1
1.1.4 Disadvantages of whole-cell based microbial biosensors .............................................................................................. 2
1.2 THE DESIGN OF WHOLE-CELL MICROBIAL BIOSENSORS .......................................................................................................................... 2
1.2.1 Reporter module............................................................................................................................................................. 3
1.2.2 Receptor module ............................................................................................................................................................ 8
1.2.3 Signal processing module ............................................................................................................................................... 8
1.2.4 Ways to stabilize microbial biosensors ......................................................................................................................... 12
1.2.5 Overview: designing a microbial biosensor involves several key steps: ........................................................................ 13
1.3 APPLICATIONS OF MICROBIAL BIOSENSORS....................................................................................................................................... 13
1.3.1 Diagnostic and therapeutic applications ...................................................................................................................... 14
H2: ENGINEERING MICROBES FOR THERAPEUTIC APPLICATIONS............................................................................................ 16
2.1 INTRODUCTION ......................................................................................................................................................................... 16
2.1.1 Microbial therapeutics? ............................................................................................................................................... 16
2.1.2 Advantages of microbiota-based cell therapy compared to traditional drug .............................................................. 16
2.2 DESIGN PRINCIPLES .................................................................................................................................................................... 17
2.2.1 Chassis development .................................................................................................................................................... 17
2.2.2 Environmental sensing module .................................................................................................................................... 18
2.2.3 Designing the intracellular signal integration and transduction .................................................................................. 20
2.2.4 Production and delivery of therapeutics ...................................................................................................................... 21
2.2.5 Kill switches .................................................................................................................................................................. 23
2.2.6 Evaluating the design principles ................................................................................................................................... 26
2.3 RECENT (PRE)CLINICAL EXAMPLES AND NEW TRENDS ......................................................................................................................... 27
2.3.1 Multiple MOs have served as chassis for engineered microbial therapies ................................................................... 27
2.3.2 Factors governing the choice of bacterial species for clinical applications .................................................................. 29
2.3.3 Programming a non-model human commensal bacterium.......................................................................................... 29
2.3.4 Challenges .................................................................................................................................................................... 30
H3: MICROBIAL SPECIALIZED METABOLITES ........................................................................................................................... 31
3.1 INTRODUCTION TO MICROBIAL SPECIALIZED METABOLITES ................................................................................................................... 31
3.1.1 What are microbial specialized metabolites? ............................................................................................................... 31
3.1.2 Ecological roles of specialized metabolites ................................................................................................................... 31
3.1.3 Four major classes of specialized metabolites .............................................................................................................. 32
3.1.4 Why interesting for humans? ....................................................................................................................................... 33
3.1.5 Natural product biosynthesis engineering ................................................................................................................... 33
3.2 ENGINEERING NATURE’S DRUG FACTORIES: POLYKETIDE SYNTHASES (PKSS) .......................................................................................... 34
3.2.1 Erythromycin polyketide synthase – biosynthesis ......................................................................................................... 34
3.2.2 Strategies for engineering the biosynthesis of polyketides .......................................................................................... 38
3.3 ENGINEERING NATURE’S DRUG FACTORIES: NON-RIBOSOMAL PEPTIDE SYNTHASES (NRPSS) ..................................................................... 40
3.3.1 Biosynthesis .................................................................................................................................................................. 40
3.3.2 Strategies for engineering the biosynthesis of non-ribosomal peptide synthases ........................................................ 41
3.4 ENGINEERING NATURE’S DRUG FACTORIES: HYBRID ASSEMBLY LINES .................................................................................................... 42
3.4.1 Hybrid polyketide-non-ribosomal peptides biosynthesis .............................................................................................. 42
3.4.2 Strategies for engineering the biosynthesis of hybrid pathways .................................................................................. 42
3.5 GENOME MINING FOR NOVEL MEDICINES ........................................................................................................................................ 43
3.5.1 “Grind and find” methodology ..................................................................................................................................... 43
3.5.2 Genome mining tools ................................................................................................................................................... 43
H4: BACTERIAL CELL BIOLOGY ................................................................................................................................................ 44
4.1 RESEARCH IN MOLECULAR GENETICS AND BIOTECHNOLOGY OF MOS ..................................................................................................... 44
4.1.1 Fundamental vs applied research ................................................................................................................................. 44
4.1.2 False dichotomy: a spectrum, not separate entities ..................................................................................................... 44
, 4.1.3 The power of fundamental research: basic discovery .................................................................................................. 44
4.2 INTRODUCTION TO BACTERIAL CELL BIOLOGY .................................................................................................................................... 45
4.2.1 What and why: (bacterial) cell biology ......................................................................................................................... 45
4.2.2 Eukaryotic spatial organization and the historic view of bacteria ................................................................................ 45
4.2.3 The three workhorses of bacterial cell biology ............................................................................................................. 46
4.2.4 Spatial organization in bacteria ................................................................................................................................... 47
4.2.5 Bacterial diversity and morphological variability ......................................................................................................... 49
4.3 METHODS FOR STUDYING BACTERIAL CELL BIOLOGY ........................................................................................................................... 50
4.3.1 Different methodologies............................................................................................................................................... 50
4.3.2 What can we extract from it and what information does it give us ............................................................................. 51
4.3.3 Other types of microscopy ............................................................................................................................................ 51
4.4 COMMON STEPS IN THE BACTERIAL CELL CYCLE ................................................................................................................................. 52
4.4.1 General introduction to the bacterial cell cycle ............................................................................................................ 52
4.4.2 Cell wall synthesis ......................................................................................................................................................... 52
4.4.3 Chromosome replication and segregation ................................................................................................................... 54
4.4.4 Cell division................................................................................................................................................................... 61
4.5 RESEARCH STORY: NUCLEOID SIZE SCALING AND INTRACELLULAR ORGANIZATION OF TRANSLATION IN BACTERIA ............................................... 63
4.5.1 Class notes.................................................................................................................................................................... 63
4.5.2 The research paper....................................................................................................................................................... 65
H5: SUGAR AND AMINO ACID SENSING.................................................................................................................................. 66
5.1 GLUCOSE ACTIVATION OF THE PKA PATHWAY IN S. CEREVISIAE: REQUIREMENT OF A DUAL INPUT SYSTEM ...................................................... 66
5.1.1 Introduction .................................................................................................................................................................. 66
5.1.2 How does yeast sense glucose? .................................................................................................................................... 67
5.1.3 Glucose activation of Ras-cAMP-PKA pathway ............................................................................................................ 69
5.2 METHIONINE ACTIVATION OF THE PKA PATHWAY IN C. ALBICANS: REQUIREMENT OF A DUAL INPUT SYSTEM ................................................... 73
5.2.1 Gpr1 is involved in morphogenesis ............................................................................................................................... 73
5.2.2 The cAMP-PKA and MAPK pathways in C. albicans ...................................................................................................... 73
5.2.3 What does activate Gpr1? ............................................................................................................................................ 75
H6: YEAST: FACTORY FOR TERPENES & TERPENOIDS ............................................................................................................... 77
6.1 ESSENTIAL OILS AND NEW ANTIFUNGAL COMPOUNDS ........................................................................................................................ 77
6.1.1 What are essential oils (EOs) and their components (EOCs)? ....................................................................................... 77
6.1.2 Screening and identifying active compounds ............................................................................................................... 78
6.2 YEAST AS A PLATFORM FOR PRODUCTION OF PLANT EOS AND EOCS ..................................................................................................... 80
6.2.1 Design-Build-Test-Learn (DBTL) approach .................................................................................................................... 80
6.2.2 Key steps in terpene biosynthesis ................................................................................................................................. 82
6.2.3 Further improvement of cytoplasmic production of acetyl-CoA in S. cerevisiae........................................................... 84
6.2.4 Construction of an orthogonal pathway for monoterpene production ........................................................................ 84
6.2.5 Expanding the chemical variety of terpenes/terpenoids .............................................................................................. 84
6.3 OVERVIEW OF DIFFERENT APPROACHES........................................................................................................................................... 85
H7: BACTERIAL PATHOGENS & COMMUNITIES ....................................................................................................................... 86
7.1 GENERAL INTRODUCTION TO BACTERIAL PATHOGENS.......................................................................................................................... 86
7.1.1 Yersinia pestis ............................................................................................................................................................... 86
7.1.2 Borrelia burgdorferi ...................................................................................................................................................... 87
7.1.3 Staphylococcus aureus ................................................................................................................................................. 91
7.2 HOW TO FIGHT PATHOGENS: ANTIBIOTICS? ...................................................................................................................................... 92
7.2.1 Antibiotic resistance and ways bacteria bypass or inactivate antibiotics ..................................................................... 92
7.2.2 Antibiotic persistence is an additional problem ........................................................................................................... 94
7.3 BACTERIAL COMMUNITIES ........................................................................................................................................................... 96
7.3.1 Bacteria rarely live in isolation ..................................................................................................................................... 96
7.3.2 Drivers behind community assembly ............................................................................................................................ 96
7.3.3 Uncovering the spatial organization of microbial communities ................................................................................... 97
7.3.4 Bacterial biofilms........................................................................................................................................................ 100
7.3.5 Research into bacterial communities ......................................................................................................................... 100
