Special Focus Topic: Synthetic Biology
Title: The Next Phase of Biology - Synthetic Biology for Synthetic
Professor Jay Keasling, Joint Bioenergy Institute, Berkeley, CA
Analysis of Aflatoxins in Pet Food by UHPLC Using PDA and Fluorescence DetectionPerkinElmer, Inc.
Commercially prepared pet foods are easy and economical ways to fulfill the nutritional requirements for pets. Dry pet food is produced with grains and cereal by-products rejected for human consumption. The contamination of these by-products, with toxigenic fungal metabolites called mycotoxins, pose a serious health threat to pets.
Aflatoxins, some of the most carcinogenic mycotoxins known, are classified as B1, B2, G1, and G2. Several aflatoxin outbreaks in commercial pet foods have been reported in the past few years. Symptoms from aflatoxin exposure include lethargy, anorexia, jaundice, and intravascular coagulation, the severity often varying based upon a pet’s breed, species, age, dose, length of exposure, and nutritional status. Even if affecting only a small percentage of commercial pet foods, problems with pet food safety impact the entire pet food industry due to recalls and loss of consumer loyalty. Such experiences have reaffirmed the need for commercial pet food manufacturers to devote extensive resources documenting product quality.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
A varicocele is an abnormal enlargement of the pampiniform venous plexus in the scrotum. This plexus of veins drains the testicles. The testicular blood vessels originate in the abdomen and course down through the inguinal canal as part of the spermatic cord on their way to the testis. More info visit: http://www.elabscience.com
Analysis of Aflatoxins in Pet Food by UHPLC Using PDA and Fluorescence DetectionPerkinElmer, Inc.
Commercially prepared pet foods are easy and economical ways to fulfill the nutritional requirements for pets. Dry pet food is produced with grains and cereal by-products rejected for human consumption. The contamination of these by-products, with toxigenic fungal metabolites called mycotoxins, pose a serious health threat to pets.
Aflatoxins, some of the most carcinogenic mycotoxins known, are classified as B1, B2, G1, and G2. Several aflatoxin outbreaks in commercial pet foods have been reported in the past few years. Symptoms from aflatoxin exposure include lethargy, anorexia, jaundice, and intravascular coagulation, the severity often varying based upon a pet’s breed, species, age, dose, length of exposure, and nutritional status. Even if affecting only a small percentage of commercial pet foods, problems with pet food safety impact the entire pet food industry due to recalls and loss of consumer loyalty. Such experiences have reaffirmed the need for commercial pet food manufacturers to devote extensive resources documenting product quality.
Overcoming Key Challenges of Protein Mass Spectrometry Sample PreparationMourad FERHAT, PhD
Overcoming Key Challenges of Protein Mass Spectrometry Sample Preparation
Bottom-up proteomics is widely accepted as a primary method to characterize proteins. To ensure efficient protein analysis researchers must optimize key steps in the workflow to avoid potential pitfalls such as poor protein sample preparation and inconsistent LC-MS instrument performance. In this presentation, we will:
• Investigate the cause of incomplete trypsin digestion and solution to this problem.
• Discuss the advantage of alternative proteases for mass spec protein analysis.
• Review the impact of mass spec compatible surfactants on protein digestion in gel and protein extraction from animal tissues.
• Detail new reference mass spec protein and peptide materials designed to optimize protein sample preparation steps and monitor key instrument performance parameters.
The presentation should prove valuable to any researcher using bottom-up proteomics, and who is concerned with improving protein mass spec sample preparation and mass spec instrument performance.
A varicocele is an abnormal enlargement of the pampiniform venous plexus in the scrotum. This plexus of veins drains the testicles. The testicular blood vessels originate in the abdomen and course down through the inguinal canal as part of the spermatic cord on their way to the testis. More info visit: http://www.elabscience.com
Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production ...rutayisirer
Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production : S. cerevisae naturally uses MVA pathway to produce isoprenoids, which constitute one of the most used natural products in pharmaceuticals (anticancer: Paclitaxel-Taxol, antimalaria: Artemisin, nutraceuticals, food additives, fragrances, etc. And this microorganism can be engineered to overproduce these chemicals, and other metabolic pathways can be incorporated for this effect.
Isoprenoids are the most abundant and highly diverse group of natural and chemical products with a variety of applications in pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. The plant extraction, chemical synthesis and in vitro enzymatic production of these compounds have been the traditional options for large-scale production and these methods have shown drawbacks and they are impractical. In response to this problem and a poor production efficiency with an increase demand in isoprenoids, the focus has been set to the microbial production, which is an excellent alternative for overcoming these limitations. Microbes require little and naturally produce the building blocks of all isoprenoids: isopentenyl diphosphate (IPP) and its isomer dimethyl allyl diphosphate (DMAPP). IPP and DMAPP can be produced by two metabolic pathways, the mevalonate pathway (MVA or MEV) and the methyl-erythritol phosphate or deoxy xylulose phosphate pathway (MEP or DXP). S. cerevisae naturally uses MVA pathway to produce isoprenoids. The availability of its entire genome sequence allows the application of metabolic engineering and synthetic biology approaches for improving its enormous biosynthetic potential.
LifeTein® is the only peptide manufacturer to offer the PeptideSynTM platform of peptide synthesis services. PeptideSynTM technology is optimized to provide a continuous flow of synthesized peptides, reducing costs while maintaining quality. PeptideSynTM has been proven to enhance the efficiency of synthesis.
Modeling Lipase Production From Co-cultures of Lactic Acid Bacteria Using Neural Networks and Support Vector Machine with Genetic Algorithm Optimization
Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production ...rutayisirer
Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production : S. cerevisae naturally uses MVA pathway to produce isoprenoids, which constitute one of the most used natural products in pharmaceuticals (anticancer: Paclitaxel-Taxol, antimalaria: Artemisin, nutraceuticals, food additives, fragrances, etc. And this microorganism can be engineered to overproduce these chemicals, and other metabolic pathways can be incorporated for this effect.
Isoprenoids are the most abundant and highly diverse group of natural and chemical products with a variety of applications in pharmaceuticals, nutraceuticals, flavors, cosmetics, food additives and biofuels. The plant extraction, chemical synthesis and in vitro enzymatic production of these compounds have been the traditional options for large-scale production and these methods have shown drawbacks and they are impractical. In response to this problem and a poor production efficiency with an increase demand in isoprenoids, the focus has been set to the microbial production, which is an excellent alternative for overcoming these limitations. Microbes require little and naturally produce the building blocks of all isoprenoids: isopentenyl diphosphate (IPP) and its isomer dimethyl allyl diphosphate (DMAPP). IPP and DMAPP can be produced by two metabolic pathways, the mevalonate pathway (MVA or MEV) and the methyl-erythritol phosphate or deoxy xylulose phosphate pathway (MEP or DXP). S. cerevisae naturally uses MVA pathway to produce isoprenoids. The availability of its entire genome sequence allows the application of metabolic engineering and synthetic biology approaches for improving its enormous biosynthetic potential.
LifeTein® is the only peptide manufacturer to offer the PeptideSynTM platform of peptide synthesis services. PeptideSynTM technology is optimized to provide a continuous flow of synthesized peptides, reducing costs while maintaining quality. PeptideSynTM has been proven to enhance the efficiency of synthesis.
Modeling Lipase Production From Co-cultures of Lactic Acid Bacteria Using Neural Networks and Support Vector Machine with Genetic Algorithm Optimization
09 CeoMeeting- Session 4- Medicines for MalariaMLSCF
Session 4: Health & Wellness
Title: The Changing Face Of Healthcare:
Where Are The Opportunities?
Special Speaker: Dr Chris Hentschel, Medicines for Malaria Venture
Session 2: Industrial Biotechnology
Title: Replacing Fossil Fuels – How Do We Scale Up Without Compromising On Food And Environment?
Mr David Ho, Hovid Bhd
Session 2: Industrial Biotechnology
Title: Replacing Fossil Fuels – How Do We Scale Up Without Compromising On Food And Environment?
Dr Reinhold Mann, Battelle Malaysia
Session 2: Industrial Biotechnology
Title: Replacing Fossil Fuels – How Do We Scale Up Without Compromising On Food And Environment?
Dr Jim Flatt, Mascoma
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
Kubernetes & AI - Beauty and the Beast !?! @KCD Istanbul 2024Tobias Schneck
As AI technology is pushing into IT I was wondering myself, as an “infrastructure container kubernetes guy”, how get this fancy AI technology get managed from an infrastructure operational view? Is it possible to apply our lovely cloud native principals as well? What benefit’s both technologies could bring to each other?
Let me take this questions and provide you a short journey through existing deployment models and use cases for AI software. On practical examples, we discuss what cloud/on-premise strategy we may need for applying it to our own infrastructure to get it to work from an enterprise perspective. I want to give an overview about infrastructure requirements and technologies, what could be beneficial or limiting your AI use cases in an enterprise environment. An interactive Demo will give you some insides, what approaches I got already working for real.
Slack (or Teams) Automation for Bonterra Impact Management (fka Social Soluti...Jeffrey Haguewood
Sidekick Solutions uses Bonterra Impact Management (fka Social Solutions Apricot) and automation solutions to integrate data for business workflows.
We believe integration and automation are essential to user experience and the promise of efficient work through technology. Automation is the critical ingredient to realizing that full vision. We develop integration products and services for Bonterra Case Management software to support the deployment of automations for a variety of use cases.
This video focuses on the notifications, alerts, and approval requests using Slack for Bonterra Impact Management. The solutions covered in this webinar can also be deployed for Microsoft Teams.
Interested in deploying notification automations for Bonterra Impact Management? Contact us at sales@sidekicksolutionsllc.com to discuss next steps.
Essentials of Automations: Optimizing FME Workflows with ParametersSafe Software
Are you looking to streamline your workflows and boost your projects’ efficiency? Do you find yourself searching for ways to add flexibility and control over your FME workflows? If so, you’re in the right place.
Join us for an insightful dive into the world of FME parameters, a critical element in optimizing workflow efficiency. This webinar marks the beginning of our three-part “Essentials of Automation” series. This first webinar is designed to equip you with the knowledge and skills to utilize parameters effectively: enhancing the flexibility, maintainability, and user control of your FME projects.
Here’s what you’ll gain:
- Essentials of FME Parameters: Understand the pivotal role of parameters, including Reader/Writer, Transformer, User, and FME Flow categories. Discover how they are the key to unlocking automation and optimization within your workflows.
- Practical Applications in FME Form: Delve into key user parameter types including choice, connections, and file URLs. Allow users to control how a workflow runs, making your workflows more reusable. Learn to import values and deliver the best user experience for your workflows while enhancing accuracy.
- Optimization Strategies in FME Flow: Explore the creation and strategic deployment of parameters in FME Flow, including the use of deployment and geometry parameters, to maximize workflow efficiency.
- Pro Tips for Success: Gain insights on parameterizing connections and leveraging new features like Conditional Visibility for clarity and simplicity.
We’ll wrap up with a glimpse into future webinars, followed by a Q&A session to address your specific questions surrounding this topic.
Don’t miss this opportunity to elevate your FME expertise and drive your projects to new heights of efficiency.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
Key Trends Shaping the Future of Infrastructure.pdfCheryl Hung
Keynote at DIGIT West Expo, Glasgow on 29 May 2024.
Cheryl Hung, ochery.com
Sr Director, Infrastructure Ecosystem, Arm.
The key trends across hardware, cloud and open-source; exploring how these areas are likely to mature and develop over the short and long-term, and then considering how organisations can position themselves to adapt and thrive.
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Transcript: Selling digital books in 2024: Insights from industry leaders - T...BookNet Canada
The publishing industry has been selling digital audiobooks and ebooks for over a decade and has found its groove. What’s changed? What has stayed the same? Where do we go from here? Join a group of leading sales peers from across the industry for a conversation about the lessons learned since the popularization of digital books, best practices, digital book supply chain management, and more.
Link to video recording: https://bnctechforum.ca/sessions/selling-digital-books-in-2024-insights-from-industry-leaders/
Presented by BookNet Canada on May 28, 2024, with support from the Department of Canadian Heritage.
Accelerate your Kubernetes clusters with Varnish CachingThijs Feryn
A presentation about the usage and availability of Varnish on Kubernetes. This talk explores the capabilities of Varnish caching and shows how to use the Varnish Helm chart to deploy it to Kubernetes.
This presentation was delivered at K8SUG Singapore. See https://feryn.eu/presentations/accelerate-your-kubernetes-clusters-with-varnish-caching-k8sug-singapore-28-2024 for more details.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
Neuro-symbolic is not enough, we need neuro-*semantic*
09 CeoMeeting- Final Talk_Jay Keasling
1. Synthetic Biology: From Bugs to Drugs and Fuels Jay D. Keasling Joint BioEnergy Institute Lawrence Berkeley National Laboratory University of California, Berkeley
2. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
3. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
4. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
5. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
6. Building a computer from off-the-shelf parts System Off-the-shelf components Idea Knowledge Base
7. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Idea
8. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Idea
9. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Idea
10. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Idea
11. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Silicon Wafer Processing Idea
16. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry
17. Synthesis of Styrene System Off-the-shelf components Idea Knowledge Base Chemistry Kinetics & Reactor Design
18. Synthesis of Styrene System Off-the-shelf components Knowledge Base Chemistry Kinetics & Reactor Design Mass Transfer Process Design Standard Connections Idea
24. A brief history of artemisinin 168 B.C. Recipes For 52 Kinds Of Diseases found in the Mawangdui Han Dynasty tomb Hemorrhoids 340 A.D. Zhou Hou Bei Ji Fang (Handbook of Prescriptions for Emergency Treatments) Fevers (malaria) 1972 Active ingredient (artemisinin) isolated
25. Artemisinin is produced in oil sacs on Artemisia annua leaves Artemisinin... ...is produced by trichomes... ...found on Artemisia annua leaves...
26. Current process Artemisinin Plant synthesis Purification Artesunate Artelinate Arteether Artemether Chemical Conversions
27.
28. Microbial synthesis of artemisinin Off-the-shelf components Knowledge Base Idea System Engineer a microorganism to produce artemisinin from an inexpensive, renewable resource.
29. Semi-synthesis of artemisinin ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism Amorphadiene Artemisinic Acid ADS p450 Synthase Hydroxylase Unit CPR OPP Artemisinic Acid Purification OPP OPP Artemisinin Chemical Conversions Artesunate Artelinate Arteether Artemether
30. Constructing an artemisinic acid-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism OPP Amorphadiene Artemisinic Acid ADS p450 Synthase Hydroxylase Unit CPR OPP OPP
35. How do we get the components we need? Genome sequencing Genome annotation Components
36. BioShack? “ Please send me your biological device as you described in your paper.” Reply: “The device you requested is in this package … please use it as described in our paper.”
37. BioShack? “ It didn’t work as described in your paper.” Reply: “In my hands, it worked as described in my paper … did you try X and Y and Z?”
38. Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism ADS amorphadiene FPP OPP OPP OPP
39. Creating an amorphadiene-producing microbe ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism EAS amorphadiene FPP “ Please send me your amorphadiene synthase as you described in your paper.” Reply: “No … you are my competitor.” OPP OPP OPP
40. 5- epi- aristolochene synthase as a model ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis ispF ispE Central metabolism 5-epi-aristolochene EAS Synthase Tobacco EAS 5- epi- aristolochene FPP OPP OPP OPP
41. Production of a model isoprenoid in E. coli Very low production of isoprenoid resulted when using the native gene 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
42. Amorphadiene synthase by design ispH ispG idi isp A dxr dxs ispD DXP Pathway FPP Pyruvate + G3P Microbial synthesis Amorphadiene ADS Synthase ispF ispE Central metabolism ADS amorphadiene FPP OPP OPP OPP
45. Microbially-derived artemisinin ispH ispG idi isp A dxr dxs ispD Amorphadiene ADS DXP Pathway Synthase FPP Pyruvate + G3P ispF ispE Central metabolism Limitations in the native biosynthetic pathways OPP OPP OPP
46. A new pathway … borrowed from nature ispH ispG idi isp A dxr dxs ispD FPP Pyruvate + G3P ispF ispE PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS FPP Mevalonate Acetyl- CoA The mevalonate pathway is responsible for cholesterol production. OPP
50. BioBricks – Genetic Legos E = Eco R1 Bg = Bgl II Ba = Bam H1 X = Xho I Restriction site destroyed in the process B0034 AMP E Bg Ba X C0010 AMP E Bg Ba X Cut with E & Ba E Bg Ba B0034 Cut with E & Bg Mix & Ligate C0010 AMP Ba X B0034 E Bg C0010 AMP Ba X E Bg
51. Rapid assembly of metabolic pathways ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK ispA idi MPD PMK MK Best producer Assemble using biobricks
54. Building a computer from off-the-shelf parts System Off-the-shelf components Knowledge Base Characterization Standard Connections Independent Devices Models, Design, & Debugging Idea
55. Systems biology for debugging synthesis System Off-the-shelf components Idea ADS p450 Synthase Hydroxylase Unit CPR PMK MPD MK idi isp A HMGS atoB tHMGR Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) FPP Mevalonate Knowledge Base Debugging Metabolomics Proteomics Transcriptomics 0 5 10 15 20 25 Time [min] Asp Phe Glu Pro Ile Leu Lys Arg Val His Met
56. Accumulation of the toxic intermediate HMG-CoA Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS FPP Mevalonate Acetyl- CoA OPP
57. HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid biosynthetic pathway OPP
58. HMG-CoA inhibits fatty acid production PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid biosynthetic pathway OPP
59. Addition of fatty acids to the growth medium restores growth PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) Synthase FPP Mevalonate Acetyl- CoA HMG-CoA Fatty acid addition to the growth medium OPP HO O
60. Supplementation with saturated fatty acids improves growth Cell density (OD 600 ) Time post-induction (hrs) Active pathway No supplement Active pathway 16:0 Supplement Inactive pathway
61. Synthetic scaffolds: another way to solve the problem Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
62. Connecting metabolic pipes with synthetic scaffolds Connecting the enzymes in some way might reduce loss of intermediate to the bulk Ac-CoA AcAc-CoA HMG-CoA Mev AtoB HmgS tHmgR tHmgR HmgS AtoB
69. Synthetic scaffolds have a dramatic effect on the mevalonate pathway Dueber. 2009. Nat. Biotech . 27:753. HmgS HmgR AtoB n = 1
70. Synthetic scaffolds can control relative enzyme ratios and optimize flux tHmgR tHmgR AtoB HmgS HmgR HmgR HmgS HmgS HmgS AtoB
71. Component optimization and debugging yields another 50 fold 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
72. Fermentation optimization pushes yields beyond 25 g/L! Fermentation and further microbe optimization done by Amyris . 0.0001 0.001 0.01 0.1 1 10 100 1000 Construct Isoprenoid (mg/L)
73. Identify final enzyme in pathway (P450/AMO) and transplant into E. coli PMK MPD MK idi isp A HMGS atoB tHMGR Amorphadiene Artemisinic Acid ADS Mevalonate pathway (TOP) Mevalonate pathway (BOTTOM) p450 Synthase Hydroxyase Unit FPP Mevalonate CPR Acetyl- CoA A. annua p450 CPR Amorphadiene Artemisinic Acid OPP
74. Proposed artemisinin biosynthetic pathway Cytochrome P450 monooxygenase Alcohol dehydrogenase Aldehyde dehydrogenase FPP Amorphadiene Artemisinic acid Artemisinin C H 2 O H O H C H O O C C H 2 O H O H C H O O C
75. Lettuce, chicory, and sunflower produce isoprenoids like artemisinin Amorphadiene ( Artemisia annua ) Artemisinin H H O H O H H O O O H H O O Germacrene A (Chicory, sunflower and lettuce) O O O O H HO OH O O O H lettucenin A niveusin A O H O O P450’s involved
77. P450 candidate produces artemisinic acid FPP Amorphadiene Artemisinic acid C H 2 O H O H C H O O C 121 248 93 188 79 105 216 136 162 173 145 55 67 201 233 121 93 248 79 188 105 136 216 162 173 145 55 67 201 233 Relative Ion Abundance Peak 1 Yeast product Peak 2 Artemisinic acid m/z
78. Completing the biosynthetic pathway in E. coli p450 Amorphadiene Artemisinic Acid 1 2 3 >25 g/L Current titer in E. coli (lab scale) > 1 g/L P450/AMO Catalyzes 3 Separate Oxidations
85. Production of advanced biofuels Idea Knowledge Base System Off-the-shelf components lmnS gerS pptS waxS fnsS
86. Acknowledgements Funding Department of Energy National Science Foundation Office of Naval Research University of California Discovery Grant Bill & Melinda Gates Foundation Keasling lab Jennifer Anthony Michelle Chang Howard Chou John Dueber Connie Kang Lance Kizer Jim Kirby Taek Soon Lee Vincent Martin Karyn Newman Farnaz Nowroozi Mario Ouellet Eric Paradise Chris Petzold Brian Pfleger Doug Pitera Dae-Kyun Ro Christina Smolke Sydnor Withers Gabriel Wu Yasuo Yoshikuni Amyris # Jack Newman Chris Paddon Kinkead Reiling Rika Regentin Neil Renninger # Jay Keasling has a financial interest in Amyris & LS9. Joint Genome Institute
Editor's Notes
Ancient Chinese medical texts dating from around 150 B.C. suggest the use of sweet Annie for people with hemorrhoids .[1] Other writings from 340 A.D. are the first known to mention sweet Annie as a treatment for people with fevers.[2] It has been used ever since for a variety of infections in Traditional Chinese Medicine . 1. Foster S, Yue CX. Herbal Emissaries: Bringing Chinese Herbs to the West. Rochester, VT: Healing Arts Press, 1992, 322. 2. Foster S, Yue CX. Herbal Emissaries: Bringing Chinese Herbs to the West. Rochester, VT: Healing Arts Press, 1992, 322.
One way you can improve expression of plant genes (and in fact genes from many different organisms) in a microbial host is to resynthesize the gene, making it look like a microbial gene even though it encodes the plant enzyme. The way we do this is to run the amino acid sequence through a computer program that exchanges all of the rare codons in the gene (at least the codons that are rare for the host (E. coli)) for codons that are used frequently by E. coli.
This new gene improved production of amorphadiene 142 fold (not shown to scale in the graph). In essence, we eliminated a bottleneck and opened up the metabolic pipes at the end of the process.
So, we decided that the best approach was to bring an entirely new metabolic pathway into the cell. The new pathway that we added is the mevalonate pathway from yeast that is responsible for cholesterol biosynthesis. Since no other growth factors for cell growth were produced by this pathway, we would not have to worry about it disrupting growth. The pathway functioned beautifully and improved production another 90 fold.
So, we decided that the best approach was to bring an entirely new metabolic pathway into the cell. The new pathway that we added is the mevalonate pathway from yeast that is responsible for cholesterol biosynthesis. Since no other growth factors for cell growth were produced by this pathway, we would not have to worry about it disrupting growth. The pathway functioned beautifully and improved production another 90 fold.
This is what is known of the artemisinin biosynthetic pathway. It begins with farnesyl diphosphate, a 15-carbon precursor that is found in every organism. Besides being the precursor to artemisinin, farnesyl diphosphate is also the precursor to cholesterol.
The goals of the Fuels Synthesis Division are to develop organisms that can efficiently produce, in high concentration, existing and next generation biofuels from the sugars of cellulose depolymerization and organisms that can withstand high concentrations of the fuels. Early deliverables from the Fuels Synthesis Division will be pathways for production of next generation biofuels enabled by our strength in synthetic biology. Understanding the toxicity and stress encountered by organisms in response to fuels, enabled by functional genomics methods developed through the Genomics:GTL programs and informatics to integrate these methods, will allow us to engineer organisms that are better able to withstand high concentrations of the fuels. Using mathematical models of metabolism and gene regulation we will engineer microorganisms to efficient convert sugars into biofuels. Our long-term goal is to develop microorganisms capable of depolymerizing cellulose into sugars and converting the sugars into biofuels in a single pot.