Synthetic Biology for Industrial Biotechnology

00:00:11.10 Hello viewers, I am Nicolas, a PhD student
00:00:15.11 from the Max Planck Institute in Marburg.
00:00:17.16 Here, at the EMBL, me and my colleagues
00:00:20.08 Gita, Joana, and Pavel, will give you an insight
00:00:23.20 into synthetic biology for industrial biotechnology.
00:00:28.00 We try to keep it rather practical than theoretical.
00:00:32.11 Synthetic biology provides a lot of tools for
00:00:36.28 metabolic engineering. There are the mathematical models,
00:00:40.23 the standard parts, and the model organisms that we
00:00:43.16 try to engineer. The first step is the mathematical model
00:00:47.14 that gives us an idea of what parts we should use
00:00:50.17 and then we implement it back into our system.
00:00:53.14 Afterwards, we analyze our system
00:00:57.02 most likely, or hopefully, it's produced
00:00:59.17 our wanted compound and feed this information back
00:01:04.16 to our mathematical model to improve
00:01:06.16 this engineering cycle tremendously
00:01:08.13 to be faster and easier in the future to produce
00:01:12.26 this compound that we want.
00:01:14.23 S. cerevisiae is a very well-known model organism.
00:01:19.02 You might know it way more under the name
00:01:22.14 of baker's yeast. It's safe, easy to manipulate and grows
00:01:26.18 rather fast and it's already used a very long time.
00:01:31.11 We decided to produce, to engineer it in a way to produce
00:01:35.22 beta-carotenoids. The beta-carotenoids give a
00:01:39.22 rather visual output, and it will look in the end like this,
00:01:42.25 fairly orange. So what is really great about this is
00:01:46.19 that beta-carotenoids are also used for a lot of fine chemical
00:01:51.22 that have a high value and are of certain importance.
00:01:54.16 Also, the pathway has already some precursors
00:01:58.01 that can be used for other applications,
00:02:00.16 some of them you can see here.
00:02:03.08 To achieve our final goal of metabolic engineering,
00:02:06.18 we have to fulfill the following steps.
00:02:11.05 The first one is actually the modeling, where
00:02:13.10 my colleague Pavel will tell you more about.
00:02:16.02 Hello, my name is Pavel and I have to tell you about modeling
00:02:18.16 is really important before you make any changes to existing
00:02:22.12 pathway. You need to plan your strategy very carefully.
00:02:24.29 Basically, what you need to do is to find a balance
00:02:27.18 between the cell fitness and the production
00:02:30.12 of your compound. However, this task can be
00:02:33.12 very hard, because as you can see, all metabolic
00:02:35.24 pathways are heavily interconnected and any
00:02:38.06 change that you make to one can be distributed
00:02:41.26 for the whole network.
00:02:43.18 Fortunately, there are many tools
00:02:45.11 that can help you with this task.
00:02:47.01 Several models have been published
00:02:49.10 and we have downloaded one of them
00:02:52.20 that is free and use it as a simple system for our modeling.
00:02:57.20 We introduce the heterologous pathway to this model
00:03:00.11 and perform the flex balance analysis.
00:03:02.13 Basically, this method can tell you in which
00:03:05.10 direction you have to optimize the flow of the
00:03:08.20 metabolites in order to get the optimal
00:03:10.29 production. At the end, we have fused the results
00:03:13.10 from the flex balance analysis
00:03:14.28 to identify the genes that should be knocked out
00:03:17.23 and genes which should be overexpressed.
00:03:20.18 Now that we know what our pathway
00:03:22.28 should look like, we have a way better idea of how
00:03:25.02 to engineer our cell. We know that we have to change
00:03:29.01 something in the natural pathway, here shown in green,
00:03:32.10 that we have to knock out certain genes to actually push
00:03:35.18 the cell into the direction to produce more
00:03:38.02 of the compound that we are looking for.
00:03:40.07 Also, we know that we have to add to the natural cell
00:03:43.06 two genes to produce beta-carotene.
00:03:48.04 These two genes are actually unique, they're
00:03:51.04 catalyzing four biochemical reactions which give us
00:03:54.18 our final product. So we can move on to the step of
00:03:58.05 DNA amplification, to get all our parts that we need.
00:04:01.25 My colleague Joana will tell you more in the lab about this.
00:04:05.01 Hi, I'm Joana and as Nicolas told you, I will show you how
00:04:08.16 we amplify the genes in the lab.
00:04:10.19 Instead of amplifying the two genes that we want
00:04:14.08 to introduce into Saccharomyces cerevisiae,
00:04:16.07 right from the strain that originally produces beta-carotene we
00:04:21.19 order them from a synthesis company, codon optimize
00:04:24.20 to maximize the protein production levels in
00:04:28.06 Saccharomyces cerevisiae. Besides amplifying these two
00:04:32.19 genes, we also have to amplify the backbone
00:04:34.25 of the plasmid and its promoter region.
00:04:37.14 We then run the fragments in the gel to check
00:04:40.19 if they have the correct sizes and later on purify them.
00:04:44.23 So, now we have amplified all our parts
00:04:47.12 that'd be the backbone with the terminuses, the promoter system,
00:04:52.04 and our two genes. Basically, we are ready to bring
00:04:55.13 them together and transform them into our cell.
00:04:57.25 This step is called DNA assembly
00:05:00.04 and my colleague Gita in the lab
00:05:01.16 will tell you more about this.
00:05:03.02 Hi, I'm Gita. I'm going to explain for you more about
00:05:06.10 the transformation and the assembly method which we are
00:05:08.26 using in this lab. Our PCR fragments are ready.
00:05:12.11 In order to produce beta-carotene,
00:05:15.20 we need to transfer them into the yeast cells
00:05:18.12 and these are yeast cells which are competent for
00:05:22.04 uptake of any heterologous fragments and in our case,
00:05:25.19 these four PCR fragments.
00:05:27.06 In fact, we are using unique property of yeast
00:05:30.16 named transformation associated recombination,
00:05:33.28 and in fact, the homology region of yeast
00:05:37.18 for PCR fragments makes it possible to
00:05:40.17 assemble them in the circular form plasmid
00:05:43.20 named episomal plasmid in the yeast cells.
00:05:46.22 Now we have constructed our final construct and we
00:05:50.01 have the genes together with the promoter cassette
00:05:52.17 and the terminuses on one plasmid.
00:05:54.01 We already transformed this into our cell.
00:05:57.05 So, we can move on to the last step, analysis.
00:06:01.01 It's actually great because our analysis is a visual
00:06:04.11 output and our cells should produce now
00:06:07.02 beta-carotene. Let us in the lab together and have a look
00:06:10.22 if they do. We show you now our results
00:06:13.29 of our little experiment. Pavel explained to you
00:06:18.05 how to model a biological system
00:06:20.10 to find the pathway that we want to implement.
00:06:22.25 Joana explained how to amplify the part that you
00:06:27.01 want to integrate into our new organism.
00:06:29.28 And Gita at the end explained to you
00:06:32.14 how to assemble them to a full construct.
00:06:35.14 Afterwards, she transformed the mutant into the cell.
00:06:39.26 Three days later, colonies appeared
00:06:42.29 on the plates. These colonies we picked and moved them
00:06:46.05 into a liquid culture that grows at 30 degrees for three days.
00:06:50.04 As you can clearly see, one of them
00:06:54.06 -- our negative control -- shows no orange color,
00:06:56.23 that means no carotene is produced.
00:06:59.08 The other one has a clear bright orange color
00:07:03.22 that means carotene is produced.
00:07:05.26 This one includes our construct in our plasmid
00:07:09.22 and therefore, our heterologous pathway.
00:07:12.05 We really hope you enjoyed this little insight into
00:07:16.20 synthetic biology for metabolic engineering.

This Talk

Audience:

Researcher

Recorded: June 2015

Talk Overview

Synthetic biology can be used in industrial biotechnology to engineer metabolic pathways to create high-value chemicals using model microorganisms such as yeast. During the Synthetic Biology in Action course, participants engineered yeast to produce beta-caretone for industrial biotechnology purposes. In this talk, they describe the steps they took to engineer an existing yeast pathway to produce the new chemical. These steps include modeling the metabolic pathway outputs, DNA design, amplification, and assembly, and analysis of the final result.

About the Speaker

Joana Guedes, PhD student at i3S-Instituto de Investigação e Inovação em Saúde, IBMC-Instituto de Biologia Molecular e Celular, Universidade do Porto Portugal

Nicolas Koutsoubelis, PhD student at the Max-Planck Institute for Terrestrial Microbiology and the Research Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany

Gita Naseri, PhD student at the University of Potsdam

Pavel Zach, PhD student at the University of West Bohemia