Algae Biotechnology

Video1: Hi, I'm Dr. Stephen Mayfield, Director of the California Center for Algae
Biotechnology and Professor of Biology at UC San Diego. I'm Doctor Ike Levine, President
of the Algae Foundation and Professor at the University of Southern Maine, Department of
Natural and Applied Sciences. The world today is facing a number of significant problems,
including climate change, deforestation, degradation of our oceans and agricultural land,
and an ever-increasing demand for food, feed, and fuel. There are no easy solutions to any
of these challenges but one theme that runs through all of these is that we need to become
much more efficient at the production and utilization of our energy, food, and consumer
products while still protecting and preserving our natural resources. Algae is the most
efficient organisms on the planet at turning carbon dioxide and sunlight into biomass. As
such, algae have the potential to play an important role in addressing many of our most
significant challenges. This course will cover the basics of algae biotechnology, from
genomes and gene regulation to transformation technologies and product development. We
will explore molecular technologies and metabolic engineering and green algae, diatoms,
and cyanobacteria, as well as traditional breeding and selection in microalgae. We will also
learn about product development and commercial technologies directly from the companies
that are making products and algae. This course is produced by the Algae Technology
Educational Consortium and UC San Diego with funding from the Algae Foundation, the
National Renewable Energy Lab, and the US Department of Energy. Welcome to the
Introduction to Algae Biotechnology.

Video 2: Potential and Challenges of Algae Biotechnology

vHello, I'm Dr. Stephen Mayfield, Director of the California Center for  Algae Biotechnology and
a professor of biology at the University of California, San Diego. Today we're going to talk
about Algae Biotechnology and specifically about the potential as well as some of the
challenges that are facing us today. There is no single challenge that I would cite as being the
most important. Instead, there are three interacting events or resources: energy, food, and
water, that  all have enormous challenges for this planet. Together, these combined to also
drive climate change. I think those, more than anything else, are ones that we really have to
address as a society. Perhaps one easy way to think about this is shown in this graph. This is a
graph of world population and energy production over the last 10,000 years. Clearly, we can
see a tight correlation between our use of fossil fuel, shown in the blue, and world population
shown in the red. There are a variety of reasons that these things interact, but one of the main
ones is that we, as a society, have used fossil fuels over just the last few 100 years to first
drive the Industrial Revolution, and more recently, the Agricultural Revolution, which is allowed
for production of vast quantities of food. This has been an enormous benefit to the planet
and kept as many as a billion people out of starvation, but it has also allowed the world
populations to Increase to the 7.6 billion people on the planet today. With that many people on
the planet, our competition for resources is really great. One of these can be shown in
this, which is there is population density and our requirement for protein specifically is directly
related to the worldwide decline in fish populations. We are simply fishing all the fish out of the
ocean. This happens to be a graph of fish caught in the North Atlantic, and it shows that those
fish catches peaked in 1975 and have decreased every year since then. The graph on the right
shows an extrapolation of this to all of the world's oceans and shows that if we continue with
our present rate of exploiting this resource without allowing it to recover, we will have fished all
of the fish out of the ocean by 2040.This demand for protein also results in an increase to
deforestation for agricultural production, primarily for the production of soybeans, which are
used to feed livestock animals like chickens, pigs, and cows. This is a map of
deforestation over the last several decades. Even today, this continues to accelerate. If the
students will simply Google, Cargill deforestation in New York Times, you will find an article
from 2017 that describes this deforestation in alarming detail. Our planet is also now covered in
plastic trash. Plastic was only invented a little more than 60 years ago and yet today there are
already 17 trillion pounds of plastic on this planet. That is 2600 pounds of plastic for every
man, woman, and child alive today. Plastic is there because it's convenient, it's cheap, and it
never degrades by design. There are up to 18 billion pounds of plastic flowing into our oceans
every year, and the pictures of the plastic products, including flip-flops and water bottles
washing up on beaches throughout the world can now be seen every day. This simply has to
change. We cannot continue to live this way. So although these challenges are great, there are
also opportunities here. One of the opportunities that we think about and that we will describe
in this course is to use algae as a new photosynthetic biomanufacturing platform. So what
does that mean? That means we are going to use algae as a conversion platform as the
engine that is going to take sunlight and carbon dioxide from the atmosphere, nutrients like
nitrogen, potassium phosphate, and water and then use those to convert into the products that
we use today, whether those are plastics or food or fuels, or animal or human feed, or even
pharmaceuticals. So this opportunity is enormous. Why do we think about algae for this? We
can define that in many different ways, but a simple one to look at is that algae have biomass
and product yields that are much greater than traditional crops and are much more
efficient. One way to look at this is the graph shown on the upper left, which is simply biomass
accumulation per acre per year. This is the way productivity is measured in all agriculture. One
of the most productive plants on the planet is sugarcane, and sugarcane can accumulate up
to 25 tons of biomass per acre per year. But about 80 percent of that biomass is
cellulose, which is not a very valuable product. Algae can accumulate at least twice that
biomass, up to 50 tons per acre per year and really beneficial. That biomass can be
accumulated as proteins or oils, two things that we can use directly as animal feed, as human
food, or to make many, many different bio-products, including plastics or fuels. A summary of
that is shown on the right, in which we've looked at just two different aspects. Oil yield in terms
of gallons per acre. The most productive plant out there is palm oil, which can produce a little
over 600 gallons per acre. Algae can now produce 2000 to 2500 gallons per acre. If we look at
protein, that is even more striking. The world's protein crop is soybean, that produces about
350 pounds per acre per year. With algae, we can hit up to 10,000 pounds per acre per
year. So what are those efficiencies and yield that really suggest the potential of algae for the
future. The other thing is that algae can now be grown at world-scale and we can utilize non-
arable land and non-potable water. I have two examples of how we're going to produce
algae, one of the open ponds shown on the left, the other is a fermentation tank shown on the
right, and both those technologies are used to produce algae, and we'll talk about it later in this
class. So what are we going to use algae to bio-manufacture? Well, there are a variety of
products that can be made. In general, there's an inverse relationship between the volume of a
product that is produced and the cost, meaning that commodities which are produced at very
low cost, have a very high volume. Other products which are produced at very high cost by
definition, have to have a very low volume. Things like shown here would be an antibody
cancer drug. So the world's supply of that is only a few kilograms yet the cost of that can be as
much as a million dollars a kilogram. Whereas if we think about fuel, 1.2 trillion with a T
gallons of fuel are consumed every year, but it's cost is less than $0.40 a kilogram. With algae,
we have an opportunity to make all of those, including food, nutraceuticals, and high-value
products. So I think one of the important aspects of algae are, can we use this for food and
nutrition? Certainly, there are many companies, and we will describe those later on in this
course, that are now using algae to either produce nutritional supplements. So these would be
low volume products but relatively high value that we would add as an ingredient to another
food, or can we actually use these to produce food itself directly that we would
consume? These are just a few examples of that, but will describe these in more detail later in
the course. We also want to think if we can make polymers from algae. As I said, fuel is only
$0.40 a kilogram, but polymers, which are also chains of carbon, which can be produced
directly from algae, can sell as much as four dollars or five dollars a kilogram. So much more
expensive, and with algae, we have an opportunity to make polymers, make those into
products like the flip-flops shown here that can then be biodegraded. So we can have an
algae polymer carbon cycle that I think can begin to address some of those issues that we see
with ocean pollution. Certainly, the biggest challenge that we have facing us is how do we get
algae production at world-scale? This is a picture of algae facility down in New Mexico which is
used now to produce a nutraceutical. Clearly, we have an ability to go to scale. There are
several companies that are using fermentation at world-scale, so the opportunity is there. But I
think one of the challenges is, how do we do this on a world scale that is sufficient for all of the
food, feed, and products we need? So thank you very much for your attention and please enjoy
the rest of the course.

Video3 :
[MUSIC] 
I'm Anthony Berndt, a postdoctoral fellow at University of 
California at San Diego in Steve Mayfield's lab. 
I'll be presenting today on an introduction to molecular biology, 
synthetic biology and 
genetic engineering with specific focus on algae biotechnology.
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So, molecular biology is an approach for staying large-scale 
biological phenomenon at the scale of life's substituent macromolecules. 
This includes nucleic acids, proteins, lipids, carbohydrates and 
intermediary metabolites that make up these macromolecules. 
Molecular biology encompasses biochemical, genetic and 
informatics techniques for interrogating biological processes.
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Molecular biology has expanded to include more than just the study of 
single molecules. 
In the -Omics era, molecular biology includes genomics, proteomics and 
metabolomics amongst others that are the large-scale collective characterization 
and quantification of biological molecules that make up an organism or organisms. 
Large-scale databases are publicly accessible and 
can be rapidly searched and analyzed. 
Sequence genomes and transcriptomes have been a recent focal point because of 
the easy interconversion of DNA sequence and 
the primary amino acid sequence of proteins.
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Large DNA datasets can help us define gene structure. 
We can infer things such as promoters, regulatory regions and 
untranslated parts of transcripts. 
In the diagram presented here, 
I have aligned mRNA sequencing data with chromatin 
immunoprecipitation of a acetylated histone. 
This acetylated histone is often associated with open chromatin regions. 
When I plot the enrichment of this acetylated histone against the aligned 
transcripts, we can see a enriched peak just upstream of the promoter 
region marked in blue that potentially indicates actively regulated chromatin and 
the outer boundaries of this regulatory region.
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Taking the sequence of this gene, 
I can compare it to many other genes found in other algae species. 
And looking at the multiple sequence alignment presented in the top figure, 
we can see regions of high evolutionary conservation with 
the evolutionary conservation track plotted in yellow underneath, 
as well as regions where the conservation is not as strong. 
Highly conserved sequence may mean that these regions are functionally important. 
We can also use informatics methods to plot phylogenetic trees and infer 
evolutionary relationships of species based upon their DNA or protein sequence.
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Molecular biology has developed new tools for manipulating the genomes of organisms. 
A commonly utilized one is a loss of function mutation 
also known as a knock out.
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This uses DNA modification techniques to mutate or even delete a gene.
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Knock outs are often used for reverse genetic analysis. 
That is we have a gene sequence and then we look for 
a phenotype of a design mutant. 
Here we're showing the use of a targeting vector 
that integrates into the host genome through homologous recombination.
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So the host vector contains homology arms representing Exon1 and 
Exon3 of the targeted gene as well as a neomycin resistance cassette. 
So this gives resistance to the antibiotic neomycin. 
Upon integration and recombination into the host's genome, 
the second Exon is deleted, generating a non-functional transcript and 
the now mutated organisms can be selected for an neomycin containing media.
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Another method for manipulating the genomes of algae and 
other organisms is a gain of function mutation or a knock in. 
This is the addition of genetic material. 
It's often used to either restore the function of a mutant or 
generate new function.
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Here we're showing the integration of an expression cassette 
into the host's genome. 
In this case, it's randomly integrating into the genome in an untargeted manner 
and generating the expression of a recombinant protein.
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There are two common transformation methods used in algae, 
the first one being electroporation. 
This is usually used for manipulating the nuclear genome. 
In this case, DNA is mixed with the cells and 
an electrical potential applied across the cells. 
This opens up pores in the cell membranes and allows the entry of DNA into the host. 
Since DNA is negatively charged, it moves toward the positive electrode, 
the DNA can then exist as a extrachromosomal, 
episomal element or integrate into the host's genome.
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Another method used to transform algae is the use of biolistic delivery or 
a gene gun. 
This is generally used for chloroplast genome transformation. 
In this case, compressed helium is used to propel DNA 
coding gold beads at high speed into a lawn of algae. 
The DNA then integrates into the chloroplast genome and 
the transformed algae can be selected for on antibiotic containing media.
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Over the years, a new field of biology has developed called synthetic biology, 
synthetic biology is primarily focused on the design, modeling and 
tuning of biological pathways. 
If molecular biology answers the question, how does it work, synthetic biology 
answers the question of now that we know how it works, what can we build with it? 
Molecular biology provides the toolkit whereas synthetic biology uses 
these tools to build new systems.
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Here we're showing an expression vector that we have used 
to express the a high-value protein of interest. 
Going left to right, we can start with the AR1 promoter. 
So this is a semi-synthetic promoter that utilizes 
the promoters of two highly expressed genes, heat shock 70A and 
Rbcs2 to generate high recombinant protein expression.
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These promoters seem to act synergistically and 
give much higher expression than either single promoter by itself.
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In yellow are the five prime and three prime untranslated regions 
of Rbcs2 that stabilize the mRNA transcript. 
The integration of this transgene is selected for 
using the bleomycin resistance cassette. 
So, this antibiotic resistance gene stoichiometrically 
binds to bleomycin or zeocin antibiotics and 
leads to the selection of only very high expressing clones. 
This antibiotic resistance gene itself contains the first intron of Rbcs2, 
this intron itself acts as a transcriptional enhancer.
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Following this antibiotic resistance gene is a viral self-cleaving peptide. 
This peptide causes the production of two separate peptides, 
the antibiotic resistance gene and the gene of interest from a single mRNA. 
Finally we have the gene of interest shown in red, 
this gene of interest has been codon optimized generating high 
translational efficiency of our gene of interest.
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To date, multiple proteins have been made in algae, 
many of these have significant commercial or biomedical applications. 
For example, 
monoclonal antibodies had been produced in the algae Chlamydomonas reinhardtii and 
these proteins may have significant application to anti-cancer treatment. 
Additionally, colostrum proteins, colostrum is the milk that mammals 
produce in the first few days to weeks after giving birth have been produced. 
These include osteopontin which has been shown to have 
significant beneficial effects to gut health. 
We can also express recombinant proteins in algae to construct new 
biosynthetic pathways, one example of this is the production of patchouli 
which is the major component of patchouli oil.
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Thanks for viewing this overview of molecular biology and 
biotechnology in algae and I hope you enjoy the rest of the course