#metabolic engineering

An undervalued part of scientific community-building and ensuring reproducibility are protocol articles and, of course, video protocols.

One of such aggregators is JoVE - Journal of Visualized Experiments.

Within it you can find a vast variety of things from across the science, so it’s a nice place to be at overall.

As for the topic of today’s digest, the focus will be more on the old-fashioned producer organisms - land plants.

Often, during bioengineering, things happen. You might get a chimeric plant, where only some part of the plant has the necessary property, and it’s not the reproductive one. You might get a sterile plant, which is, however, otherwise perfect. Your plant might get infected with all sorts of viruses.

So, usually to deal with these issues you use clonal propagation. In case of potato tubers it might sound trivial, but what with the other plants?

Potato tubers are stems specialized for starch storage and clonal propagation. It's what they are. There is zero sex involved in a healthy potato tuber.

Well, the thing about plants is that all of their living cells (we remember here that the word ‘cell’ initially referred to empty cell wall chambers of dead cells within the wood) are omnipotent in the sense that they can give rise to a whole plant.

Unless you have some sort of a trick to alter the genetic makeup of generative organs, the size of an explant - a tissue or a group of cells which you choose for propagation - is in reverse dependency with the genetic diversity (derogatory) within the regenerated plant.

Of course, work precision requirements and time to regeneration increase as we go from ‘a functional chunk of plant’ to ‘a single plant cell’ in this system.

So let’s start from the plain example - clonal propagation using big explants.

Yeah, I know that cannabis contains a dangerous psychoactive drug, banned in The Federation and all, but people do research it and they make a good job explaining how they do it.

So, like, watch the video, and I will comment on the .pdf attached.

However, unintended cross-

pollination is an inherent risk when performing sexual

reproduction, causing undesirable offspring, which leads to

the potential loss of desirable traits or an introduction of

unwanted traits. An example of this unintended pollination is

highlighted by hemp growers receiving hemp seed pollinated

with THC- producing pollen resulting in significant economic

loss due to the non-compliant plants (>0.3% total THC w/

w).

So, we have a problem here: natural reproduction seeks to increase diversity within reproducing group, but for hemp the definition is homogeneity in its capability to produce THC, or, precisely, the lack thereof.

w/w is a weight per weight indication of percentage, used alongside w/v (weight per volume) and v/v(volume per volume). You might note that w/v can not be put as a percentage since it has a dimension of kg*m^(-3), or, if you are an astrophysicist, g*cm^(-3). In that case the convention is that you normalize the value by the value of water density, which nobody really cares to look up so they just use 1000kg*m^(-3) or 1g*cm^(-3). “Why not use partial density then?” to avoid undergrads from going insane immediately.

A common form of

asexual Cannabis reproduction is to cut and insert small

portions of a female plant into a soilless substrate which

is covered by a humidity dome to induce root formation.

Although this method has proven successful, a common

drawback is the accumulation of a high level of humidity

(usually 80% or higher) inside the dome, providing an

ideal growth environment for fungal pathogens, which can

be detrimental to new, sensitive cuttings.

So, the issue is that non-axenically grown plants contain quite a bit of foreign entities, which under high humidity can destroy the plantlets, and sterilizing every single piece of plant you are isolating is not feasible.

Another form

of asexual propagation is micropropagation using tissue

culture, where sterile techniques allow for the propagation

of insect, microbe, and virus-free Cannabis plant material in

limited space12 . This process, however, is expensive, time-

consuming and requires trained laboratory technicians which

are generally inaccessible for large-scale Cannabis facilities.

Well, this isn’t entirely true, protocol optimization of latest decade allows us to do micropropagation for low cost and without any skill, BUT. It is time-consuming indeed.

Okay, this here is basically the point where the upstream work enters this protocol. You did some selection, you got a plant, and you are propagating it.

This here is another side - what you have to get ready for your protocol. The light day duration is important for establishing the vegetatibe phase, meaning that the growth will go into the new shoots for plantlets and not into bloom. The water here exists only for the humidity, so tap water is acceptable.

You might find yourself thinking “this all seems pretty straightforward so far“, and yes. Yes it is. It's all straightforward, forever and always. The actual horror of science.

So, here we combine our ready mother plant with many plantlets with our propagation setup. Indolylbutyric acid is an artificial auxin, a plant hormone that induces root initiation. It, however, inhibits root growth, so the plantlets should be dipped and not "left soaking overnight".

Light is usually measured in micromol of photons×m^(-2)×s^(-1), because all of actual plant photochemistry starts with reactive chlorophyll relaxation same for both photosystems and the same endpoint energy regardless of the energy of the incoming photon. Fun fact also: since plant use photons and not total light flux energy for photosynthesis, it means that by using red light only you can get more photosynthesis products per joule theoretically.

Next up is maintenance, which is fairly regular. Of note is introducing nutrient solution after the initial period of acclimation, as to avoid excessive acidification of the wound due to nutrient uptake and the loss of moisture.

Hypochlorous acid means “buffered bleach”.

The next article, by popular demand, covers the limonene synthesis.

When you search for ‘limonene biosynthesis‘ either in PubMed or in Google Scholar, the two articles that pop out first are a review on advances in production of limonene in a huge variety of organisms and a research article on prototyping metabolic pathways in vitro before putting them into the cell.

They are both kinda hard to read, even if they are good.

The other article showcases how even good journals sometimes publish things that would be more fitting into an advertising email than in a research journal. In this case, authors are pushing forward their neural network, while diminishing the importance of actual human ingenuity in these sorts of things.

So, for today’s journal club we chose this article for its elegant approach and important insights.

Engineering of cyanobacteria for the photosynthetic production of limonene from CO2

Let’s start with the abstract!

Isoprenoids, major secondary metabolites in many organisms, are utilized in various applications. We constructed a model photosynthetic production system for limonene, a volatile isoprenoid, using a unicellular cyanobacterium that expresses the plant limonene synthase. This system produces limonene photosynthetically at a nearly constant rate and that can be efficiently recovered using a gas-stripping method. This production does not affect the growth of the cyanobacteria and is markedly enhanced by overexpression of three enzymes in the intrinsic pathway to provide the precursor of limonene, geranyl pyrophosphate. The photosynthetic production of limonene in our system is more or less sustained from the linear to stationary phase of cyanobacterial growth for up to one month.

Writing is considerably worse here than in the previous article, so, like, the first sentence says basically nothing.

The second sentence tries to catch up as a result of this, and ends up rushed. They constructed model system for producing limonene in a photosynthetic bacterial system by using an enzyme found in plants.

spoiler: the photosynthetic bacterial system here is Synechocystis sp. PCC 6803 again

limonene is a valuable compound actually! Known for giving pines and oranges their distinct flavour, it also found use in cleaning industry as a renewable solvent for removing highly hydrophobic dirt, machine oil and bad odor. Being a nearly saturated aliphatic (meaning that it has no aromatic nuclei) hydrocarbon, it also can be used as fuel.

The next sentence mostly repeats the previous points, except it offhandedly points to researchers making a gas-stripping device that continuously extracts limonene from the culture.

Now, we should note that limonene in plants is an antimicrobial and antifungal compound. Does it affect our microbial culture? The next sentence says ‘no’.

This lack of strain on cells is then demonstrated as the culture kept producing limonene for a month.

The main text is written slightly better, so it's going to be a bit easier.

So, let’s move to introduction.

Fun fact also, while limonene is a monoterpene, it is still of isoprenoid biosynthetic nature. All sterols, including steroid hormones, such as cortisol, testosterone or estradiol, are triterpenes, i.e. also isoprenoids! Meaning that they can be produced entirely within the isoprenoid pathway. However, aside from making them from petroleum, plant sources are very slow to grow. Marker degradation process, for example, one that revolutionized the steroid chemical synthesis and allowed HRT to even exist for both cis and trans people, uses yams as a plant material for example.

So, of course people tried to make them in the genetically engineered organisms. And of course they started from E. coli and yeast to do it, but, then again, heterotrophic organisms live by turning organic matter into CO2, making organic matter using them is not very rational.

So, they chose a cyanobacterium for it, and used the most studied one.

So, the results are written in a very dry way. Thus, it is imperative that we look at figures. This is a metabolic pathway.

You probably remember the G3P and pyruvate from the previous article, and you might remember that phytol and carotenoids are structural moieties in the light-harvesting systems of photosynthetic organisms. Specifically, phytol is a part of chlorophylls, and carotenoids are these super-active antioxidants that give carrots their color and allow you to see at all. So, like, everything from Calvin cycle (it's the CO2 fixation cycle) and the lower right corner has to occur in the chassis organism no matter what.

Calvin cycle is when you use the carbon you got by affixing it to the ribulose to make more ribulose to affix carbon to. It's literally it. It might be called after Some Guy, but it's nothing conceptually hard. Just moving carbons around like in those children's problems of "how do you measure 5 liters of water having only a 3-liter and 1-liter bucket?"

The arrow pointing to limonene starts at GPP, standing for geranyl pyrophosphate. GPP is made by combining two "activated isoprene" blocks (DMAPP and IPP) , hence the name "isoprenoids". Isoprene is an old name for a gas that you get if you decay natural rubber, which is also an isoprenoid. It has two double carbon-carbon bonds, so it can be 'activated' by pyrophosphate group in two ways as a result. Limonene synthase is a 'magic enzyme' here, which takes the intermediate product of what is called 'primary metabolism' (you will die a lot if you do not do it) into a 'secondary metabolite' (everything else).

Well, Figure 2 is a biologist's version of those "this is going to be so tasty!!!" pictures on the culinary blogs where they post the gray dough in the least appetizing light spread thin on the ugliest dish in existence.

Of note: Cm^R and Km^R signify cassetes of antibiotic resistance to chloramphenicol and kanamycin. They can be used as selective markers, letting the strain that has it survive in the presence of said antibiotic.

Ptrc is a trc promoter. It is strong, making lots of protein. And constitutive, meaning it works always.

6xHis-tag is. Hm, it's a bit of satanic magic actually. If you put six pentagram-like amino acids (protein constituents) in a row, they will be able to bind nickel or cobalt ions, both named after demons (Nick and kobold respectively).

The second colorful sausage shows that they also expressed a cassete of usual isoprenoid pathway enzymes under a strong promoter, but like. It doesn't matter, just as insertion sites (blue arrows on a string) do not really matter. Now, for the black and white part of the figure. The pic labeled (B) is "PCR analysis of the integration of the limonene synthase using the primers 2031-9 and 2031-12". Which means that they amplified using Polymerase Chain Reaction a select part of the bacterial genome, which should contain their insert, and this part became bigger after the insertion, confirming that the insertion really happened. In the pic labeled (C) they confirm that their insert makes protein and that this protein is not immediately degraded by the cell. Pic labeled (D) sucks ass, so I am not commenting on it. So, now we know that their construction makes protein in Synechocystis, but is this protein functional?

I have absolutely no idea why they didn't put this result into the previous figure. They basically just show that an ion of the weight of limonene appears at the retention time of limonene, and is absent in the wild type strain. Whatever.

But there are many other ions here! How do we isolate limonene from this mess? And they solve it very elegantly, but also completely undersell the ingenuity of the approach. Air that we breathe is actually not very polar. Oxygen and nitrogen are monoelemental gases, carbon dioxide is perfectly symmetrical, argon is a noble gas. This means that the non-polar compounds like limonene actually would prefer to be in the air than in the water solution!

So the authors connect the air output of the culture to the cold trap (cold trap is a vessel on the path of gas that is cold and traps wanted/unwanted things by being too cold for them to be in gaseous phase) filled with octane. And all the limonene goes to the cold trap and does not poison the algae. This is genius, but also they really tried to make it look the worst way possible.

Hip Dadaist(@hypdadaist) — WHY ARE SCIENCE PAPERS LIKE THIS???

You take the stupidest and the craziest people that can still be functional, put them into an underfunded institution on the diet of deadlines, and pay them on the basis of how much papers they write. I am actually surprized that we as civilization lasted that long since the fall of the Soviet Union.

The other two figures are basically "number go up", so commenting on them seems pointless.

Discussion also sucks, aside from the calculation that they used 2.5% of cell isoprenoid synthesis by sloppily putting an unmodified enzyme under the "eh, it's going to be fine anyway" promoter. The coding sequence for the enzyme was isolated from the backyard japanese catnip their lab neighbor studied.

This is really cool because of THINK OF THE POSSIBILITIES, but also it seems like authors despaired at some point.

Like, there is probably some science fan out in the wild that will be like "you can make saturated hydrocarbons in your backyard with just sun, air and fertilizer????" and there are scientists that are like, "yeah, yeah, the clean energy ready to go whenever, we've all seen it".

That’s it for now!

So, BioMedCentral, or BMC is a group of journals of very high quality within Nature Publishing Group. The name of the journal, Microbial Cell Factories, probably speaks for itself. The article is quite recent, published in the end of 2020.

The name, as it is common, is nearly incomprehensible. "Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC" Thankfully, it's a good journal, so it immediately provides a background:

PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW).

PHB is a biodegradable plastic alternative. And a good one at that!

Synechocystic sp. PCC 6803 is a long name for a common lab beast, sometimes nicknamed "green E.coli" for being green and easy to genetically manipulate.

Cell Dry Weight is basically a stand-in for “material that goes downstream“ (in this case towards getting actual plastic granules or fibers). It’s a bit of a technicality, since while in the eukaryotic algae wet weight does not correlate with the actual amount of biomass capable of synthesizing things, in cyanobacteria the relationship is actually quite direct, but since we have to compare with eukaryotic algae, here it is.

The biggest thing in abstract is the conclusion, which says "The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB".

PPT1 is the name of the strain they made.

And, without reading hard into the results section of abstract, we find that this amount is 81% of cell dry weight. This is a lot!

Now, let’s read the text proper

So, the introduction starts well, from the issue of plastic contamination. Next, it goes into consideration of the microbe E.coli as a production organism.

E. coli is a human symbiote, therefore it eats what humans eat, and is very hungry for external organic matter, i.e. it is a heterotroph

Like, explicitly: "However, these production processes require crop-derived organic carbon sources for growth and production and pose a threat to human food-supply."

This is a very important consideration, as you probably should not let your plastic sources compete with your food sources. In fact, you would like your resources as ontologically far away from each other as possible.

And what is more further ontologically from human food than rocks! Cyanobacteria are a class of bacteria that use photosynthesis to turn mineral compounds into living matter. The model cyanobacterium is usually Synechocystis sp. PCC 6803, it is easily modifiable and well-described.

Then the introduction delves into the physiological conditions under which cyanobacteria produce PHB. Basically, PHB occurs during the nitrogen starvation when the energy stored from photosynthesis is put into a usage network.

Then they provide a table of attempts to increase the production capacity, and it looks miserable!

[Table 1 from the article proper, showing no more than 40% yield of PHB from the algae, even though quite sophisticated constructions were inserted]

Then they talk about their previous work, which identified a genetic switch PirC to aforemented stress physiological conditions under which cyanobacteria produce PHB.

It also should be noted, that this switch frees "building carbon blocks", acetyl-CoA, not only for PHB production, but also for a variety of other metabolis processes, so it can be of use in other production processes.

The next part is results, so it has Pictures.

The wording is hard if you aren't used to it, so let's mostly skip it and look directly at the picture. Everybody does this. An overworked scientist is more likely to look at your figures or at your protocol in methods than to try to understand your Genius Idea.

[Image: Main carbon metabolic pathways involved. CO2 is the major inorganic carbon source, usually present in the atmosphere causing liberals to freak out as a medium-power greenhouse gas. It is fixed into the ribulose-bis-phosphate by RuBisCO enzyme, which is the most abundant and possibly the most important protein on Earth, since it is the only major protein that irreversibly fixes carbon. Ribulose-bis-phosphate(RuBP) then immedialely self-cleaves into two molecules of 3-PGA, usually called just "the phosphoglycerate", as it is immediately transformed into 2-PGA,  which is a precursor of phosphoenolpyruvate, the Highest Energy Phosphate Bond Compound in Living Organisms Ever. So, in this picture you see that breaking the PirC, which inhibits the enzyme that makes a 2-PGA out of 3-PGA, shifts the metabolism from fixing stuff and storing it as sugars, ending in glycogen in the upper right corner (such processes are commonly referred as catabolism) to spending stuff onto Chemistry (commonly referred as anabolism). The lower part of the picture demonstrates formation of PHB through more or less universal carbon blocks - acetyl-CoA]

The next figure is basically "we made a strain and it does not suck".

[Image of Number Go Up for their strain and wild type]

OD700 is a common measure of cyanobacterial culture density. Unlike OD600 for E.coli, however, it is actually based on absorption of light by the photosynthetic apparatus and not on light scattering, so it is easier to compare on equipment with different adjustments for light scattering.

"WT" is usually "wild type", an entity that was Not Engineered In Any Way.

Figure 3 shows that in a non-complicated starvation conditions the engineered strain makes significantly more PHB than the wild type strain.

Then they, like, adjust medium and get the 80% and 80% dry weight looks like an inflation fetish art.

So, now onto the how it's done.

The very first paragraph of Results gives us this description of their work:

To test if the PHB content of a Synechocystis sp. PCC 6803 pirC mutant strain (ΔpirC) could be further increased, we cloned and overexpressed phaA and phaB from the PHB producer strain Cupriavidus necator (formerly known as Ralstonia eutropha) into ΔpirC. We used these genes, since C. necator is known as a highly efficient PHB synthesizing organism. Furthermore, the expression of heterologous enzymes ensures that they are not inhibited by intracellular post-transcriptional regulatory mechanisms. Both genes were cloned into a pVZ322 vector under the control of a strong promotor, PpsbA2. The plasmid was then transformed into the strain ΔpirC, thereby creating the strain ΔpirC-REphaAB (Fig. 1). For the sake of clarity, the strain is referred to as PPT1 (for PHB Producer Tübingen 1) from here on.

Usually when the results say "this was done like that" it clarifies nothing. So you have to go to the methods and see what was actually done.

The promoter psbA2 and the phaAB genes were amplified from genomic DNA of Synechocystis and Cupriavidus necator, respectively.

For this, the primer psbaA2fw2/psbA2rv2 or RephaABA2fw/RephaABA2rv were used (Table 3). A Q5 high-fidelity polymerase (NEB) was used to amplify the DNA fragments.

The latter were subsequently assembled into the pVZ322 vector [12], which was beforehand linearized with XbaI.

The resulting vector was propagated in E. coli Top10 and isolated using a NEB miniprep kit. The plasmid was subsequently sequenced to verify sequence integrity.

The correct plasmid was then transformed into Synechocystis using triparental mating [44], resulting in the strain REphaAB. The REphaAB plasmid was also transformed in the strain ΔpirC, resulting in the strain PPT1 (ΔpirC-REphaAB).

So, this is a relevant part of a method. In order to understand it, however, you need to understand prokaryotic gene structure and the Terminal Central Dogma.

So, the central dogma is a statement about a predominant flow of information in the cell.

Life is the mode of existence of protein bodies

- Friedrich Engels

Catalytic functions are performed by proteins. Proteins are assembled from amino acids by the ribosome from a sequence provided by mRNA. mRNA is transcribed from a gene. Genes are loci in DNA that do things.

Of course, such a view of life lacking feedback modes is bullshit, and was debunked in, like, five years after its formulation, but it’s still pretty useful as a sketch, as it makes it obvious that the gene elements have to be nested.

So, let’s look at the prokaryotic gene structure.

(image from Wikipedia)

A "promoter" is a part of a gene that facilitates transcription. Since in most bacteria protein synthesis is cotranscriptional, promoters are single most important feature determining expression strength of a protein.

The promoter psbA2 and the phaAB genes were amplified from genomic DNA of Synechocystis and Cupriavidus necator, respectively. For this, the primer psbaA2fw2/psbA2rv2 or RephaABA2fw/RephaABA2rv were used (Table 3). A Q5 high-fidelity polymerase (NEB) was used to amplify the DNA fragments. 

So, they took the DNA pieces corresponding to the parts of genome of organisms, amplified it using PCR and put a cloning adapter for them to be inserted into a vector.

A vector is a piece of DNA that carries the insert of interest, hence the name. 

Q5 polymerase is a good PCR polymerase for use! PCR has gotten quite a name as a method to amplify any chosen fragment of DNA, including viral cDNA, in a sample, but is also a preparative method.

Most commonly a vector is a plasmid, i.e. a circular DNA with its own machinery allowing it to multiply in the cell. The fact that a vector is a plasmid is usually denoted by a lowercase "p" in front of a name, so in this light the next part makes more sense:

The latter were subsequently assembled into the pVZ322 vector [12], which was beforehand linearized with XbaI. 

The map and the sequence of pVZ322 vector can be found on the internet to play around.

Some manipulations require DNA to be linear, hence the need for linearization. To make it site-specific, DNA cutting enzymes called "restrictases" are employed. XbaI is one of them.

The following parts are very straightforward in light of this,

The resulting vector was propagated in E. coli Top10 and isolated using a NEB miniprep kit. The plasmid was subsequently sequenced to verify sequence integrity.

Vector propagation is basically letting bacteria make more of the vector.

Kits are an evil capitalist ploy created so that labs get addicted to their utilities and products. The evil became apparent when the Qiagen kits ended in the midst of COVID-19 pandemic, when screening for viral RNA was really important.

Sequencing is a process of “reading” DNA so that its exact nucleotide sequence becomes known

The correct plasmid was then transformed into Synechocystis using triparental mating [44], resulting in the strain REphaAB. The REphaAB plasmid was also transformed in the strain ΔpirC, resulting in the strain PPT1 (ΔpirC-REphaAB).

PCC 6803 is a naturally competent strain, so “transforming” here means “pouring the DNA prep onto slightly starved cells for the uptake to happen“.

Competence usually refers to DNA uptake/transformation competence of cells

Triparental mating is simply a mating process that involves three parents.

So, this is an introductory post on the “Metabolic Engineering of Green Dudes” sort-of-a-Journal-Club! This is going to be a Tumblr activity not unlike roleplay blogs, but with added benefit of me getting to improve my ability to communicate things to people who do not constantly think about said things, and also to provide some recent views and techniques to make stuff that is useful in a household/community. Whether you actually put this knowledge to use is up to you, and I hope that the format will be accessible enough for people to hop on and off, though I do hope to reach and engage enough of a constant audience to have other people post. So, let’s go!

A lot of things that we encounter in our life are bound by laws of chemistry. A great russian scientist M.V. Lomonosov has said that “Chemistry spreads its influence far and wide into the human deeds” in 1751 and the influence has only increased since then.

Water has properties dictated by its chemistry, food has properties dictated by chemistry of its constituents and which reactions happen between them, machines have chemical properties from both the materials of the constituents and the sort of things that they are pushing through themselves to do useful work.

A lot of chemical compounds that we encounter in our life are of biological origin. And many chemical reactions that we see are purely biological, like souring milk and leavening bread. But also treating wastewater and tending to plants. But also, like, our own body’s metabolism, like taking medication and processing food.

[A picture of a half-loaf of bread (product of yeast fermentation of plant seed derivatives) superimposed on “Regular insulin“ (a mammalian peptide commonly produced in bacteria such as E.coli) and an antigen test (product of conjugation of latex nanoparticles with recombinant antibody on a paper matrix). All bolded things are products of biochemistry!]

Quite a lot of biological tinkering falls into the domain of synthetic biology, which is mostly either making things up from scratch, or introducing things that were somewhere else but not here into an organism you need. 

There is also metabolic engineering that, until fairly recently, was about hitting the metabolic network with a crowbar until you get nice yields. Pretty much all of traditional selection and breeding is this.

the crowbar is radiation or toxic mutagens pretty much always! Nobody is doing the same kind of heroic feat as Vavilov’s nowadays.

However, relatively recently the methods of molecular and synthetic biology finally reached the metabolic engineering, and this was rather productive. 

So, in words of Liu and Nielsen, 2019:

Briefly, metabolic engineering can be defined as the process of improving cellular properties toward specific biochemical production using recombinant DNA technology. 

Recombinant DNA technology is an unnecessary and a bit outdated term for any manipulation with DNA where the DNA has more than one “source”. People usually say ‘assembly’ nowadays but *shrug*

This is a rather broad definition, however, in this case of wikipedia definition that some people use we get this:

Metabolic engineering is the practice of optimizing genetic and regulatory processes within cells to increase the cell's production of a certain substance.[...] The ultimate goal of metabolic engineering is to be able to use these organisms to produce valuable substances on an industrial scale in a cost-effective manner.

...Which is absolutely an unhinged way to view a scientific field during three ongoing pandemics, Climate Change Crisis and several quite active wars.  It is both overly specific, talking about ‘optimization’, specifying that the process should be within cells, and puts way too much emphasis on the cell being an unchangeable constant in this process. We might further elaborate on why these constraints might be detrimental, but so far there’s not really a way to do it without going deep. 

As you can guess, the scope of what you can engineer cells to produce is theoretically at least as wide as what cells produce for us already, and is really only limited by the highest energy requirement step, which can not exceed the energies that occur in the cell. 

(From Liu and Nielsen, 2019 again)

And the energies that occur in the cell, in case of a photosynthetic organism, are like 1.8 eV per single electron. For comparison, a TNT explosive has an energy of detonation of 10 eV per the whole seven-carbon molecule, and carbons can have up to four valent electrons. We’re talking these amounts of energy processed in photosynthetic organisms, so of course the most fun metabolic engineering is in photosynthetics.

The requirements for metabolic engineering are usually what you would expect from normal lab/industry practice of handling the producent organism, but there are many, MANY lifehacks to reproduce or even improve these using incredibly cheap and/or household things. With photosynthetic organisms growing a culture is something that just happens. Like weeds on the backyard, or algae blooms.

Practically it does entail quite a bit of thinking and some work with hands, but it can be both rewarding AND fun.

The first article will cover the very memeable nowadays topic of dealing with microplastics, specifically not, like, removing the existent microplastics, but building a foundation for such an issue to not happen again.

It’s scheduled to appear on Wednesday, 2022-05-25 23:55 MSK on this blog!

The long-term outlook for the Muconic Acid Market is characterized by technological convergence, strain optimization breakthroughs, and strategic corporate consolidation among leading biotechnology startups and multinational chemical giants. Major chemical producers are investing heavily in engineered microbial chassis, continuous flow fermentation systems, and automated AI-driven metabolic simulation software. These technological advancements enable firms to reduce production costs, enhance long-term yield stability, and accelerate commercial delivery timelines for demanding industrial polymer clients. Sustainable circular economy initiatives, such as waste-to-chemical conversion technologies, also enhance the industry's environmental profile.

Strategic mergers, acquisitions, and joint ventures among independent biotech innovators and multinational chemical corporations will likely define future corporate growth strategies. By pooling financial resources and proprietary fermentation patents, major market players can secure multi-year bio-intermediate supply contracts and expand their global footprint more efficiently. Furthermore, ongoing research into direct-to-polymer conversion pathways that bypass intermediate processing steps allows companies to engineer next-generation sustainable material architectures.

Institutional investors and private equity firms continue to monitor the sector closely, drawn by strong recurring demand profiles and steady cash flow generation across green chemistry supply chains. As global climate mandates drive continuous consumer and corporate spending on sustainable materials, muconic acid commands attractive profit margins. Companies that successfully balance biotechnological efficiency with cutting-edge environmental sustainability will capture significant competitive advantages.

Today on Rensselaer Polytechnic Institute Week on The Academic Minute - Robert Linhardt, professor of biocatalysis and metabolic engineering, explores a new approach to treating Lyme Disease.