I am completely bias when I say that a tolerant organism is a "better" organism for whatever reason. I also think that a tolerant human being is much more capable of performing certain activities. After all, most of the people from the World have faced harsh conditions and historically we have needed to adapt… to evolve for survive. Same with all living organisms on Earth (and the Universe...?).
What is this tolerance anyway?
Paraphrasing Charles Darwin's Theory of Evolution in "On the origin of Species" book published in 1859, evolution is the process carried out by natural selection in a particular organism that resulted in better capabilities for the organism to survive and reproduce.
Survival and reproduction are in fact an intrinsic part of tolerance. In the context of Biology, a typical tolerance experiment includes a measurement of growth given a harsh condition or a challenge that the organism is exposed to. Tolerant organisms are those that survive/reproduce better in these conditions.
You can think of tolerance as the consequence of adaptation to X environment (output) given particular traits selected in a particular way (input).
How are these traits picked?
When natural selection is on charge of picking of the "better" traits we call it evolution (slow but very effective), when these particular traits are selected by a human being with some type of knowledge, we could call it rational engineering (fast but moderate effectiveness).Regardless of the process for the selection of traits, tolerance is an essential quality for the survival of any organism.
Let’s focus on rational engineering since it’s what I pretend to become an expert of. I’d say there are four approaches within the rational design of a tolerant organism that I could think of.
Selection based on existing and known tolerant organisms
High-throughput selection and random events
Selection based on a fundamental structure
A shot in the dark
1) Selection based on existing and known tolerant organisms
The variety of different environments allowed life to flourish in practically all conditions that we know. This bloom of living organisms can be attributed to particular physical characteristics. For instance, in the World of Microbiology where thousands of species exists, there are microbes that can tolerate up to 120 ºC (i.e., thermophiles) and other that can live just fine at very acidic (i.e., acidophiles), salty (halophiles) or high-pressure (piezophiles) conditions. Researchers still have tons of work to do in order to characterize these microorganisms and their special “super-powers”. In contrast to typical super-powers from the comics where some of them aren’t tangible (e.g., the telepathy used by Professor X or the super speed employed by Quicksilver),
the “super-powers” of microbes must be a consequence of something tangible, physical and real.
Considering that all living organisms on Earth utilize amino-acids, fatty acids, and sugars as building blocks for creating their structures, some special re-arrangement of these should give them the special capabilities isn’t it?. Also, all structures are basically coming from some sort of machines called proteins and all of these are coming from a genetic code where all information is stored in each cell of each organism.
How is this genetic code within super-organisms related to their capabilities of tolerance?
The genetic code within each cell of each organism (with and without “superpowers”) contains the instructions (e.g., ATGGTAAATG) written in a special molecule called DNA. This molecule is composed of four nucleotides and each of them are formed by a sugar (deoxyribose), a nitrogenous base and a phosphate group. The variation of only 4 nucleotides (A,T,G,C for Adenine, Thymine, Guanine an Cytosine respectively) is the language that the cell utilizes to assemble its “machines” or proteins (Figure1).
Figure 1.- Genetic code. Each nitrogenous base together with a phosphate group and a sugar (deoxyribose) makes a nucleotide. The four nucleotides A,T,G,C are utilized in sequences for writing the information of each gene that will be translated into a protein with a specific function within the cell
Thus, understanding of these instructions is essential for discovering special traits for tolerance and identify when (and how) these sets of proteins/machines get activated and functional against the harsh conditions. I am sure you have heard of genes. Well, they are nothing more than an instruction of the cell utilizing this mentioned code. For example, let’s assume that the machines (here, I’ll start to refer them as proteins) to produce ethanol are called PDC and ADHB. Well, each of these proteins has a DNA sequence. These two DNA sequences with the required instructions for assembling these two proteins within the cell and produce ethanol are called genes.
Some work has been done for understanding these DNA instructions (genes) and utilize them to increase tolerance of organisms. Here, I’ll refer to microorganisms in the context of biorenewables since a special interest for researching them has been ongoing for the last decades. A great example of utilizing traits from a known tolerant organism in another was the insertion of the cti gene from Pseudomonas putida (a solvent tolerant bacterium) in E. coli (the most studied microorganism although without “super-powers” of interest for biorenewables at least). For more information you could read the scientific article published in an indexed journal (see references) published by Tan Z in 2016(1). The insertion of this single gene gave to E. coli the capacity of being more tolerant to solvents. The selection of this gene was possible because of the existing knowledge of Cti protein (in this case the protein is an enzyme because it catalyzes a reaction just like a nano-machine) from P. putida and its involvement in solvent resistance(2).
2) High-throughput selection and random events
The development of technology has enabled us to study the genetic code very rapidly. There are multiple tools for doing even whole-genome engineering and these tools are applicable to all biological science fields with numerous applications. One particular application of high-throughput tools for gene selection is of course the increase of tolerance. This type of approach for selection of genes relies on methods that can test numerous combinations of genes. This capability is useful especially when you don’t know the instructions of the tolerant gene (i.e., the DNA sequence). These methods can generate tons of data of DNA sequences that must be processed with high-throughput sequencing methods (coding in software). Thus, there is an existing (and very good) overlap between informatics and experimental work within rational engineering. In the future, we might have numerous studies that utilize both ways to select traits: rational engineering approaches in the lab and deep learning. Another characteristic of these methods is the creation of libraries. These methods generate a very large combination of genes and these combinations might be even random. Therefore, there is a stochastic component when different combinations are being both created and tested for selection of a trait (e.g., tolerance). The example I chose was published by a research group in Colorado and was executed for finding tolerant furfural genes. In general, the method consisted in creating lots of DNA sequences of different length (this is possible because there are “machines” or enzymes that cut DNA at certain sites in the genome). The enzyme cuts very frequently in the genome thus it is feasible to cut most of the genome in single pieces. Not necessarily knowing how many DNA sequences will be produced after the cutting and how many of these will contain a tolerant gene, all this DNA library is put in host bacteria. The bacteria are grown on furfural and those bacteria with the furfural tolerant genes would grow faster (Figure 2).
Figure 2.- Schematic representation of enrichment of tolerant genes utilizing DNA libraries.
Once the more tolerant genetically modified bacteria (also called strains) are grown, the DNA sequence can be tracked down and the furfural genes identified. This methodology identified lpcA gene as a furfural tolerant gene (3).
3) Selection based on a fundamental structure
At this point, the mentioned rational design approaches for selection of genes have considered only the DNA sequence and its phenotype (an observable property such as tolerance). However, analogies, abstract thinking and I’d say common sense are good allies when a researcher comes up with ideas too. Dr. Jarboe realized that cell membrane genes appeared often times in high-throughput methods (such as transcriptomics) for identifying tolerance. She also made an analogy about the cell membrane of a microbe with a production vessel. The vessel is damaged by its contents and by external conditions limiting productivity. Considering that the cell membrane is what contains all reactions happening in the cell, and what defines a cell as an entity different from the environment...
Why shouldn’t us consider the cell membrane as the key cellular structure for tolerance?
In addition, the cell membrane is involved in respiration processes and the viability of the organism (Figure 3).
Figure 3.- Cell membrane. The cell membrane besides phospholipids it has membrane proteins with vital functions such as respiration and transport of nutrients.
Thus, the cell membrane is a fundamental cellular structure and a modulation of membrane components might impact tolerance too. The exposure to different challenges might promote various mechanisms of autoregulation and responses in the cell but regardless of which of them get activated, all processes must begin from the cell membrane since it’s the first barrier of defense just as our skin is our first barrier against foreign compounds (part of the innate immunity). Genes involved in cell membrane are fewer compared to all genes from the genome, likewise, genes involved in membrane composition and properties are also fewer compared to all genes from the cell membrane. Thus, the selection of membrane genes involved in membrane properties and membrane lipid composition is an adequate approach for looking tolerant genes that confer of “superpowers” to organisms. There are numerous examples of this approach. One of them is the redistribution of phospholipids from the cell membrane that improved tolerance in E. coli towards several challenges (Figure 3)(4).
Figure 3.- Gram negative cell membrane. E. coli cell membrane is a gram negative. This means it contains a thin layer of peptidoglycan between the bilayer of phospholipids in addition to sugars on the outer membrane protein. The engineered strategy changed the distribution of a particular type of phospholipid (purple) in the cell membrane which promoted changes in membrane properties and tolerance.
4) A shot in the dark
This approach could be applied for basically all research questions and I’d say might be hard to know whether it’s been applied. In the context of selection of tolerant genes, I’d say this would be an approach that is not necessarily different from the other three approaches discused above, but it certainly has a much bigger component of uncertainty and unknown. This could be related to daily life events such as winning the lottery, going out for dinner at certain time and location and that someone random takes care of your check (it happens, at least in Ames IA), or simply by wearing the right shoes for an unexpected event such as a rainy/snowy afternoon after a sunny morning. This approach is indeed unexpected. It turns out that all elements were in the right place in the right time and your intuition or luck or whatnot put them together to produce something again unexpected. As an approach for selection of tolerant genes this wouldn’t be that you will try to select random genes, neither that you are selecting genes that you already have full knowledge of their functionality. For the selection of these tolerant genes utilizing a “shot in the dark” approach, it would be something in between the full certainty (fully enlightened data) and the completely random experiment (lack of structure in train of thought). Some structure in your train of thought is necessary but not necessarily previous studies that confirm it. Although this might sound very weird, it isn’t different compared to the other three approaches, the only difference is that right before performing the experiment the thought of “let’s give it a try” or “you never try, you never know” is mainly based on an analogy, an idea, an observation rather than data of previous studies. If you think about it all experiments, and all approaches within rational engineering started as a “shot in the dark” approach.
Even though a shot in the dark approach might most probably result in waste of time and resources, the benefit might be giant.
If successful, this approach might contribute with new alternatives which could be greatly appreciated by other people and impact society. An example of this approach, although it wasn’t for the discovery or picking of tolerant genes is the development of CRISPR-Cas9 as a gene editing tool invented by both Dr. Charpentier and Dr. Doudna in 2012(5). This is an example of shot in the dark approach because previous scientist discovered CRISPR arrays, but they didn’t give them use as molecular biologists and chemical engineers are doing nowadays (Figure 4).
Figure 4.- CRISPR-cas9 gene editing tool. This system is composed of a gRNA (red and purple) which contains a sequence typically called N20 (purple) which recognized the site of gene editing in the target DNA (light blue). The editing is possible because the gRNA is bound to both the target DNA and the Cas9 enzyme (boomerang-like shape structure shown). The Cas9 enzyme cuts the DNA at the site of editing. Typically another set of foreign DNA called donor DNA (not shown) is inserted in this site which produces an edited DNA.
Note: it is not necessary to invest all time looking for shot in the dark approaches, science and the use of resources must be responsible.
Even ground-breaking discoveries had previous studies that supported them.
The selection of instructions that increase tolerance against harsh conditions is necessary since all organisms even humans aren't used to live in all type of environments. The flexibility of adaptation and the way of picking up these instructions might vary but the result is beneficial. In terms of Microbiology, a characterized, and tolerant microbe is more robust thus it can be employed for producing a variety of chemicals of interest. The interest for studying tolerance and sensitivity in diverse type of cells might produce different applications across fields.
References
(1) Tan, Z.; Yoon, J. M.; Nielsen, D. R.; Shanks, J. V.; Jarboe, L. R. Membrane Engineering via Trans Unsaturated Fatty Acids Production Improves Escherichia Coli Robustness and Production of Biorenewables. Metab. Eng. 2016, 35, 105–113.
(2) Junker, F.; Ramos, J. L. Involvement of the Cis / Trans Isomerase Cti in Solvent Resistance of Pseudomonas Involvement of the Cis / Trans Isomerase Cti in Solvent Resistance of Pseudomonas Putida DOT-T1E. 1999, 181 (18), 5693–5700.
(3) Glebes, T. Y.; Sandoval, N. R.; Reeder, P. J.; Schilling, K. D.; Zhang, M.; Gill, R. T. Genome-Wide Mapping of Furfural Tolerance Genes in Escherichia Coli. PLoS One 2014, 9 (1), e87540.
(4) Tan, Z.; Khakbaz, P.; Chen, Y.; Lombardo, J.; Yoon, J. M.; Shanks, J. V.; Klauda, J. B.; Jarboe, L. R. Engineering Escherichia Coli Membrane Phospholipid Head Distribution Improves Tolerance and Production of Biorenewables. Metab. Eng. 2017.
(5) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science (80-. ). 2012.