Leaders in Pharmaceutical Business Intelligence (LPBI) Group

From Genomics of Microorganisms to Translational Medicine

Reporter and Curator: Demet Sag, PhD

Pharmacogenomics needs new materials that are inert against the host and specifically  active to modulate molecular metabolism towards wanted homeostasis of the physiological system.  These can come from natural resources or men-made.  That is why we must know the origin  to  improve.     Recently, Synthetic Biology, even though it is a developing upcoming field, it is generating mile stones for applications in the clinic, the biotechnology industry and in basic molecular research. As  a result, it created a multidisciplinary expertise from scientists to engineers.  Among other things extending the search to first life on Earth is one of the many alternatives.  Here I like to present how synthetic biology can be initiated onto Translational Medicine from adiscovery of molecules from the sea.

Microorganisms played a role in evolution to start a life.  99 % of our genome is related to microbial organisms. initially there was a classical  Microbiology, then evolved to Industrial Microbiology and Biotechnology then Microbial Genomics and now Microbiome and Health became the focus.  Finally,  the circle is getting tide into how microbiome involved with healthy and disease state of human? How they can be used that is what it really means to include microorganisms into human health for diagnostics and targeted therapies?

Or should we start from  scarcity?

Microbiology is my first formal education and  building block.  Simple but help to understand system biology and  the mechanism of life in a nut shell.   The closest field is embryonic stem cell biology for building “synthesizing” a whole new organism.  Then  system biology and developmental biology also gain interest.

The real  remember the month of October in 2001 when DOE reported that they sequenced 23 organisms in Walnut Creek.  Having seen presentation to identify microorganisms through complex crystal structure assays through chemical pathway  at the Microbial Genomics Meeting organized by ASM in Monterey, CA in 2001.

Discovery of microorganisms in marine life like in Mediterranean Sea, containing 38% salt,is very similar with finding circulating disease making cells.   Yet, they are similar since both search for a specific needle in the pile.  Furthermore, the unique behavior of enzymes from microbial organisms such as Taq polymerase or restriction enzymes made it possible for us to develop new technologies for copying and propagating significant sequences.  When these early molecular biology methods are combined with the power of genomics and knowledge of unique structures in molecular physiology, it is possible to design better and sensitive sensors or build an organism to rptect or fix the need of the body.  neither sensors nor synthesized organism model are complete since one is missing the basic element of life “transformation of information” the other is missing the integrity that once nature provided in a single simple cell.

Having sensory smart chip/band/nanomolecule to redesign the cells may also possible if only we know the combination.  Thus, we have options to deliver if we know what to be carried.

(Figure: The combined strategy of gene-based screening and bioactivity-based screening for marine microbial natural products (MMNPs) discovery, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3705366/figure/marinedrugs-11-00700-f002/)

As we come across, novel pathways or primary pathways of physiology gain significant interest to determine marine microbial compound for therapeutics since they are further away from the evolution three that gives an advantage for biomedical/translational scientist to avoid most part of th eimmune responses such as inflammation, toxicity. Yes, indeed these are not scientific tails but true since currently, 16 of 20 marine antitumor compounds under clinical trial are derived from microbial sources because marine microorganisms are a major source for MMNP discovery.  However, isolation of these organisms.  For example, pretreatment methods, enrichment, physical, and chemical techniques (e.g., dry heat, exposure to 1%–1.5% phenol, sucrose-gradient centrifugation, and filtration through cellulose membrane filters) can be applied to increase especially the less abundant specific groups of marine microorganisms, . A variety of pretreatment methods including recovery of these microorganisms.  This reminds me ecosystem of the soil, since in soil the trouble is identifying the specific culture among millions of others.

Regardless of the case,  nutrients are the key for selecting and isolating any organisms but specifically, as a result any marine microbes have specific nutrient requirements for growth (e.g., sponge extract ) or chemical (e.g., siderophores, signal molecules, non-traditional electron donors, and electron acceptors.  This also should remind us subject of Biology 101 Essential Vitamins and Minerals.  What we eat who we are.

For example, Bruns et al. employed technique where they employed different carbon substrates (agarose, starch, laminarin, xylan, chitin, and glucose) at low concentrations (200 μM each) so that they can  improve the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea. As a result of this growth medium they were able to elevate yield, which is created higher cultivation efficiencies (up to 11% in fluid media) compared to other studies.

Yet, another component must be addressed that is culture medium such as ionic strength for a microbila growth. For example, Tsueng et al. study on marine actinomycete genus Salinispora that can produce bioactive secondary metabolites such as desferrioxamine, saliniketals, arenamides, arenimycin and salinosporamide.  However, they observed that  three species of Salinispora, S. arenicola, S. tropica, and S. pacifica require a high ionic strength but  S. arenicolahas a lower growth requirement for ionic strength than S. tropica and S. pacificaUsing after assaying them against sodium chloride-based and lithium chloride-based media. As  aresult, there is a specificity for growth. 

In addition, energy must be supported imagine that in marine organisms the metabolism is very unique, may be slow and possibly.  However, the main criteria is  most of them grow under low oxygen conditions like tumors.  Warburg effect posed a  problem for human but helped microorganisms to survive and evolve.  One’s weakness the other’s strength make a great teamwork for solving diseases of human kind es especially for cancer. 

This reminds us to utilize minerals, electrons specifically after all the simplest form of carbon metabolism based on biochemical pathways like Crebs cycle, one carbon metabolism and amino acid metabolism etc. Even though 90% of human body made up off microbial origin there are microorganisms that are not cultured yet.

The irony is less than 1% of microorganisms can be cultured.  Furthermore, they are not included for representing the total phylogenetic diversity. Therefore, majority of work concentrated on finding and cultivating the uncultured majority of the microbial world for MMNPs’.  For example,  an uncultivated bacterial symbiont of the marine sponge Theonella swinhoei  producing many antitumor compounds such as pederin, mycalamide A, and onnamide A.

In any conditions if any living needs to be recognized and remembered, their place would be either on top or the bottom of the stack. Microbiome searches for specificity among tone of other organisms to recognize the disease, changes in cell differentiation and pathways or marine microbiologist search for uncommon scarce organisms. Yet, both of them are beneficial with their unique way.

Then what is the catch or fuss?  The catch is screening to identify what makes this organism unique that can be use for human health. Translational medicine may start from the beginning of life from microorganisms created.  This can be called with its newly coined named”synthetic biology” but if we go further than the conventional screening methods which include bioactivity-guided screening and gene-guided screening  and increase the power with genomics we may call it “synthetic genomics”.

As  a result these signature sequences establishes the “unique” biomarkers  or therpaeutics to be used for drug discovery, making vaccines, and remodulating the targeted cells. How?

These microorganisms secrete these metabolites or proteins to their growth medium just like a soluble protein, if you will like a inflammation factor or any other secreted protein of our human body cells. Collecting substrate or extract the pellet could be the choice.   in a nut shell this require at least three steps: First, finding the bioactivity, apply bioactivity-guided screening for direct detection of  the activity such as antimicrobial, antitumor, antiviral, and antiparasitic activities.  Second, a bioinformatic assessment of the secondary metabolite biosynthetic potential in the absence of fully assembled pathways or genome sequences. Third, application on cell lines and possible onto model organisms can improve the process of MMNP discovery so that allow us to prioritize strains for fermentation studies and chemical analysis. 

In summary, establish the culture growth, analyze bioactivity and discover the new gene product to be used.  Here is an example, first they  isolated Marinispora sp from the saline culture.  Next step,  identify new sources of bioactive secondary metabolites, gene-guided screening has been deployed to search target genes associated with NPs biosynthetic pathways, e.g., the fragments between ketosynthase and methylmalonyl-CoA transferase of polyketides (PKS) type I, enediyne PKS ketosynthase gene, O-methyltransferase gene, P450 monooxygenase gene, polyether epoxidase gene, 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene, dTDP-glucose-4,6-dehydratase (dTGD) gene, and halogenase gene. The, apply bioinformatics that includes synthesizing the knowledge with  homology-based searches and phylogenetic analyses, gene-based screening  to predict new secondary metabolites discovered by isolates or environments.  Finally, identify the sequnce for PCR and use against a cell line or model organisms. In this case,  CNQ-140 based on significant antibacterial activities  against drug-resistant pathogens (e.g., MRSA) and impressive and selective cancer cell cytotoxicities (0.2–2.7 μM of MIC50 values) against six melanoma cell lines provided significant outcome. They are recognized as antitumor antibiotics with a new structural class, marinomycins A–D

This is a great method but there are two botle necks: 1. 99% of microbial organisms are not cultured in the labs. 2. Finding the optimum microbial growth and screening takes time. Thus, assesments can me done through metagenomics.  However, metagenomics has its shortcomings since on face of living unless applications applied in vivo in vitro results may not be valid.  The disadvantage of  metagenomics can be listed as:  1. inability of efficient acquisition of intact gene fragment,  2. incompatibility of expression elements such as promoter in a heterologous host.  On the pther hand, there can be possible resolution to avoid these factors  so metagenomics-based MMNP discovery can be plausable such as development  in  synthetic biology by large DNA fragment assembly techniques for artificial genome synthesis and synthetic microbial chassis suitable for different classes of MMNP biosynthesis.

However, many gene clusters have been identified by combined power of genomics and biioinformatics for MNP discovery.  This is  mainly necessary since  secondary metabolites usually biosynthesized by large multifunctional synthases that acts in a sequential assembly lines like adding carboxylic acid and amino acid building blocks into their products.  

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