**6. Future**

having only the sequence itself. New biological parts (genes, promoter sequence, terminators, etc.), devices (gene networks), and modules (biosynthetic pathways) are only limited by our imagination and the drive to create them. Also, without the advancement of methods for analyzing large amounts of data, bioinformatics, codon optimization software, genome mining, and user-friendly databases, synthetic biology creations are permeating in many laboratories around the world. With this in mind, we will review the current technologies for synthetic genes and genomes, and how this technology can be applied in generating novel regulatory circuits and even whole genomes. In this regard, *E. coli* is the key organism for such endeavors. Why? Well, in this section, we provide some examples that we consider may be helpful in the future of mankind and are in our opinion relevant in the field of genetic engineering and

264 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Synthetic biology is a relatively new branch of molecular genetics that incorporate engineering principles for modifying several aspects of cell physiology, rewiring genetic circuits, creating novel circuits, and synthesizing custom-made DNA sequences and even genomes [87]. This particular branch of biology needs to be supported by an extensive knowledge of the organism that modifications or even whole genome synthesis is attempted, several novel tools for analyzing big datasets and molecular tools for that particular organism, for the generation of sequences and the computational design of DNA molecules, and a goal that can be achieved with the desired organism. *E. coli* along with *Saccharomyces cerevisiae* are the most studied and well-comprehended organisms in science, and diverse phenotypes have been

Multiplexing is the novel approach for redesigning organisms to do desired tasks [90]. By cycling through design-build–test framework we can achieve novel features in existing proteins and can further the advancement of genetic engineering. Thus far the most complicated and time-consuming part of this framework is the testing of the novel designs. Highthroughput approaches have led to the development of fast and reliable screening methods and advances in this area, such as designed biosensors for the screening of metabolite producing strains or high-throughput methods for product screening, where the use of fluorescent proteins, colorimetric assays, and mass spectrometry are cornerstones for the development of screening methods for assessing success in strain engineering [90, 91]. DNA sequencing and synthesis coupled with good screening methods is the platform for future tools for the devel-

With all the technologies available, the advancement of using *E. coli* for biotechnological applications based on synthetic approaches have led to the development of strains capable of synthesizing several novel compounds. In the following lines, we provide some examples that we find important for improving environmental conditions and human well-being.

Synthesis of important metabolites can be difficult, and researchers must face the stubbornness of microorganisms to redirect carbon flux to their own processes, rendering the production of relevant molecules costly and inefficient. But *E. coli* is a flexible platform for the efficient production of molecules for the pharmaceutical industry, metabolites and molecules relevant for food additives, pigments, and more recently complex aliphatic molecules [92].

synthetic biology.

identified that are helpful for bioengineering [88, 89].

opment of designed microorganism that can do desired tasks.

With all the research that is currently conducted in *E. coli*, we envision that the future for this versatile microbe is bright. Gained knowledge on *E. coli* is overwhelming, nearly 340,000 research papers with the "*Escherichia coli*" keyword available in Pubmed, versus 115,000 using "*Saccharomyces cerevisiae*" as a keyword for an example. Several genomes from environmental sources have been sequenced. Thousands of research papers aimed to assess the metabolic and genetic potential of this organism have been published. Now we need to start joining forces for boosting the true potential of this wonder microbe.

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With all the data available, we are on the verge of really important findings and novel biotechnological procedures using *E. coli*. Recently, with an extensive analysis of human microbiota, we are approaching exciting times where all the knowledge can be applied to help people during certain diseases related to colon microbiota imbalances [104]. Findings suggest that microbiome can be manipulated to improve certain metabolic pathways [105].

In the rest of the biotechnology fields, we have enough information that suggests *E. coli* will be in the spotlight for quite some time. Even with novel organisms that have been proposed to substitute it, like *Vibrio natriegens*. Recently, Lee and collaborators analyzed the genome sequence and growth properties of the fast-growing bacterium *V. natriegens* [106], where authors created a novel platform for genetic engineering in one of the fastest growing bacteria. In previous reports, it has been shown that *V. natriegens* are capable of being transformed with *E. coli* plasmids [107], rendering that all the molecular tools available thus far may be compatible with this new system. This bacterium as *E. coli* renders only one important drawback: it may be pathogenic to oysters [108]. But as with old *E. coli*, we should be careful when managing genetically modified organisms. With recent efforts to generate lab-contained organisms, *E. coli* mutants that can only survive in the presence of synthetic amino acids have been created [109]. Despite all the molecular tools developed so far for *E. coli*, the future is still open for a novel molecular toolbox. Until a novel toolbox is standardized and incorporated into the everyday life of the laboratories around the world, we will keep exploiting the capabilities of *E. coli*, one wonderful bug.
