**5. Genetic engineering and synthetic biology of** *E. coli*

With the avenue of *in vitro* DNA synthesis to generate larger fragments with increased fidelity along with novel assembly methods, we are now capable of generating large and custommade DNA molecules with the desired properties or even without the source of a DNA sample 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 synthetic biology.

Several aromatic compounds have been successfully synthesized in *E. coli* due to their biological activities (vitamins and antioxidants, for example), pigmentation (applied in different industrial processes), and fragrance etc. [93]. In the case of the perfume industry, the synthesis of other relevant compounds such as precursors for Ambrox, a highly appreciated odorant for the perfume industry, or the synthesis of Geraniol, a valuable acyclic monoterpene alcohol, is also used in the perfume industry and pharmaceutical

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Another relevant area was *E. coli* stepping in to biofuel production. The twentieth century is characterized by the human dependence on fossil fuels. They participate in a myriad of processes, and the demand is increasing. In order to alleviate the demand, scientists have turned to the development of novel technologies for biofuel production by the conversion of carbon sources into usable fuel. There are several reports where *E. coli* have been successfully engineered for the synthesis of branched-chain fatty acids or short-chain fatty acids that can ultimately lead to the mass production of fuel precursors or useful materials derived from oil [96–98]. Perhaps, the most promising future is a fully replicated fossil fuel, i.e., a mixture structurally and chemically identical to the fossil fuels that are currently under use, which is a mixture of aliphatic n- and iso-alkanes of various chain lengths [99]. Also, a more complete metabolic atlas of *E. coli* is needed, and recent efforts have mapped the metabolic flux from this

Another important field where *E. coli* is making an important contribution powered by synthetic biology is the antibiotic production. From the biomedical standing point of view, increasing antibiotic resistance in pathogens is a burden for humankind, and the discovery of novel compounds is a time-consuming task. Recent efforts with known polyketides have started to give good production rates in *E. coli*, favoring the process of antibiotic production from different sources, eliminating the need for host growth standardization, and inducing

Due to the modularity of polyketide synthases, they are excellent candidates for engineering, for either the production of novel compounds from existing gene clusters (through module shuffling, mutagenesis or deletions), or by the introduction of novel environmentally

Therefore, the production of diverse metabolites or biological or industrially relevant molecules can be successfully achieved in *E. coli*. Success stories have been published more frequently. With the advancement of high-throughput technologies, more sensitive detection,

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

and analytical tools, and better DNA synthesis, the future for *E. coli* is brilliant.

sequenced gene clusters and heterologous production [102, 103]

applications [94, 95].

bacterium further [100].

conditions [101].

**6. Future**

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

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 development of designed microorganism that can do desired tasks.

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].

Several aromatic compounds have been successfully synthesized in *E. coli* due to their biological activities (vitamins and antioxidants, for example), pigmentation (applied in different industrial processes), and fragrance etc. [93]. In the case of the perfume industry, the synthesis of other relevant compounds such as precursors for Ambrox, a highly appreciated odorant for the perfume industry, or the synthesis of Geraniol, a valuable acyclic monoterpene alcohol, is also used in the perfume industry and pharmaceutical applications [94, 95].

Another relevant area was *E. coli* stepping in to biofuel production. The twentieth century is characterized by the human dependence on fossil fuels. They participate in a myriad of processes, and the demand is increasing. In order to alleviate the demand, scientists have turned to the development of novel technologies for biofuel production by the conversion of carbon sources into usable fuel. There are several reports where *E. coli* have been successfully engineered for the synthesis of branched-chain fatty acids or short-chain fatty acids that can ultimately lead to the mass production of fuel precursors or useful materials derived from oil [96–98]. Perhaps, the most promising future is a fully replicated fossil fuel, i.e., a mixture structurally and chemically identical to the fossil fuels that are currently under use, which is a mixture of aliphatic n- and iso-alkanes of various chain lengths [99]. Also, a more complete metabolic atlas of *E. coli* is needed, and recent efforts have mapped the metabolic flux from this bacterium further [100].

Another important field where *E. coli* is making an important contribution powered by synthetic biology is the antibiotic production. From the biomedical standing point of view, increasing antibiotic resistance in pathogens is a burden for humankind, and the discovery of novel compounds is a time-consuming task. Recent efforts with known polyketides have started to give good production rates in *E. coli*, favoring the process of antibiotic production from different sources, eliminating the need for host growth standardization, and inducing conditions [101].

Due to the modularity of polyketide synthases, they are excellent candidates for engineering, for either the production of novel compounds from existing gene clusters (through module shuffling, mutagenesis or deletions), or by the introduction of novel environmentally sequenced gene clusters and heterologous production [102, 103]

Therefore, the production of diverse metabolites or biological or industrially relevant molecules can be successfully achieved in *E. coli*. Success stories have been published more frequently. With the advancement of high-throughput technologies, more sensitive detection, and analytical tools, and better DNA synthesis, the future for *E. coli* is brilliant.
