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The highly diverse chemical structures of Natural Products isolated from microbes or derived semisynthetically from natural intermediates allowed the development of a broad range of different drug activities, including antibiotics and chemotherapeutics.

Genome sequencing data facilitated by the development of Next Generation sequencing platforms indicate that microbial genomes contain an untapped resource of biosynthetic gene cluster that can be exploited to generate novel functions.

Unfortunately, most of these gene clusters are not expressed under normal laboratory growth conditions even when it is possible to grow the natural host in lab environment. In addition, the size of the Biosynthetic gene clusters (reaching up to 200kb) renders the in vitro manipulation of this large clusters difficult (Smanski et al. 2016).

Recombineering or Recombineering‐derived strategies have therefore been an ideal method to characterize and to engineer long gene clusters. In fact, specific gene clusters can be inserted in an heterologous host to facilitate the genetic manipulation of the genes present in these clusters.

An alternative strategy would be the use of endogenous recombineering systems from different hosts to manipulate the particular genes present in the gene cluster (Yin et al. 2015).

Recombineering is rapidly becoming the method of choice to manipulate biosynthetic gene clusters but it is also increasingly used to evolve the bacterial genome as pioneered by the work of Church´s group. Hang HH, Isaacs FJ et al. in their landmark paper of 2019 described the use of recombineering to accelerate bacterial genome evolution by an automated multiplex recombineering strategy that they named MAGE (Wang et al. 2009). MAGE is using a pool of ssDNA oligonucleotide coupled with the expression of a ssDNA‐binding protein to install thousands of functionalized genome variants in E. coli genome. MAGE has been recently applied to lower Eukaryotes such as Saccharomyces cerevisiae (Si et al. 2017).

One of the problems associated with high‐throughput Recombineering is the need to select for the functional variant because there is usually no selective advantage for the recombined bacterial cell. To overcome this limitation, several strategies have been developed to combine the precision and efficiency of Recombineering with the strong selection pressure that occurs in bacteria after the generation of DSB caused by endonucleases like Cas9/CRISPR (Jiang et al. 2013; Jiang et al. 2015; Baker et al. 2016). Despite the large success of Recombineering applied to E. coli genetic engineering, the engineering of large biosynthetic pathways is still inefficient in endogenous hosts where a Recombineering system is not efficient or where Homologous Recombination is not efficient. Therefore, alternative strategies need to be developed to overcome this limitation. One possibility would be to exploit alternative pathways (Su et al. 2016) of DNA recombination/repair such as non‐homologous end joining (NHEJ) as previously done in mammalian cells (Maresca et al. 2013). A more promising approach relies on the recently described integration system by Transposon‐encoded CRISPR‐Cas (Klompe et al. 2019; Strecker et al. 2020). These strategies can facilitate genome engineering of bacteria and can possibly be implemented for the genetic manipulation of Eukaryotic genomes that are less prone to homologous recombination as described in the next section.