## 7 Synthetic Biology Methodologies to Enhance Biofuel Yields
### Clustered Regularly Interspaced Short Palindromic Repeats/Cas9  
This is an advanced, adaptable molecular tool that has gained importance in recent times for its efficient and rapid engineering of genomes related to different organisms. This technique employs a Cas9 endonuclease to cleave the target site, and the target site is accurately reached with the help of a guide RNA that has \(-20\) nucleotides attached to Cas9. Ever since the inception of this approach, it has been used in various organisms and for numerous applications [156; 157]. Genome engineering of the host can be performed by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated cleavage of the double strand, either with homology-directed repair or with non-homologous end joining. [PERSON] et al. and [PERSON] et al. have used the CRISPR/Cas9 method for engineering _E. coli_ to adjust the production of n-butanol and \(\beta\)-carotene, respectively, in the fed-batch fermentation [158; 159]. [PERSON] et al. have used the same approach but in a multiplexed fashion to engineer _Clostridium tyrobutyticum_ in batch fermentation for regulating the n-butanol pathway, yielding huge n-butanol produce (26.2 g/L) [160]. Likewise in _S. cerevisiae_, this approach was used to knockout the contending pathway genes and introduce heterologous genes in required loci to enhance the production of fatty alcohols [11]. Not just a single locus, but multiple loci can be targeted with different genes to be inserted or deleted (multiplex CRISPR/Cas9) using this approach [161; 162]. In a study by [PERSON] et al., CRISPR/Cas9 was multiplexed in _S. cerevisiae_ with five targets, and it ended up with a 42-fold increase in its production of mevalonate. Similarly, [PERSON] et al. have used CRISPR/Cas9 multiplexing in yeast genome to introduce 2,3-butanoli as well as xylose consumption pathways, resulting in the utilization of xylose and production of 2,3-butanoli [162]. 2-phenylethanol is an aromatic compound that is widely used in personal care products, foods, and beverages. It can be blended with gasoline and can be used as a biofuel additive. It was reported by [PERSON] et al. that engineering Ehrlich as well as Shikimate pathways using the CRISPR-mediated multigene integration system in _Kluyveromyces marianus_ improved stress tolerance and 2-phenylethanol production via the Shikimate pathway [163].  
The rewiring leads to the creation of assorted pathways in the host that in turn create a new network of metabolites. This produces desired characters by streaming the cellular resources towards the target production rather than the cell's growth. This approach has been effectively applied to produce many biofuel precursor compounds like isoprene [164], acetone [165], fatty acids [166], 1-butanol [167], ethylene [168], and 2-methyl-1-butanol [169]. All the above-mentioned studies depict the importance of artificial metabolic pathways for achieving the enhancement of biofuel production. Recently computational resources and algorithms have also been developed like the Pathway Prediction System [170], Retro-Biosynthesis Tool [171], and Biochemical Network Integrated Computational Explorer algorithm [172; 173].