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Bioremediation for Sustainable Environmental Cleanup

2022). TALENs and ZFNs can recognize one and three base pairs with modular regulatory proteins,

respectively. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas is an

adaptive immune system that protects bacteria from foreign nucleic acids. CRISPR-Cas systems

are a type of adaptive immune defence mechanism found in prokaryotes. This mechanism can be

used to create specific double-stranded DNA breaks. NHEJ or HDR oversee repairing these sites

(Sharma and Shukla 2022). CRISPR has several benefits, including high precision, convenience of

design, fast turnaround time and is inexpensive. The disadvantage of CRISPR is that it may cause

cell death in bacteria that lack the NHEJ repair system. To combat this, recombinase proteins such as

SSr and -Red are used (Caldwell and Bell 2019). For example, 2,5--furandicarboxylic acid (FDCA)

is a renewable alternative to petroleum-based terephthalic acid and is used to make polyurethanes,

polyamides and polyesters (Pham et al. 2020). As a result, FDCA is a critical component of industrial

production. A number of enzymes convert 5-hydroxymethyl furfural (HMF) to FDCA (Caldwell

and Bell 2019, Pham et al. 2020 Zhang et al. 2002, Biot-Pelletier and Martin 2014). Recombinant

P. putida S12 expressing HMF/furfural oxireductase can produce FDCA where CRISPR-Cas was

used to induce double-stranded breaks. Double-stranded breaks were then used to recombine

HMF/furfural oxidoreductase through Red-mediated recombineering. The recombinant strain was

extremely efficient, converting approximately 88–85% of HMF to FDCA and frequent one-step

stable gene integration was possible into all chromosomes of polyploid P. putida S12. As a result,

the most stable strain of P. putida S12 was produced for the synthesis of FDCA. Although the

stable integration of genes and efficient degradation was observed in this recombinant strain, higher

concentration HMF (100 mM) was affecting the conversion. Initial increased density of biomass

rescues the inhibition with increased conversion 100 mM and 150 mM HMF to 96 to 75% of FDCA

(Hsu et al. 2020).

Genome shuffling is another method usually used for strain improvement at the industrial level.

It is a combination of DNA shuffling and directed evolution used to improve strains (Zhang et al.

2002). DNA shuffling has successfully achieved directed molecular evolution of several genes and

pathways (Biot-Pelletier and Martin 2014). For genome shuffling, the protoplast fusion method is

employed where the fusion of two cells with different genetic traits achieves the desired modified

phenotype. Even intergenic hybridization between Aspergillus niger and Penicillium digitatum

results in increased verbenol production (Rao et al. 2003). The consequence of protoplast fusion is

a library of strains with accumulated mutations. These strains can now be screened for phenotypes

of interest. As a result, it is a very useful tool, and more hybrid strains can be obtained than through

protoplast fusion as more parents are involved and saves a lot of assays and time (Zhang et al. 2002).

Pseudomonas are the gram-negative saprophytic in most of the soil dwelling bacteria. One

of the most important features of Pseudomonas sps., is degradation of almost all type of carbon

skeletons. The Recombinant DNA Advisory Committee has approved Pseudomonas putida as one

of the popular bacteria for genetic engineering and certified it as a biosafe host for heterologous gene

expression (Nelson et al. 2002). They exhibit rapid and robust growth. P. putida is resistant to very

high concentrations of toxic especially xenobiotic compounds due to solvent resistance mechanisms

mediated by the interaction of cellular factors (Ramos et al. 2002). Some examples include fine-

tuning lipid fluidity to adjust membrane functions, activating stress response systems and inducing

efflux pumps. P. putida is also metabolically extremely adaptable. It has a ED/EMP cycle, a distinct

metabolic pathway that employs enzymes from the Entner-Doudoroff (ED), pentose phosphate

and Embden-Meyerhof-Parnas (EMP) pathways (Nikel et al. 2015). Due to the availability of the

ED/EMP cycle, it is now possible to build completely different biochemistries using new

methodologies. In this chapter, the use of genetically modified Pseudomonas sp. especially P. putida

for compound synthesis and degradation to aid in pollution control will be described.