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Microorganisms found in nature may have some restriction on degradation of substrate and

synthesis of a product. Such microorganism especially bacteria can be improved by making a

change in the genes using different tools available today. Furthermore, utilization of constitutive

promoter libraries can modulate gene expression. They have a wide range of expression levels and

can control multiple genes at the same time (Alper et al. 2005b, Jensen and Hammer 1998). This

kind of system also eliminates the need for inducible systems. It is extremely useful in large-scale

product production. Secondary structures of mRNA, RNase cleavage sites and RBS sequestering

sequences are the post-transcription modulators and control the expression of multiple genes under

the control of single promotor. Tunable Intergenic Regions (TIGR) placed between coding regions

aid in post-transcriptional manipulation of phenotype expression (Pfleger et al. 2006).

8.2 Techniques for strain Improvement

Microorganisms with/without desired characters can be improved by using various methods

modulating sequence in the genome by adding or removing the nucleotides. Such changes can

be made using evolutionary engineering and genetic engineering. The concept of evolutionary

engineering is based on the idea that organisms naturally evolve and undergo mutations in order

to do so (Sauer 2001). As the frequency of mutation is very low in evolutionary engineering,

mutagenic agents, physical or chemical can cause changes in an organism’s DNA. This includes

the use of nitrosoguanidine, ultraviolet light, etc., to introduce point mutations and increase the

population’s variation (Adrio and Demain 2006). Site directed mutation is carried out due to prior

knowledge of functioning of the gene. Required mutation can be implemented using different

methods revolving around thermocycler mediated polymerizing reactions. Using recombinant DNA

technology, random or site-specific genetic variations can be introduced into the microorganism.

This can be accomplished through transposon mutagenesis. Transposition is the process of moving a

chromosomal segment from one location to another. For example, the incorporation of an antibiotic

marker sequence into the organism’s genome via transposon (Alper et al. 2005a). Tn5 transposon

is used for random chromosomal integration, and Tn7 transposon is used for targeted chromosomal

integration, homologous recombination (RecET-mediated recombination can be used for greater

efficiency), and Cre/loxO integration (Aoyama et al. 2005). Pseudomonas putida KT2440 rDNA

is a favourable locus for biosynthetic gene cluster expression (Bojanovič et al. 2017). Antisense

RNA inhibition is another approach (antisense cDNA is used to downregulate the gene expression)

to control the expression of a particular gene (Bojanovič et al. 2017). In some cases, instead of

down regulating the gene expression, overexpression is necessary. Gene overexpression can be

accomplished by inserting the expression vector plasmid into the organism. In plasmid vectors,

single or multiple biosynthesis genes or operons are introduced (Aoyama et al. 2005).

Site specific recombination can generate a scar after deletion mutation in the form of FRT

(Flp/Flp recombination target) or lox sites. Thus, the recognition sites recombine in the chromosome

generating deletion or inversion mutations. Furthermore, the selection method fails to select

resistance cassette deleted mutants. A counter-selective marker is a method of genetic modification

that does not use markers. This technique can be used to positively select double recombinants for

plasmid excision. Counter-selective marker systems employ sucrose, fusaric acid and streptomycin

as substrates for mutant selection (Reyrat et al. 1998, Graf and Altenbuchner 2011). A novel

counter-selective marker system is the upp gene, which codes for UPRTase from Bacillus subtilis,

Enterococcus faecalis and other bacteria. UPRTase converts 5-flurouracil to 5-fluro-UMP, which

is then converted to 5-fluro-dUMP. The latter is harmful to cells. This is particularly useful for

the deletion or insertion of large DNA fragments. Meganucleases, Zinc Finger Nucleases and

Transcription activator-like effector nucleases are homing endonucleases that recognize and cleave

longer DNA sequences (12 to 40 base pairs). ZFNs and TALENs are fusion proteins made up of

a DNA binding domain engineered by scientists and a nonspecific nuclease (Sharma and Shukla