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growth by the product vanillin due to toxicity at high concentrations. Due to these major issues, there is a significant decrease in the vanillin yield (Stentelaire et al., 1998). To prevent these bottlenecks and hence to increase the vanillin yield, metabolic engineering and genetic manipulation were applied so far. Table 2.3 enlists several genetically engineered microorganisms, which were used to enhance the vanillin yield from various substrates. Genetic engineering or genetic modification (GM) is a direct manipulation or modification of the organism's genome using recombinant DNA technology. Due to its high production rate, the vanillin production of GM organisms has gained much attention (Barghini et al., 2007; Ni et al., 2015). The metabolic pathway of eugenol was investigated in many species of bacteria and fungi such as Pseudomonas, Byssochlamys, Penicillium, and Rhodococcus. To improve the yield of vanillin, genetic engineering was introduced and in this context many attempts have been made so far. For example, Overhage et al., 1999 constructed a mutant Pseudomonas sp. strain HR199 by the insertion of a regulatory element (omega element) into the normal vanillin dehydrogenase gene, which was found to accumulate significantly higher concentrations of vanillin (2.9 mM; 0.44 g/l). Rhodococci are well known for their metabolic versatility in utilizing various xenobiotic compounds, and thus they are extensively used in wide range of biotransformation processes. Plaggenborg et al. (2006) investigated the potential of Rhodococcus sp. for the biotechnological production of vanillin from eugenol by overexpression of vaoA gene corresponding to VAO from P. simplicissimum CBS 170.90 for the hydrolysis of eugenol to coniferyl alcohol along with calA and calB genes corresponding to dehydrogenases of coniferyl alcohol and coniferyl aldehyde from Pseudomonas sp. HR199, respectively. The transformant efficiently catalyzed conversion of eugenol to vanillin at a rate of 1.22 mmol/h/l of culture. Likewise, vanillyl alcohol oxidase gene (vaoA) from P. simplicissimum CBS170.90 has been expressed in Amycolatopsis sp. HR167, which efficiently converted eugenol to coniferyl alcohol with a maximum yield of 4.7 g/1 after 16 hour biotransformation (Overhage et al., 2006). Such innovative idea of metabolic engineering for vanillin production has been further evaluated with the recombinant strains of Ralstonia eutropha H16 (Overhage et al., 2002), E. coli XL1‐Blue (Overhage et al., 2003) and it is found to have significant increase in vanillin yield. It has been reported previously that isoeugenol is one of the ideal substrate candidates for metabolized into vanillin through an epoxide‐diol pathway involving oxidation of side chains of propenylbenzenes. The biotransformation products of isoeugenol are higher than those obtained from eugenol. Later, it is also evaluated that the production of vanillin using metabolically engineered E. coli cells, where isoeugenol monooxygenase of P. putida IE27 is overexpressed that produced 28.3 g/l of vanillin from 230 mM of isoeugenol with a molar conversion efficiency of 81%. It also has been reported that no or less accumulation of unwanted byproducts, such as vanillic acid or acetaldehyde, are observed during this conversion (Yamada et al., 2008). Tang et al. (2018) has generated a recombinant E. coli expressing a 9‐cis‐epoxycarotenoid dioxygenase gene from Serratia sp. (SeNCED), which is known for catalyzing the cleavage of isoeugenol and 4‐vinylguaiacol to vanillin. The recombinant E. coli produced 0.53 g/l of vanillin (3.47 mM) in eight‐hour reaction by utilizing isoeugenol (4 mM) as substrate.

Table 2.3 Genetically modified (GM) microorganisms for synthesis of biovanillin.

Substrate	Microorganism	Yield (g/l)	References
Eugenol	Pseudomonas sp. HR199	0.44	Overhage et al. (1999)
	Two‐step process: E. coli XL1‐Blue and E. coli (pSKechE/Hfcs)	0.3	Overhage et al. (2003)
Isoeugenol	Pseudomonas putida IE27	16.1	Yamada et al. (2007)
	E. coli BL21(DE3)	28.3	Yamada et al. (2008)
Ferulic acid	E. coli strain JM109/pBB1	0.851 mol/l	Torre et al. (2004)
	E. coli strain JM109/pBB1	2.52	Barghini et al. (2007)
	E. coli XL1‐Blue (pSkechE/Hfcs)	Trace amount	Overhage et al. (2000)
	E. coli (pDAHEF)	0.58	Yoon et al. (2005a)
	Recombinant E. coli	1.1	Yoon et al. (2005b)
	E. coli DH5α (pTAHEF)	1.0	Yoon et al. (2007)
	E. coli (pTBE‐FP)	2.1	Song et al. (2009)
	E. coli NTG‐VR1	2.9	Yoon et al. (2007)
	E. coli DH5α (pTAHEF‐gltA)	1.98	Lee et al. (2009)
	E. coli BW25113 (pTAHEF)	5.14	Lee et al. (2009)

Ferulic acid is well known as a potential substrate for bioconversion to vanillin, which is available abundantly in the natural sources. Ferulic acid is converted to vanillin by the catalytic activity of feruloyl‐CoA synthetase (fcs) and enoyl‐CoA hydratase/aldolase (ech). Yoon et al. (2005a) generated a recombinant E. coli expressing with fcs gene of Amycolatopsis sp. HR104 and ech gene of Delftia acidovorans under the control of P_BAD promoter that produced the highest yield of vanillin (580 mg/l) under the optimized conditions in LB medium, which is 3.6‐folds higher than the yield obtained before media optimization. Meantime, another E. coli genetically engineered to express fcs gene of Amycolatopsis sp. HR104 and ech gene of D. acidovorans under the control of IPTG‐inducible trc promoter in complex medium (2YT) supplemented with ferulic acid (0.2% w/v) has produced highest amount of vanillin (1.1 g/l) (Yoon et al., 2005b). Improvement of the vanillin production has been investigated further by Yoon et al., 2007 by constructing recombinant E. coli carrying plasmid pTAHEF cloned with both fcs and ech genes of Amycolatopsis sp. and tested with ferulic acid (2.0 g/l) that produced a maximum of 1.0 g/l vanillin in 48 hours. In due course, bacterial growth and vanillin yield decreased due to vanillin toxicity. In order to avoid vanillin toxicity, generation of NTG mutant and removal of excess vanillin from the culture medium using polystyrene resin XAD‐2 has been followed. The vanillin production by NTG mutants has increased three times higher when compared with its wild‐type strain. Following

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