Eggplant
2.4 Strain Development for Improved Production of Vanillin
2.4.1 Metabolic and Genetic Engineering
The characteristics property like rapid growth rates and less complexions to molecular genetics, microorganisms are innovative targets for biotechnology research and due to its ability to grow on versatile carbon sources, microorganisms are preferred so far as good candidates for the production of vanillin. Microbes such as bacteria, fungi, and yeast have been used for the small as well as large‐scale production of vanillin from various substrates, such as eugenol, isoeugenol, ferulic acid, and other organic compounds. A drawback of microbial production of vanillin is the excess oxidation and reduction of end product to vanillic acid and vanillyl alcohol, respectively as well as inhibition of microbial 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
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