# D-Limonene: Promising and Sustainable Natural Bioactive Compound
## 2 Production of D-Limonene
### Biotransformation: A New Direction for Future Development  
Biotransformation to high-value natural products is a direction to explore as an alternative to extraction from natural plants, usually using metabolic engineering and biosynthetic engineering to process microorganisms for sustainable production [59]. For example, using the methylerythtold 4-phosphate pathway or the mevalonate-dependent pathway [60], the introduction of a specific gene fragment into the test bacterium and induced expression for the synthesis of D-limonene resulted in D-limonene production up to 0.15 g/(L-h) under ideal conditions [61]. The yield of D-limonene obtained by metabolic engineering depends on the strain, the type of exogenous gene introduced, the incubation temperature, and other factors [29, 62, 63]. In addition to the methylerythtitol 4-phosphate pathway and mevalonate-dependent pathways, the isopentenol utilization pathway and the isoprenoid alcohol pathway have also been used to produce D-limonene, with reported maximum yields of approximately 0.7 ug/(L-h) and 10.42 ug/(L-h), respectively [64, 65, 66, 59]. Alternatively, the alcohol-dependent hemiterpene pathway has been suggested as a promising pathway for terpene production;  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Plant (Essential Oil)** & **Parts** & **Composition (\%)** & **Ref.** \\ \hline _Citrus \(\times\) sinensis_ & Fruit peels & 98.54 & [45] \\ _Citrus \(\times\) aurantium_ & Fruit peels & 93.70 & [46] \\ _Citrus reticulata_ & Fruit peels & 91.65 & [45] \\ _Citrus delicosa_ & Fruit peels & 91.27 & [45] \\ _Tetraudin damilli_ & Leaves, Fruits & 72.71 & [47] \\ _Hyprtis laq_ & Leaves, Fine stems & 72.60 & [48] \\ _Cymbogenic citratus_ & - & 71.00 & [49] \\ _Citrus unxilin_ & Fruit peels & 70.22 & [50] \\ _Citrus maxima_ & Fruit peels & 67.58 & [51] \\ _Citrus \(\times\) limon_ & Dry leaves, Fruit peels & 62.97 & [52] \\ _Citrus \(\times\) laftiglia_ & Fruit peels & 54.71 & [53] \\ _Bunera grauelens_ & Fruits & 43.60 & [54] \\ _Citrus medica_ & Leaves, Fruits & 39.77 & [47] \\ _Psilium guajava_ & Leaves & 38.01 & [55] \\ _Citrus bergania_ & - & 21.47 & [56] \\ _Zantharylum schinifoliaium_ & - & 21.24 & [57] \\ _Liltest cubelu_ & Fruits & 14.30 & [58] \\ _Zanthacylum armatum_ & Leaves, Fruits & 10.70 & [47] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Source and composition of D-limonene.  
however, its use for D-limonene production has not been reported to date [63]. Biotransformation as an alternative method to extract D-limonene from natural plants has the advantages of high concentration and yield and has great potential for expanding production. However, D-limonene has a certain degree of cytotoxicity, which greatly reduces the adaptability of engineered cells, and the specificity and inefficiency of the biosynthetic enzyme pathway in heterologous hosts hinder the large-scale production of D-limonene by microorganisms. In addition, because of public health, environmental protection, and other considerations, some regions, such as the European Union, have set the most stringent scrutiny standards for the application of genetically modified organisms, which has limited the promotion of genetically modified microorganisms, so plant essential oil extraction is still the main method to produce D-limonene [59, 67, 68].