The LJH19 strain was successfully engineered to produce limonene. In the plant growth media, the limonene production of the engineered LJH19 increased from 0.40 to 3.40 mg/l. However, the limonene yield is still relatively low, indicating that the limonene yield is closely related to the precursor supply. To increase limonene yield, a combination of engineering approaches with the rerouting of the MEP pathway, biosynthetic pathway engineering, and the enhancement of limonene concentration in the cell are needed. The engineered LJH19 strain was used to confirm the potential of limonene production in this study, whereas its application in plant growth media requires further evaluation. However, the limonene production by the engineered LJH19 strain in the plant growth media showed the potential of using LJH19 strain for limonene production, either as a biofactory for limonene production or a direct limonene application. As shown in Table 2, the LJH19 strain has a high tolerance to limonene and its enzymatic reaction products. A previous study reported that limonene inhibited microbial growth under aerobic conditions37. Therefore, the engineered LJH19 strain may show superior bacterial growth under limonene production in plant growth media.
In this study, we adopted an overexpression strategy for gpps gene and used the supernatant broth from the engineered LJH19 strain as plant growth media to demonstrate the potential of limonene production. In the current work, the limonene production was compared to the endogenous level of LJH19 strain. In the absence of the precursor of limonene, gpps, LJH19 showed a limonene production of 0.40 mg/l, which is similar to the limonene production of the parental LJH19 strain (Table 1). After the introduction of the exogenous gpps gene, limonene production increased to 3.40 mg/l. This is 2.0-fold higher than that of the parental LJH19 strain and indicates that the gpps gene is effective for limonene production in LJH19 strain (Table 1).
The finite element analysis was carried out for both axial and in-plane bending load cases. The results from the finite element analysis are compared with the measured values obtained from the manufacturer’s representative operating plant. The difference between the measured values and the finite element analysis results is less than the allowable variation between the manufacturer’s limits. It is shown that the finite element analysis results are accurate. 827ec27edc