3.3 Construction and characterization of a 6HA producing strain
Whereas P. taiwanensis VLB 120 (pSEVA_CL_2) showed promising properties regarding cascade activity and stability, the presence of host-intrinsic hydrolases still led to a product mix consisting of ε-CL and 6HA (Figure 3BD). An industrial production process always relies on an efficient DSP, which in turn demands the avoidance of excessive byproduct accumulation. One possibility to prevent ε-CL hydrolysis is the knockout of the respective hydrolase(s) in the host strain P. taiwanensis VLB120. However, its genome encodes over 100 enzymes with hydrolytic activity. Consequently, identification and inactivation of the responsible enzyme(s) would be very challenging, especially as several enzymes may be involved in this reaction, possibly even in a cooperative manner. The more promising alternative is to focus on 6HA as the only reaction product, which can also serve as a monomer to produce PCL [12]. Furthermore, 6HA is significantly less toxic to the cells as compared to ε-CL (Figure 4D).
Whereas concentrations of up to 20 mM 6HA did barely affect the growth, 20 mM ε-CL reduced the growth rate by ~50%. For 6HA, a half-maximal growth rate was observed at ~ 100 mM, which in turn led to complete growth inhibition in the case of ε-CL.
To push the reaction towards 6HA, an additional lactonase was included in the pSEVA_CL_2 construct, originating from the cyclohexane degradation pathway of Acidovorax sp. CHX100 (see Supplementary Information, Section 5, for the nucleotide sequence), resulting in pSEVA_6HA_2 (Figure 4A). This construct indeed enabled the exclusive production of 6HA to a concentration of 1.74 ± 0.17 mM after 2 h of reaction (Figure 4B). The high initial specific activity of 52.5 ± 5.0 U gCDW-1 (in the first 5 min) dropped by 50 % within 30 min and then remained stable. Lactonase gene expression led to a detectable lactonase band and was found to enable a high ε-CL hydrolysis activity of 836.6 ± 16.5 U gCDW-1, but did not influence Cyp, CDH, or CHMO levels and activities nor the active Cyp concentration (Figure 4C, Tables 2 and S4). The growth rate during expression (0.37 ± 0.01 h-1) also remained comparable to that of the empty vector control (Table S4). A construct pSEVA_6HA_1 with all genes under the control of only one promoter also was established. It again led to less favorable properties such as slower growth, (transient) cyclohexanol accumulation, and lower initial activities (Tables 2 and S4, Figure S3), confirming the superiority of the two promoter approach.
Finally, the two strains containing two-promoter constructs for the 3- or 4-step pathway were tested for the conversion of 5 mM cyclohexane on a 40 mL scale. Both strains enabled complete conversion within 3 h with the 4-step pathway being superior regarding selectivity (100 % for 6HA) than the 3-step pathway (80 % towards ε-CL) (Figure 4E). The initial specific activities of P. taiwanensis VLB120 harboring pSEVA_CL_2 or pSEVA_6HA_2 were high and in the same range (68.4 ± 6.5 or 61.5 ± 3.2 U gCDW-1, respectively). The activities showed a decrease over time, most probably due to the decreasing substrate availability, giving overall activities of 30.8 ± 5.8 and 33.2 ± 0.7 U gCDW-1, respectively. Consequently, complete conversion of cyclohexane to 6HA via the in vivo 4-step cascade was found to be feasible and efficient without serious impediments by enzyme kinetics or biocatalyst instability.
Overall, P. taiwanensis VLB120 (pSEVA_6HA_2) can be considered a highly promising production strain for the conversion of cyclohexane to the PCL monomer 6HA.