3.2 Assembling caprolactone-producing strains
To assess CDH and CHMO gene expression to different levels, we generated two ε-CL producers based on the platform organism for Cyp gene expression developed recently [18]. First, CDH and CHMO genes were placed downstream of the Cyp genes on the same operon inP. taiwanensis VLB120 pSEVA_CL_1 (Figure 3A). Consequently, one mRNA is produced, harboring all 5 genes sequentially. To enhance CDH and CHMO levels, a second strain harboring pSEVA_CL_2 was created. pSEVA_CL_2 contains a second Ptrc promoter upstream of the CDH and CHMO genes giving rise to increased expression rates of the respective genes.
In bioconversions applying resting P. taiwanensis VLB120 (pSEVA_CL_1), ε-CL accumulated up to 1.46 ± 0.01 mM within 120 min, after which the reaction was stopped (Figure 3B). Besides the desired product ε-CL, also the intermediate cyclohexanol was detected to a maximal concentration of 42 µM after 60 min. Additionally, 6HA, the hydrolysis product of ε-CL (Figure 1), accumulated in the culture (especially in the second hour of bioconversion) and reached a final concentration of 0.77 ± 0.07 mM after 120 min. The specific overall product formation rate considering ε-CL and 6HA remained quite stable at a high level (37.3 ± 1.9 U gCDW-1). The same experiment employing P. taiwanensis VLB120 (pSEVA_CL_2) (Figure 3D), resulted in ε-CL accumulation to a 20 % higher concentration of 1.80 ± 0.01 mM after 120 min and a higher specific product formation rate (43.4 ± 1.9 U gCDW-1). In contrast to pSEVA_CL_1, the insertion of the second promoter completely prevented the emergence of cyclohexanol, whereas 6HA accumulated to a comparable concentration of 0.7 mM within 120 min. The activity increase observed in the first 10 min of both experiments (Figure 3BD) may be attributed to the direct addition of liquid cyclohexane into the bacterial culture resulting in high local and thus toxic/inhibitory cyclohexane concentrations, which then were attenuated upon cyclohexane redistribution among gas and liquid phase.
The direct comparison of both strains carrying either pSEVA_CL_1 or pSEVA_CL_2 via SDS-PAGE showed that Cyp levels were similar (Figure 3CE). CDH and CHMO levels were close to the detection limit in P. taiwanensis VLB120 (pSEVA_CL_1), whereas the insertion of the second promoter in the construct pSEVA_CL_2 significantly enhanced CDH and CHMO levels (Figure 3E). Assessing the initial specific activities of pSEVA_CL_1 containing enzymes for cyclohexane (37 U gCDW-1), cyclohexanol (39 U gCDW-1), and cyclohexanone (44 U gCDW-1) conversion revealed similar values for all three reaction steps (Table 2) with the CHMO activity being slightly higher than the other two. The higher CDH and CHMO content of P. taiwanensis VLB120 (pSEVA_CL_2) directly translated into higher alcohol (76 U gCDW-1) and ketone (84 U gCDW-1) conversion activities, respectively (Table 2). The introduction of the second promoter doubled the CDH and CHMO activities without affecting the amount of active Cyp in the cells (Table S4). Coexpression of CDH and CHMO together with Cyp genes resulted in a 20 % growth rate reduction from 0.37 ± 0.01 (pSEVA_Cyp) to 0.29 ± 0.01 h-1 (pSEVA_CL_1), indicating a metabolic burden (Table S4). Concomitantly, the active Cyp content decreased by 30 %. Interestingly, such decreases in growth rate and active Cyp content were not observed with pSEVA_CL_2 (Table S4). These results indicate that higher CDH and CHMO levels are crucial to prevent the accumulation of cascade intermediates, especially of the CHMO inhibitor cyclohexanol, and thus to drive the cascade towards ε-CL formation. Furthermore, the two-operon approach reduced the metabolic burden as indicated by the growth rate of the respective strain compared to the one-operon approach.
To further characterize cyclohexanol conversion efficiencies, different cyclohexanol concentrations were added to P. taiwanensis VLB120 cells containing pSEVA_CL_1 or pSEVA_CL_2 (Figure 3F). With pSEVA_CL_1, increasing cyclohexanol led to a decrease in the initial specific ε-CL formation rate and the accumulation of cyclohexanone in the culture (Figure 3F). This correlated with CHMO inhibition and only 15 % of the produced cyclohexanone were converted to ε-CL when 1 mM of cyclohexanol was added as substrate. For a similar cyclohexanol amount (1mM), the elevated CDH and CHMO levels in cells carrying the pSEVA_CL_2 construct resulted in a stable activity of the overall cascade, giving rise to higher cyclohexanol and, subsequently, cyclohexanone conversion with 35 % being converted to ε-CL (Figure 3F). Cyclohexanone accumulation was only observed for initial cyclohexanol concentrations of ≥ 0.4 mM.
In conclusion, both tested strains exhibited decent specific whole-cell activities for the entire cascade. The main difference consisted in the production of small amounts of cyclohexanol with pSEVA_CL_1. Due to CHMO inhibition and CDH kinetics, cyclohexanol was found to potentially disrupt the cascade in a self-enforcing manner. However, the high CDH and CHMO expression levels in P. taiwanensis VLB 120 (pSEVA_CL_2) efficiently prevented cyclohexanol accumulation. Furthermore, the two-operon approach involved a lower metabolic burden, auguring for stable biocatalytic activities.