4.1 Efficient design of in vivo cascades
We developed a production strain harboring an efficient cascade for the conversion of cyclohexane to 6HA with a decent activity in the 50-60 U gCDW-1 range. It has been shown that detailed analyses of enzyme kinetics and respective reaction engineering for a three-step cascade could efficiently enhance the conversion of several unsaturated cyclic alcohols to the corresponding lactones in vitro [1, 30]. Scherkuset al. analyzed the kinetic parameters of an alcohol dehydrogenase and a CHMO to produce 6HA from cyclohexanol[31]. Similarly to the CDH investigated in our study, the KM value was significantly higher for the reverse reaction, and CHMO was severely inhibited by cyclohexanol. Establishing a kinetic model enabled the setup of an efficient fed-batch process.
Whereas the balancing of enzyme ratios in vitro is a rather straight-forward approach [1], in case of whole-cell biocatalysis, this requires fine-tuning of expression levels which, furthermore, should not drive the demand of resources beyond cellular capacities [7]. The so-called metabolic burden arises from the change in demand for (biomass) building blocks and energy (ATP, NAD(P)H) and is system- and condition-dependent[32, 33]. In this study, we observed a gradual decrease in the growth rate with increasing operon size (Table S4). For the cascade investigated, the two-operon- compared to the one-operon approach not only enabled faster growth indicating low metabolic burden, but also led to higher CDH and CHMO expression levels and cascade activities. The relation between gene organization and gene expression is poorly understood. It has been found for E. coli that gene expression increases with the length of the operon resulting in more cotranscriptional translation [34]. Increased translation can result in metabolic burden and misfolded or otherwise non-functional proteins, which was found for the Cyp in our previous study [18]. Although without a terminator after the Cyp genes (Figure 3A), RNA polymerase dissociation may have been promoted by the transcription initiation machinery occupying the downstream promoter region and thereby opening up the DNA[33]. Thus, mRNAs with shorter average length can be expected for the two promoters- as compared to the one promoter constructs. Shorter mRNAs, in turn, have been found to show increased stability in E.coli cells [35] and to recruit fewer ribosomes [34], thus decreasing the metabolic burden. In general, the metabolic burden increases with gene and operon size and with the plasmid copy number. It is further enhanced by some antibiotics such as kanamycin and thus tends to be high for plasmid-based expression, especially when antibiotic resistance genes are used as selection markers [36]. The two operon approach may have profited from shorter but more stable mRNAs and thus reducing metabolic burden and can be considered suitable for efficient expression of the designed pathways in P. taiwanensis VLB120. For further optimization, metabolic modeling of cascades and combinatorial pathway engineering taking into account metabolic burden effects may become interesting, although they still suffer from incomplete knowledge[37-39].