Rational Engineering of a Multi‐Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis

The current industrial production of polymer building blocks such as ε‐caprolactone (ε‐CL) and 6‐hydroxyhexanoic acid (6HA) is a multi‐step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco‐efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions. Based on kinetic and thermodynamic analyses of the individual enzymatic steps, a 4‐step enzymatic cascade in Pseudomonas taiwanensis VLB120 is rationally engineered via stepwise biocatalyst improvement on the genetic level. It is found that the intermediate product cyclohexanol severely inhibits the cascade which could be optimized by enhancing the expression level of downstream enzymes. The integration of a lactonase enables exclusive 6HA formation without side products. The resulting biocatalyst shows a high activity of 44.8 ± 0.2 U gCDW−1 and fully converts 5 mm cyclohexane to 6HA within 3 h. This platform organism can now serve as a basis for the development of greener production processes for polycaprolactone and related polymers.


Introduction
In synthetic applications, cascade reactions allow for streamlined product formation via multiple reaction steps with the advantage to avoid the isolation of intermediates, thus saving resources, reagents, and time. [1] Multi-step biocatalysis employing whole cells emerged as a powerful tool for the synthesis of value-added compounds. [2][3][4]5] Precise and delicate fine-tuning of gene expression is required to balance individual enzyme amounts and activities for the construction of "designer cells." [1,6,7,8] Especially "artificial cascades" employing heterologous genes of diverse DOI: 10.1002/biot.202000091 origin constitute a major challenge as they introduce novel enzymatic functions into the host. [4,9] On the one hand, it is crucial to provide sufficient enzyme amounts to sustain reasonable rates. On the other hand, too much overexpression, especially of more than one gene, can severely hamper host metabolism and interfere with its stability. [10,11] Moreover, optimizations regarding the choice of the host strain, substrate uptake, and pathway flux can systematically improve in vivo cascade. [11][12][13]14] A holistic approach comprising catalyst and reaction engineering allows controlling the product formation patterns. [15,16] Nowadays, plastics are ubiquitous in human life and cause severe litter problems. Thus, biodegradable polymers such as polycaprolactone (PCL), polylactic acid, and polyhydroxyalkanoate have gained importance. [17] PCL can either be synthesized by the ring-opening polymerization of -caprolactone ( -CL) or by the polycondensation of 6-hydroxyhexanoic acid (6-HA). [18] Industrially, -CL is produced from cyclohexane through the Union Carbide Corporation (UCC) process, which suffers from serious environmental issues, a low cyclohexane conversion of 10-12%, and only moderate selectivity of 85-90%. [19,20] General advantages of biocatalysis, such as high selectivities and operation at moderate temperature and ambient pressure, have a big potential to design a more eco-efficient process. Recently, two biocatalytic approaches to synthesize -CL from cyclohexane have been published. Pennec et al. demonstrated a onepot reaction applying purified enzymes, [21] which, however, suffered from a low conversion of 3%. Recently, Au-doped TiO 2 and graphitic carbonitride photocatalysts catalyzing the oxidation of cyclohexane to cyclohexanone were combined with E. coli cells expressing a Baeyer-Villiger monooxygenase in a proof-of-ofconcept study producing 0.4 mm -CL from cyclohexane, but with a low conversion (200 mm cyclohexane employed). [22] Karande et al. generated a whole-cell biocatalyst showing superior total turnover numbers (TTN), [23] depending, however, on the expensive growth substrate citrate and the volatile inducer dicyclopropylketone. Thereby, they established a 3-step cascade in P. taiwanensis VLB120 by introducing cytochrome P450 monooxygenase (Cyp), cyclohexanol dehydrogenase (CDH), and Baeyer-Villiger cyclohexanone monooxygenase (CHMO) genes from The cascade is composed of a cytochrome P450 monooxygenase (Cyp), a cyclohexanol dehydrogenase (CDH), and a cyclohexanone monooxygenase (CHMO) for the production of -caprolactone ( -CL). [23] Optionally, a lactonase (Lact) catalyzes the ring-opening reaction to yield 6-hydroxyhexanoic acid (6HA).
Acidovorax sp. CHX100 (Figure 1). Respective cascade development mainly focused on the monomer -CL and gave rise to a maximal activity of 22 Ug CDW −1 , with the first Cyp-catalyzed step being rate-limiting. The successive enzymes, CDH and CHMO exhibited much higher activities of 80 and 170 Ug CDW −1 , respectively. In a separate study, the activity of cells containing only the Cyp could be more than doubled by expression system engineering. [24] In this study, we set out to amend the latter system with CDH and CHMO genes and thereby achieve high respective activities, utilize glucose instead of citrate as carbon and energy source, prevent the accumulation of intermediates, and keep the expression related metabolic burden reasonably low to allow for stable catalysis. Explicitly, oxygenases such as Cyps or Baeyer-Villiger monooxygenases are prone to form reactive oxygen species via uncoupling reactions, which may hamper the catalytic prowess of the cells. [11,13] Another point to be considered is that, due to the accumulation of the hydrolysis product 6HA, the approach of Karande et al. [23] suffered from restricted cascade selectivity. We aimed to tackle all these points by rational pathway engineering including the characterization of the involved enzymes as the basis for the rational assembly of the expression system. It is thereby crucial to balance enzyme activities without dissipating the cell's resources.

Results
The construction of an efficient biocatalytic in vivo cascade necessitates a balanced expression of the cascade genes to avoid side product accumulation. Besides the well-characterized initiating Cyp, [24,25] CDH, and CHMO have been employed for PCL monomer synthesis from cyclohexane, [23] but nothing is known about respective reaction kinetics and possible inhibitions by pathway intermediates as they have been observed before, for example, for Baeyer-Villiger monooxygenases. [26,27] Consequently, CDH and CHMO were characterized as the first step in this study to support the rational engineering of a productive 3-step cascade based on the optimized Cyp-containing whole-cell biocatalyst. [24]

Characterization of CDH and CHMO
CDH and CHMO were cloned separately into the pSEVA244_T vector [24] to characterize their in vivo activity. As CDH catalyzes an equilibrium reaction, the kinetic parameters were assayed for both reaction directions ( Table 1). For the reverse reaction with cyclohexanone as substrate, CDH showed a 10 times lower v max  [28] c) Based on an estimated 5 g CDH in 100 g total cell dry weight (CDW).
compared to the forward reaction. On the other hand, the K S values, corresponding to the apparent substrate uptake constant (concentration at which whole cells show half-maximal transformation rates) as it is used in the Monod-kinetics for microbial growth, [28] differed by a factor of almost 100 in favor of the reverse reaction (0.05 for cyclohexanone, 3.57 mm for cyclohexanol). The concentration of cyclohexanol and cyclohexanone in the system determine if v max or K S prevail. Furthermore, we theoretically and experimentally assessed the cyclohexanol/cyclohexanone concentration ratio at equilibrium. Utilizing the group contribution method [29] assuming a physiological intracellular NADH to NAD concentration ratio of 10.6 under aerobic conditions, [30] this ratio was determined to be 1.9 (Section S4, Supporting Information). It was confirmed experimentally by applying different initial alcohol and ketone concentrations giving a cyclohexanol/cyclohexanone concentration ratio of 1.95 ± 0.29 after 16 h ( Figure S2, Supporting Information). This thermodynamic preference of the backward reaction, together with the low K S value for cyclohexanone emphasizes the necessity of an efficient cyclohexanone withdrawal by the successive enzyme in the cascade, that is, CHMO. Substantial research has been conducted with a cyclohexanone monooxygenase originating from Acinetobacter sp. [31] Generally, the substrate as well as product toxicity, are features of Baeyer-Villiger monooxygenase-catalyzed reactions. [26,27] Substrate toxicity was generally observed at aqueous concentrations in the mmrange, which should thus be avoided during the cascade reaction. Furthermore, CHMO may be inhibited by the cascade intermediate cyclohexanol and its product -CL. Acidovorax CHMO indeed was found to be highly prone to inhibition by cyclohexanol (Figure 2A). At a cyclohexanol concentration as low as 0.4 mm, the high initial CHMO activity of 160.3 ± 0.1 Ug CDW −1 was found to be reduced to half this rate. Cyclohexanol concentrations ≥ 1.7 mm completely abolished CHMO activity. However, up to an -CL concentration of 17 mm, no product inhibition was found for CHMO ( Figure 2B). Inhibition studies with P. taiwanensis VLB120 (pSEVA_CHMO). The influence of A) cyclohexanol or B) the product -CL on specific CHMO activity was investigated in resting cell bioconversions. Cells were cultivated in M9* medium with 0.5% (w/v) glucose, induced by isopropyl -d-1-thiogalactopyranoside (IPTG) for 6 h, harvested, and resuspended in KPi-g buffer (100 mm potassium phosphate buffer supplemented with 1% (w/v) glucose) to a biomass concentration of A) 0.25 g CDW L −1 in 1 mL liquid volume or B) 0.5 g CDW L −1 in 10 mL liquid volume. Reactions were started by adding 3 mm cyclohexanone. Graphs represent average values and standard deviations of two independent biological replicates. The average experimental errors over all measurements for the activities in Panels A and B are 3.4% and 18.2%, respectively.
Summing up, these results emphasize that the produced cyclohexanol needs to be directly converted by CDH to avoid CHMO inhibition. High intracellular CDH and CHMO activities are important to avoid any accumulation of alcohol and ketone intermediates, as already low alcohol amounts can be expected to inherently reinforce such accumulation.

Assembling Caprolactone-Producing Strains
To assess CDH and CHMO gene expression to different levels, we generated two -CL producers based on the platform organism for Cyp gene expression developed recently. [24] First, CDH and CHMO genes were placed downstream of the Cyp genes on the same operon in P. 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 P trc 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), -CL accumulated up to 1.46 ± 0.01 mm within 120 min, after which the reaction was stopped ( Figure 3C). Besides the desired product -CL, also the intermediate cyclohexanol was detected to a maximal concentration of 42 µM after 60 min. Additionally, 6HA, the hydrolysis product of -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 -CL and 6HA remained quite stable at a high level (37.3 ± 1.9 Ug CDW −1 ). The same experiment employing P. taiwanensis VLB120 (pSEVA_CL_2) ( Figure 3E), resulted in -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 Ug CDW −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 3C,E) 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, with pSEVA_CL_1 showing leaky expression ( Figure 3B,D). 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 3D). Assessing the initial specific activities of pSEVA_CL_1 containing enzymes for cyclohexane (37 Ug CDW −1 ), cyclohexanol (39 Ug CDW −1 ), and cyclohexanone (44 Ug CDW −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 Ug CDW −1 ) and ketone (84 Ug CDW −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, Supporting Information). 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, Supporting Information). Concomitantly, the active Cyp content decreased by 30%. Interestingly, such decreases The graphs depict cyclohexanone and -CL concentrations as well as the whole-cell activity ( -CL formation) for an assay time of 10 min. Graphs represent average values and standard deviations of two independent biological replicates. The average experimental errors over all measurements for the concentrations of cyclohexanol, cyclohexanone, -CL, and 6HA are 6.9%, 4.7%, 6.7%, and 35.2%, respectively, and 7.2% for the whole-cell activities.  in growth rate and active Cyp content were not observed with pSEVA_CL_2 (Table S4, Supporting Information). 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 -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 -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 -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 -CL ( Figure 3F). Cyclohexanone accumulation was only observed for initial cyclohexanol concentrations ≥ 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.

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 -CL and 6HA ( Figure 3C,E). An industrial production process always relies on an efficient DSP, which in turn demands the avoidance of excessive byproduct accumulation. One possibility to prevent -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. [18] Furthermore, 6HA is significantly less toxic to the cells as compared to -CL ( Figure 4D).
Whereas concentrations of up to 20 mm 6HA did barely affect the growth, 20 mm -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 -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 Section S5, Supporting Information, 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 4C). The high initial specific activity of 52.5 ± 5.0 Ug CDW −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 -CL hydrolysis activity of 836.6 ± 16.5 Ug CDW −1 , but did not influence Cyp, CDH, or CHMO levels and activities nor the active Cyp concentration ( Figure 4B, Table 2 and  Table S4, Supporting Information). The growth rate during expression (0.37 ± 0.01 h −1 ) also remained comparable to that of the empty vector control (Table S4, Supporting Information). 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 (Table 2 and Table S4, Figure  S3, Supporting Information), 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  Figure 3. D) Relative growth rate of P. taiwanensis VLB120 in the presence of varying amounts of -CL (green) or 6HA (orange) (a growth rate of 0.47 ± 0.01 h −1 represents 100%). E) Conversion of 5 mm cyclohexane by P. taiwanensis VLB120 containing pSEVA_CL_2 or pSEVA_6HA_2. Resting cell bioconversions were performed in 40 mL KPi-g buffer containing 1.05 g CDW L −1 of cells in closed 250 mL screw-capped and baffled shake flasks. Graphs represent average values and standard deviations of two independent biological replicates. The average experimental errors over all measurements for -CL concentrations, 6HA concentrations, whole-cell activities, and relative growth rates are 11.7%, 11.6%, 17.7%, and 1.9%, respectively. regarding selectivity (100% for 6HA) than the 3-step pathway (80% towards -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 Ug CDW −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 g CDW −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.

Discussion
The development of eco-efficient sustainable production processes has been one of the major objectives of biotechnology research over the last decade. Such promise is based on the inherent biodegradability, selectivity, and specificity of biocatalysts. [32] Biotechnological solutions already have replaced chemical processes for the production of biosurfactants, amino acids, and even complex heterocyclic compounds. [33,34] The research presented in this study aimed to set a basis for the replacement of the highly polluting UCC process [19] by developing a direct route from cyclohexane to the PCL monomer 6HA enabling full conversion.

Efficient Design of In Vivo Cascades
Various factors including functional expression and stability of the cascade enzymes, toxicity of the reactants and products, equilibrium thermodynamics, cofactor regeneration, and byproduct formation should be considered for the design and construction of efficient whole-cell biotransformation pathways. [4] The final production strain developed in this study for the conversion of cyclohexane to 6HA enabled a decent activity in the 50-60 Ug CDW −1 range for the whole cascade without side product formation. It has been shown that detailed analyses of enzyme kinetics and respective reaction engineering for a 3-step cascade could efficiently enhance the conversion of several unsaturated cyclic alcohols to the corresponding lactones in vitro. [2,35] Scherkus et al. analyzed the kinetic parameters of an alcohol dehydrogenase and a CHMO for the production of 6HA from cyclohexanol with isolated enzymes. [36] Similarly to the CDH investigated in our study, the K M value of the alcohol dehydrogenase was significantly lower for the reverse reaction, and CHMO was severely inhibited by cyclohexanol. Establishing a kinetic model enabled the setup of an efficient fed-batch process. Increasing the expression and stabilizing the activity of Baeyer-Villiger monooxygenases also led to higher 9-hydroxynonanoic acid and C11 nylon monomer concentrations in 4-and 5-step catalytic cascades, respectively. [7,16] Whereas the balancing of enzyme ratios in vitro is a rather straight-forward approach, [2] 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. [11] One possibility is the use of different plasmids to adjust the gene copy number, [37] which also can influence plasmid stability. [7] In our previous study, we varied copy number, RBS, and regulatory systems for Cyp gene expression. [24] The best system in terms of activity, stability, and metabolic burden was used in this study to engineer multi-gene operons. The so-called metabolic burden arises from the change in demand for (biomass) building blocks and energy (ATP, NAD(P)H) related to plasmid maintenance, gene expression, and enzyme activity and is system-and condition-dependent. [38,39] Our results indicate a gradual decrease in the growth rate with increasing operon size (Table S4, Supporting Information). 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 co-transcriptional translation. [40] Increased translation can result in metabolic burden and misfolded or otherwise non-functional proteins, which was found for the Cyp in our previous study. [24] Also 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. [39] Thus, mRNAs with shorter average length can be expected for the two-promoter-as compared to the one-promoter constructs. Shorter mRNAs, in turn, have been found to show increased stability in E.coli cells [41] and to recruit fewer ribosomes, [40] thus decreasing the metabolic burden. In general, the metabolic burden increases with gene and operon size. 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. [42] The two-operon approach may have profited from shorter but more stable mRNAs 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 these approaches still suffer from incomplete knowledge. [43][44][45]

Production of PCL Precursors
The biocatalytic production of PCL or its precursors has been heavily investigated over the last years ( Table 3). Approaches based on isolated enzymes, [21,[46][47][48][49][50] as well as whole cells, [23,37,[51][52][53] have successfully been established. However, most of these approaches relied on cyclohexanol as a substrate, [37,[47][48][49][50][51][52][53] which needs to be synthesized from cyclohexane employing an ecologically critical process. [54] Additionally, inhibition of CHMO by cyclohexanol or substrate inhibition necessitated the development of suitable reaction concepts, for example, two-liquid phase [46] or fed-batch systems. [49] The highest productivity of 1.87 g L −1 h −1 was obtained with isolated enzymes by employing an appropriate feeding strategy for the complete conversion of 283 mm cyclohexanol to 6HA [49] (Table 3). The CHMO from Acinetobacter heterologously expressed in E. coli showed the highest TTN with almost 70 000 mol -CL mol CHMO −1 . [52] In general, whole-cell approaches show lower yields on biocatalyst, as target enzymes constitute only about 1-10% (w/w) of cells, but avoid the enormous effort to purify the enzymes. The highest -CL yield of 1.2 g -CL g biocatalyst with a high initial activity of 15 Ug CDW −1 was observed by Ménil et al. in complex medium. [37] Compared to cyclohexanol, cyclohexane is an even more challenging substrate due to its high volatility and toxicity. In comparison to solvent-sensitive E. coli employed to convert cyclohexanol to 6HA, [53] we obtained a tenfold higher specific whole-cell activity and a similar yield on biocatalyst (Table 3). P. taiwanensis VLB120 is known to tolerate low-logP solvents and can, therefore, be considered suitable for the biotransformation of the more toxic substrate cyclohexane. [55][56][57]  Possible prolongation of the reaction with an appropriate substrate feeding and the application of a high-cell density setup hold big potential to further improve the product titer and the volumetric productivity. This study, for the first time, demonstrates a whole-cell approach directly converting cyclohexane to the PCL precursor 6HA. The biotransformation to -CL presented by Karande et al. [23] could be optimized by enhancing the conversion, yield on biocatalyst, TTN, and specific activity (Table 3). In comparison with this strain, the two strains developed in this study (Table 3, entries 13-15) showed a 3-times higher specific whole-cell activity allowing for lower cell concentrations to achieve a full conversion of 5 mm cyclohexane and thereby higher yields on biocatalyst and TTN. The chemo-biocatalytic approach presented by Li et al. constitutes a proof-of-concept and suffered from low conversion and very low activities. [22] The use of isolated enzymes to convert cyclohexane to -CL suffered from low conversion and TTN, which can be attributed to mass transfer limitations or inherent instability of P450 monooxygenases. [11,21] The cellular environment allows for more stable catalytic activities with superior productivities. As a result, the main limitation for future process development is considered not to lie necessarily in the catalyst itself, but rather in the reaction engineering with cyclohexane feeding/cyclohexane mass transfer and cell toxification as critical points. This may be solved by cyclohexane feeding potentially via the gas phase. The achieved increase in whole-cell activity and conversion, however, can be considered a huge step forward towards the establishment of an economically viable process. [58]

Conclusion
In this study, we developed the strain P. taiwanensis VLB120 (pSEVA_6HA_2) that expresses 6 genes encoding 4 enzymes able to fully convert 5 mm cyclohexane to the PCL monomer 6HA. Accumulation of intermediates and byproducts was successfully prevented, and a high cascade activity was achieved. Our study demonstrated that a balanced expression of pathway encoding genes guided by enzyme characteristics (kinetics, inhibition) allowed for streamlined production of -CL and, especially, 6HA. The constructed orthogonal pathway/cascade also can serve as a template to be amended by additional enzymes to synthesize nylon monomers such as adipic acid and 6-aminohexanoic acid. This in combination with their solvent tolerance [56,59,60] and versatility regarding reactor setups-including biofilm approaches [61][62][63] -qualify VLB120 strains harboring pSEVA_CL_2 or pSEVA_6HA_2 as a promising platform organism for greener polymer production routes.

Experimental Section
Bacterial Strains, Plasmids, Media, and Chemicals: Microbial strains and plasmids used in this work are listed in Table S1, Supporting Information. Cells were grown in lysogeny broth (LB) medium [64] or M9* medium [65] with a pH of 7.2 supplemented with 0.5% (w/v) glucose as sole carbon and energy source. Kanamycin (50 µg mL −1 ) was applied for selection when necessary. Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) or Carl Roth (Karlsruhe, Germany) in the highest purity available and used without further purification. 6HA was acquired from abcr (Karlsruhe, Germany). Molecular biology methods are explained in detail in Section S1, Supporting Information.
Strain Construction: The strains and plasmids used in this study are listed in Table S1, Supporting Information. The plasmids pSEVA244_T and pSEVA_Cyp constituted the basis for the constructs engineered in this study and originate from Schäfer et al. . [24] The primers used for such engineering are listed in Table S2, Supporting Information.
For the construction of plasmids pSEVA_CDH and pSEVA_Cyp_CDH, the CDH gene was amplified from pCapro [23] using the primers PLS013 and PLS014. The resulting purified fragment was fused either to KpnIdigested pSEVA244_T or pSEVA_Cyp by Gibson Assembly [66] to yield pSEVA_CDH or pSEVA_Cyp_CDH, respectively.
Plasmids pSEVA_CHMO and pSEVA_CL_1 were assembled accordingly. The CHMO gene was amplified from pCapro with primers PLS015A and PLS016 or PLS015B and PLS016. The forward primer was different for the two constructs due to the adjustment of the 5' region (overhang to the plasmid backbone). The resulting purified fragments were fused via Gibson Assembly either to XbaI-digested pSEVA244_T or pSEVA_Cyp_CDH to yield pSEVA_CHMO or pSEVA_CL_1, respectively.
Acidovorax sp. CHX100 was cultivated for 4 days in nutrient broth (NB) medium [64] for DNA isolation performed with the peqGOLD Bacterial DNA Mini Kit (PeqLab, Erlangen, Germany). The lactonase gene was amplified with the primers PLS017B and PLS018, and Gibson Assembly of the resulting fragment and HindIII-digested pSEVA_CL_1 gave rise to pSEVA_6HA_1.
The P trc promoter was amplified from pSEVA244 [67] with primers PLS021 and PLS022. It was fused to EcoRI-digested pSEVA_CL_1 or pSEVA_6HA_1 via Gibson Assembly to yield pSEVA_CL_2 and pSEVA_6HA_2.
Growth of Bacterial Cultures: Cultivations were carried out at 30°C and 200 rpm in a Multitron shaker (Infors, Bottmingen, Switzerland). Microorganisms were cultivated in an LB pre-culture for ≈20 h, from which an M9* pre-culture (1% v/v) was inoculated and incubated for another 12-16 h. From this culture, an M9* main culture was inoculated to a starting OD 450 of 0.2. Heterologous gene expression was induced with 1 mm isopropyl -d-1-thiogalactopyranoside (IPTG) when the cultures reached an OD 450 of ≈0.5. Incubation was continued for another 4-6 h, and cells were harvested for SDS-PAGE analyses, CO spectra analyses, and/or activity or toxicity assays (see below). One OD 450 unit corresponds to a biomass concentration of 0.186 g CDW (grams of cell dry weight) L −1 . [68] CO difference spectra (see Section S2, Supporting Information, for details) were recorded to determine the active Cyp concentrations.
Toxicity Assay: P. taiwanensis VLB120 was cultivated as described above but without induction. Different concentrations of -CL or 6HA were added 2 h after inoculation, and the growth rate was determined from this time point on for at least 7 h.
Resting Cell Bioconversions: The cells were cultivated as described above, harvested by centrifugation (10 min, 5000 × g, RT), and resuspended to a specific cell concentration in 100 mm potassium phosphate buffer (pH 7.4) supplemented with 1% (w/v) glucose (KPi-g buffer). The cells were transferred to baffled Erlenmeyer flasks (100 mL) or microcentrifuge tubes (2 mL) with liquid volumes of 10 or 1 mL and biomass concentrations of 0.5 or 0.25 g CDW L −1 , respectively, equilibrated at 30°C for 10 min (flasks in a water bath at 250 rpm; microcentrifuge tubes in a Ther-moMixer C (Eppendorf, Hamburg) at 2000 rpm), then provided with the substrate (as stated in the Table and Figure legends). Biotransformations were stopped by the addition of 0.5 mL ice-cold diethyl ether containing 0.2 mm n-decane as an internal standard to 1 mL sample. After 2 min extraction by vortexing and short centrifugation, the organic phase was dried over water-free Na 2 SO 4 before it was transferred to a GC vial for analysis. The aqueous phase was removed with a syringe from the microcentrifuge tube and stored at −20°C for HPLC analysis. The activity is given in Ug CDW −1 , where 1 U corresponds to 1 µmol product formed within 1 min reaction time.
For the conversion of 5 mm cyclohexane ( Figure 4E), 250 mL screwcapped and baffled Erlenmeyer flasks were used with a liquid volume of 40 mL and a biomass concentration of 1.05 g CDW L −1 . The caps contained two septa, a Teflon septum facing the inner side of the flask, and a silicon septum facing outwards. Pure cyclohexane (21.8 µL) was added to start the reaction and the flasks were tightly closed. For each sampling point, 1.5 mL liquid volume was removed through the septa using a syringe. From this sample, 1 mL was extracted with diethyl ether for GC analysis as described above and 0.5 mL was used for HPLC analysis.
For details on analytical methods refer to Section S3, Supporting Information.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.