Chemo-enzymatic cascades to produce cycloalkenes from bio-based resources

Engineered enzyme cascades offer powerful tools to convert renewable resources into value-added products. Man-made catalysts give access to new-to-nature reactivities that may complement the enzyme’s repertoire. Their mutual incompatibility, however, challenges their integration into concurrent chemo-enzymatic cascades. Herein we show that compartmentalization of complex enzyme cascades within E. coli whole cells enables the simultaneous use of a metathesis catalyst, thus allowing the sustainable one-pot production of cycloalkenes from oleic acid. Cycloheptene is produced from oleic acid via a concurrent enzymatic oxidative decarboxylation and ring-closing metathesis. Cyclohexene and cyclopentene are produced from oleic acid via either a six- or eight-step enzyme cascade involving hydration, oxidation, hydrolysis and decarboxylation, followed by ring-closing metathesis. Integration of an upstream hydrolase enables the usage of olive oil as the substrate for the production of cycloalkenes. This work highlights the potential of integrating organometallic catalysis with whole-cell enzyme cascades of high complexity to enable sustainable chemistry.


Supplementary
Reaction conditions: 6 (20 mM), E. coli (OhyA2) or E. coli (OhyA2-FadL) (5 g l -1 ), KP buffer (200 mM pH 8.0, 1% glucose), 30 °C, 250 rpm, 5 h. The progress of the reaction was monitored by GC-MS and the conversions were estimated using the relative peak areas on the total ion chromatogram. Source data are provided as a Source Data file. Data are mean values of triplicate experiments with error bars indicating standard deviations (n = 3). Figure 12. A strain library for the production of 9-hydroxynonanoic acid from 6. a, Construction of a plasmid library for the co-expression of PpBVMO, MlADH, OhyA2, and TLL with different expression levels, and screening of the resulting E. coli library to identify the most effective strain for the production of 9-hydroxynonanoic acid. All E. coli strains included an additional plasmid harboring the fatty acid transporter FadL. b, Initial screening of 576 strains in six 96-well plates for production of 9hydroxynonanoic acid. c, Further validation and comparison of 24 strains (best four from each plate) for the production of 9-hydroxynonanoic acid. The reactions were performed using oleic acid (6, 5 mM) and Supplementary Figure 13. A strain library for the production of azelaic acid (4a) from oleic acid (6). a, Construction of a plasmid library for the co-expression of ChnD, ChnE and FadL with different expression levels, and screening of the resulting E. coli library to identify the most effective strain for the production of azelaic acid (4a). All E. coli strains included an additional plasmid PpBVMO-MlADH-OhyA2-TLL from the strain P11-F9 in Figure S12. b, Initial screening of 192 strains in two 96-well plates for production of azelaic acid (4a). c, Further validation and comparison of 8 strains (best four from each plate) for the production of azelaic acid (4a). The reactions were performed using oleic acid (6, 5 mM) and

Chemicals and Materials
All the chemicals were purchased from commercial suppliers and used without further purification. The key chemicals are listed below.
Fast digest restriction enzymes and T4 DNA ligase were purchased from Thermo Fisher.
Q5 high fidelity DNA polymerase and NEBuilder HiFi DNA assembly master mix were purchased from New England Biolabs Plasmid miniprep-kit and gel extraction-kit were bought from Macherey-Nagel.
DNA primers were ordered from Microsynth.
LB (Lysogeny broth) medium was used for genetic engineering of E. coli.

Engineering of E. coli with Combinatorial RBS Libraries
The RBS Calculator (https://salislab.net/software/) 11,12 in the 'Design: RBS Sequences' mode was used to generate context-specific RBSs with a target translation initiation rate of 100,000 for each gene in its specific genetic context within the operons. The resulting synthetic RBSs were used as a starting point to generate RBS prediction data using the RBS Library Calculator 11,13 in the 'Predict: RBS Library' mode.
For this, the eight base positions with a high impact on TIR were selected and fully randomized with degenerate bases (8N). These 8N libraries containing 65,536 sequence-TIR pairs for each gene were used as input for the RedLibs algorithm 14 . The target library size was set to four and the target library distribution was set to a uniform distribution between the minimum and maximum TIR values for each gene's 8N library. The resulting partially degenerate sequences coding for close-to-uniformly distributed TIR values (Supplementary Figure 20) were used to design primers for library construction (Supplementary Table 1).
All amplifications of DNA fragments were performed by PCR using the Q5 DNA polymerase (New England Biolabs). The gene fragments for operon library construction were amplified with primers containing the corresponding degenerate RBS in a primer overhang. Primers RBS4-PfBVMO-F and PfBVMO-RBS4-R were used to amplify E6-PfBVMO from pRSF-E6-PfBVMO. Primers RBS4-MlADH-F and MlADH-RBS4-R were used to amplify MlADH from pET28a-MlADH. Primers RBS4-OhyA-F and OhyA-RBS4-R were used to amplify OhyA2 from pET28a-OhyA2. Primers RBS4-TLL-F and pRSF-RBS4-R were used to amplify pRSF-TLL from pRSF-TLL. These four fragments containing the degenerate RBSs were assembled to generate the operon PfBVMO-MlADH-OhyA2-TLL on a pRSFduet-1 plasmid backbone using the NEBuilder HiFi DNA assembly master mix (New England Biolabs) and transformed into electrocompetent cells of E. coli (FadL) containing pACYC-FadL.