3.2 Screening of fatty acyl-CoA reductases
We further screened four fatty acyl-CoA reductases (FARs) to identify the reductase with the highest activity and selectivity towardsZ 11-14:acid. The four reductases were: HarFAR from cotton bollworm H. armigera [34], SlitpgFARII from African cotton leafworm Spodoptera littoralis [35], and two reductases fromO. nubilalis , OnuFAR_E and OnuFAR_Z [36]. First, we expressed the four reductases individually in the strain ST7982. The resulting strains were cultivated as follows: first, the cells were grown for biomass propagation for 22 h, followed by a production phase in media supplemented with equal amounts of E andZ 11-14:Me (500 mg/L of each isomer) lasting for 28 hours. Fatty alcohols were extracted from the broth and quantified on GC-MS. HarFAR and SlitpgFARII expressing strains produced around 40 and 14 mg/L ofE 11-14:OH and Z 11-14:OH from the corresponding methyl esters, respectively, and did not exhibit a preference towards any isomer. OnuFAR_E produced very low amounts (<1 mg/L) ofE /Z 11-14:OH, which were below the quantification limit. However, manual inspection of the chromatograms showed that this reductase had a bias towards E isomer, as expected. No fatty alcohols were detected in the strain expressing OnuFAR_Z (Figure 2, Figure S3).
Next, for de novo production of Z 11-14:OH, we expressed the four reductases in the strain ST9992, which contained Fas2pI1220F mutation and expressed desaturase Lbo_PPTQ. Interestingly, while HarFAR resulted in 2.8-fold higher product titer than SlitpgFARII in the feeding assay above, in this experiment with de novo production, a higher Z 11-14:OH titer was obtained in strain with SlitpgFARII, 37.7±2.6 mg/L versus 29.2±1.6 mg/L for HarFAR strain.
However, the strain expressing SlitpgFARII additionally produced 402.5±17.3 mg/L of 14:OH and 54.7± 4.0 mg/L of Z 9-16:OH, which is 2.2 and 1.8-fold higher compared to HarFAR, respectively (Figure 2B). Very high activity towards tetradecanoate of SlitpgFARII was also observed by Antony et al. [35], who reported that among a wide variety of saturated, mono-, and di-unsaturated fatty acyl substrates, tetradecanoate was converted into alcohol most efficiently.
We chose HarFAR for further strain engineering due to its high activity and better selectivity towards Z 11-14:CoA than SlitpgFARII.