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The reduction of oximes was recently identified as a promiscuous activity of Old Yellow Enzymes (OYEs). This reaction involves a two‐step reduction of α‐oxime‐ß‐ketoesters to the corresponding amines, which spontaneously dimerise to yield pyrazine derivatives. This biotransformation is currently limited to substrates with small substituents like methyl/ethyl on the keto moiety. We used a structure‐based approach to engineer 12‐oxophytodienoate reductase 3 (OPR3) from Solanum lycopersicum as a prototypical OYE to accept oximes with bulkier substituents. To this end, three single and two double variants were prepared and tested on six oxime substrates. The engineered variants indeed showed activity on some of the bulkier substrates, which had not been converted at all by the wild‐type enzyme, including the diester compound diethyl‐2‐(hydroximino) malonate. While we were unable to identify variants capable of converting substrates with branched and aromatic substituents, the results demonstrate the validity of our engineering approach, suggesting potential pathways for expanding the substrate scope of OYEs. [ABSTRACT FROM AUTHOR]

Microbial oil obtainable from the fermentation of lignocellulose hydrolysates represents a promising and sustainable alternative to first generation biodiesel. Among the lignocellulosic crops, giant reed (Arundo donax) is attracting interest due to the impressive biomass yield and the low input requirement. However, a delignification step is needed to facilitate the fermentable sugar release from the lignocellulosic matrix, paying attention to the production of growth inhibitors that represent a bottleneck in the development of microbial oil production. The aim of this study was to evaluate the suitability of an enzymatic hydrolysate of alkali pre-treated A. donax biomass, as a substrate for the growth of the oleaginous yeast Lipomyces starkeyi. Specific attention was also paid to possible inhibitory effects of compounds generated by the alkaline pre-treatment (10% slurry, NaOH 0-1.5% w/w, 120 °C, 20 min). Increasing NaOH levels enhanced the release of phenolic compounds and increased the fermentable sugar yield after enzymatic hydrolysis of the washed pre-treated fiber. Saccharification yields reached a plateau in correspondence to NaOH 1.2% dose, which gave 407 mg of sugars per g of dry biomass. A medium containing 30 g/L of reducing sugars from the hydrolysate resulted suitable for the growth of L. starkeyi and for lipid accumulation, achieving 12.2 g/L of dry cell biomass with 43% w/w of total lipids. The pre-treatment produced soluble inhibitors that affected moderately the yeast growth in an initial phase, followed by a recovery. Thus, extensive washing of the fiber could be avoided, while a thorough filtration after the pre-treatment would be recommended. [ABSTRACT FROM AUTHOR]