Effect of the western conifer seed bug <i>Leptoglossus occidentalis</i>, an invasive alien insect, on seed production reduction in Japanese black pine <i>Pinus thunbergii</i>

To determine the potential reduction of the seed productivity of Japanese black pine (Pinus thunbergii) by the western conifer seed bug (Leptoglossus occidentalis), two feeding experiments were conducted in the center of Kyushu Island, southwestern Japan, in fall of 2020. First, L. occidentalis individuals and a preharvested P. thunbergii cone were enclosed in a nonwoven fabric bag for 2 weeks, which yielded a mean filled seed rate of 18.8% compared with that of 35.6% in the control. Second, L. occidentalis individuals and harvested cones were enclosed in a plastic case for 1 week, affording a mean filled seed rate of 25.7% compared with that of 36.4% in the control. These results suggest that L. occidentalis feeding reduces the filled seed rate of P. thunbergii and damages the seed orchards of this species. Additionally, P. thunbergii seeds from cones damaged by L. occidentalis could be distinguished by their germination ability when sorted using an ethanol floatation technique. Overall, these findings suggest the importance of carefully monitoring L. occidentalis infestation in P. thunbergii seed orchards.</p

Refined Regio- and Stereoselective Hydroxylation of l‑Pipecolic Acid by Protein Engineering of l‑Proline <i>cis</i>-4-Hydroxylase Based on the X‑ray Crystal Structure

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe²⁺/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of l-proline into hydroxy-l-prolines (Hyps). l-Proline <i>cis</i>-4-hydroxylases (<i>cis</i>-P4Hs) from <i>Sinorhizobium meliloti</i> and <i>Mesorhizobium loti</i> catalyze the hydroxylation of l-proline, generating <i>cis</i>-4-hydroxy-l-proline, as well as the hydroxylation of l-pipecolic acid (l-Pip), generating two regioisomers, <i>cis</i>-5-Hypip and <i>cis</i>-3-Hypip. To selectively produce <i>cis</i>-5-Hypip without simultaneous production of two isomers, protein engineering of <i>cis</i>-P4Hs is required. We therefore carried out protein engineering of <i>cis</i>-P4H to facilitate the conversion of the majority of l-Pip into the <i>cis</i>-5-Hypip isomer. We first solved the X-ray crystal structure of <i>cis</i>-P4H in complex with each of l-Pro and l-Pip. Then, we conducted three rounds of directed evolution and successfully created a <i>cis</i>-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward <i>cis</i>-5-Hypip