Production of Cinnamaldehyde through Whole-Cell Bioconversion from trans-Cinnamic Acid Using Engineered Corynebacterium glutamicum.

Cinnamaldehyde (CAD) has various applications in foods and pharmaceuticals and has gained prominence as a potent nematicide in agricultural research owing to its nematicidal activity. However, conventional methods of CAD production, including extraction from plants or organic chemical synthesis, are environmentally hazardous and limit its utilization for downstream applications. Here, we engineered Corynebacterium glutamicum as a whole-cell biocatalyst for the efficient bioconversion of trans-cinnamic acid (t-CA) into CAD. An expression module of Mycobacterium phlei carboxylic acid reductase was constructed for the conversion of t-CA to CAD. Additionally, the putative dehydrogenase-related genes (dkgA, adhC, and cg1176) responsible for the conversion of CAD to cinnamyl alcohol were deleted from the engineered C. glutamicum strain to prevent the loss of CAD. Furthermore, as the conversion is NADPH-dependent, we investigated the conversion efficiency by exchanging the putative promoter region for the zwf gene, which encodes glucose-6-phosphate dehydrogenase, with a strong promoter to increase the NADPH pool. Finally, a bioconversion platform using C. glutamicum as a whole-cell biocatalyst was developed by deleting the vdh gene, which is involved in the reverse conversion of CAD to t-CA. Taken together, a 100% conversion yield of 1.1 g/L CAD from 1.2 g/L t-CA was obtained within 30 min.

Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.