Background trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of l-phenylalanine (l-Phe) into t-CA and developed a repeated bioconversion process. Results An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of l-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of l-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. Conclusions We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

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.

Recent advances in the RNA delivery system have facilitated the development of a separate field of RNA therapeutics, with modalities including mRNA, miRNA, antisense oligonucleotide (ASO), siRNA, and circular (circRNA) that have been incorporated into oncology research. The main advantages of the RNA-based modalities are high flexibility in designing RNA and rapid production for clinical screening. It is challenging to eliminate tumors by tackling a single target in cancer. In the era of precision medicine, RNA-based therapeutic approaches potentially constitute suitable platforms for targeting heterogeneous tumors that possess multiple sub-clonal cancer cell populations. In this review, we discussed how synthetic coding and non-coding RNAs, such as mRNA, miRNA, ASO, and circRNA, can be applied in the development of therapeutics. Significance StatementWith development of vaccines against coronavirus, RNA-based therapeutics have received attention. Here, we discuss different types of RNA-based therapeutics potentially effective against tumor that are highly heterogeneous giving rise to resistance and relapses to the conventional therapeutics. Moreover, we summarized recent findings suggesting combination approaches of RNA therapeutics and cancer immunotherapy.

Kirsten rat sarcoma viral oncogene (KRAS) mutations are present in 26% of non-small cell lung cancer (NSCLC) patients (The Cancer Genome Atlas data). Studies have reported the presence of spatial or intra-tumoral heterogeneity of KRAS mutations. Though resistance to mutant KRAS inhibitors targeting a specific form of major KRAS mutations (e.g, G12C or G12D) is multifaceted, intra-tumoral heterogeneity of KRAS mutation is one of the crucial factors for the intrinsic resistance to the inhibitor. Multi-targeting of different KRAS mutations can prevent resistance due to the heterogeneity of KRAS mutations within a tumor. To achieve this, we incorporated an mRNA-based cancer vaccine containing transcripts for multiple KRAS mutant antigens. Our KRAS cancer vaccine (CV) candidate showed significantly attenuated tumor growth by 37% in the syngeneic KRAS G12C-expressing LL/2 tumor-bearing mice. CV treatment group showed significantly decreased tumor size by 45.4% after biopsy. Moreover, the mice treated with KRAS CV showed spleen enlarged by 42.6% indicating increased immune responses. To confirm, the expansion of T cells in the cancer vaccine-treated group, we collected tumor tissues and analyzed proportions of CD4+ and CD8+ T lymphocytes in the tumor. We found a 14.5-fold increase in infiltration of CD8+ T cells and a 0.5-fold increase in CD8+ CD44+ memory/effector T cells in the tumors from mice treated with KRAS CV. Whereas CD4+ Foxp3+ Treg cells were decreased by 3.9-fold in the tumor. HLA-A*02 is the most common MHC class I allele. To investigate the changes in MHC expression, we conducted FACS analysis using HLA-A*02 antibodies after ex vivo treatment of KRAS CV on the human peripheral blood mononuclear cells (hPBMCs). KRAS CV treatment facilitated higher proportions of HLA-A*02+ monocytes (CD14+) and B cells (CD19+). Though LL/2 tumor model has been considered an immunosuppressive model, our results suggest that the KRAS CV can significantly enhance immune responses, thereby suppressing tumor growth. Citation Format: Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Yong Ho Heo, Seongju Jeong, Yu-Yon Kim, In Young Choi. An mRNA-based cancer vaccine multi-targeting KRAS mutations inhibits tumor growth by increasing immune response in KRAS mutant LL/2 mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB222.