Selection of iPSCs without mtDNA deletion for autologous cell therapy in a patient with Pearson syndrome.

Screening for genetic defects in the cells should be examined for clinical application. The Pearson syndrome (PS) patient harbored nuclear mutations in the POLG and SSBP1 genes, which could induce systemic large-scale mitochondrial genome (mtDNA) deletion. We investigated iPSCs with mtDNA deletions in PS patient and whether deletion levels could be maintained during differentiation. The iPSC clones derived from skin fibroblasts (9% deletion) and blood mononuclear cells (24% deletion) were measured for mtDNA deletion levels. Of the 13 skin-derived iPSC clones, only 3 were found to be free of mtDNA deletions, whereas all blood-derived iPSC clones were found to be free of deletions. The iPSC clones with (27%) and without mtDNA deletion (0%) were selected and performed in vitro and in vivo differentiation, such as embryonic body (EB) and teratoma formation. After differentiation, the level of deletion was retained or increased in EBs (24%) or teratoma (45%) from deletion iPSC clone, while, the absence of deletions showed in all EBs and teratomas from deletion-free iPSC clones. These results demonstrated that non-deletion in iPSCs was maintained during in vitro and in vivo differentiation, even in the presence of nuclear mutations, suggesting that deletion-free iPSC clones could be candidates for autologous cell therapy in patients. [BMB Reports 2023; 56(8): 463-468].

Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs), microbial secondary metabolites with ultraviolet (UV) absorption properties, are promising natural sunscreen materials. Due to the low efficiency of extracting MAAs from natural producers, production in heterologous hosts has recently received attention. Shinorine is a well characterized MAA with strong UV-A absorption property. Previous, we developed Saccharomyces cerevisiae strain producing shinorine by introducing four shinorine biosynthetic genes from cyanobacterium Nostoc punctiforme. Shinorine is produced from sedoheptulose 7-phosphate (S7P), an intermediate in the pentose phosphate pathway. Shinorine production was greatly improved by using xylose as a co-substrate, which can increase the S7P pool. However, due to a limited xylose-utilizing capacity of the engineered strain, glucose was used as a co-substrate to support cell growth. In this study, we further improved shinorine production by attenuating glucose catabolism via glycolysis, which can redirect the carbon flux from glucose to the pentose phosphate pathway favoring shinorine production. Of the strategies we examined to reduce glycolytic flux, deletion of HXK2, encoding hexokinase, was most effective in increasing shinorine production. Furthermore, by additional expression of Ava3858 from Anabaena variabilis, encoding a rate-limiting enzyme 2-demethyl 4-deoxygadusol synthase, 68.4 mg/L of shinorine was produced in an optimized medium containing 14 g/L glucose and 6 g/L xylose, achieving a 2.2-fold increase compared with the previous strain.