RESUMO
BACKGROUND: Betalains, comprising red-violet betacyanins and yellow-orange betaxanthins, are the hydrophilic vacuolar pigments that provide bright coloration to roots, fruits, and flowers of plants of the Caryophyllales order. Betanin extracted from red beets is permitted quantum satis as a natural red food colorant (E162). Due to antioxidant activity, betanin has potential health benefits. RESULTS: We applied combinatorial engineering to find the optimal combination of a dozen tyrosine hydroxylase (TyH) and 4,5-dopa-estradiol-dioxygenase (DOD) variants. The best-engineered Saccharomyces cerevisiae strains produced over six-fold higher betaxanthins than previously reported. By genome-resequencing of these strains, we found out that two copies of DOD enzyme from Bougainvillea glabra together with TyH enzymes from Abronia nealleyi, Acleisanthes obtusa, and Cleretum bellidiforme were present in the three high-betaxanthin-producing isolates. Next, we expressed four variants of glucosyltransferases from Beta vulgaris for betanin biosynthesis. The highest titer of betanin (30.8 ± 0.14 mg/L after 48 h from 20 g/L glucose) was obtained when completing the biosynthesis pathway with UGT73A36 glucosyltransferase from Beta vulgaris. Finally, we investigated betalain transport in CEN.PK and S288C strains of Saccharomyces cerevisiae and identified a possible role of transporter genes QDR2 and APL1 in betanin transport. CONCLUSIONS: This study shows the potential of combinatorial engineering of yeast cell factories for the biotechnological production of betanin.
RESUMO
Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.
