Engineered reversal of function in glycolytic yeast promoters

ABSTRACTPromoters are key components of cell factory design, allowing precise expression of genes in a heterologous pathway. Several commonly-used promoters in yeast cell factories belong to glycolytic genes, highly expressed in actively-growing yeast when glucose is used as a carbon source. However, their expression can be suboptimal when alternate carbon sources are used, or if there is a need to decouple growth from production. Hence, there is a need for alternate promoters for different carbon sources and production schemes. In this work, we demonstrate a reversal of regulatory function in two glycolytic yeast promoters by replacing glycolytic regulatory elements with ones induced by the diauxic shift. We observe a shift in induction from glucose-rich to glucose-poor medium without loss of regulatory activity, and strong ethanol induction. Applications of these promoters were validated for expression of the vanillin biosynthetic pathway, reaching production of vanillin comparable to pathway designs using strong constitutive promoters.

Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Alternate Promoters Direct Tissue-Specific Expression of Erythrocyte Ankyrin Transcripts with Novel NH2-Termini.

Mutations in erythrocyte ankyrin, ankyrin-1, are the most common cause of typical hereditary spherocytosis. Co-inheritance of cardiac, muscular, and neurologic diseases such as cardiomyopathy, psychomotor retardation, and spinocerebellar abnormalities with hereditary spherocytosis has been described. In the nb/nb mouse, an ankyrin-1 mutation manifests in erythroid cells with ankyrin deficiency and a spherocytosis phenotype, and in neural cells with an age-dependent psychomotor disorder due to loss of cerebellar Purkinje cells. These observations highlight the importance of understanding ankyrin-1 structure, function, and regulation in erythroid and nonerythroid cells. In erythroid cells, ankyrin expression is directed by a compact promoter controlled by a single GATA-1 site. Nonerythroid ankyrin-1 isoforms have been described with diversity arising from alternate splicing, alternate polyadenylation, and, in skeletal muscle, use of an alternate, tissue-specific promoter. Using 5′ RACE, we identified 2 additional alternate first exons of the ankyrin-1 cDNA. One encoded a first exon with an initiator methionine followed by 12 amino acids, designated exon 1A, that spliced in-frame to erythroid exon 2 sequences. The other, designated 1B, encoded a novel initiator methionine followed by 40 highly charged amino acids that also spliced in-frame to the erythroid exon 2. Both exons, found in human and mouse, link directly to the downstream exons encoding the ankyrin repeat and spectrin binding domains of ankyrin-1. Exon 1B mapped to a location 98.5 kb 5′ of erythroid exon 1 (1E) and exon 1A mapped 30.1 kb 3′ of exon 1E. Northern blot and quantitative RT-PCR analyses demonstrated that 1B was expressed in heart, skeletal muscle, and brain. Similar to what we previously reported for the promoter of the erythroid-specific exon 1, 1E, DNase I hypersensitive site (HS) mapping identified a pair of HS in genomic DNA, one immediately 5′ of exon 1B and one that mapped 6.6 kb downstream of this site (chr8:41,873,229 and 41,866,593, UCSC assembly, Mar 2006). Luciferase-reporter gene expression studies with plasmids containing either a 700bp or a 278bp fragment of the 1B flanking sequence directed high-level luciferase expression in RD cells (human rhabdomyosarcoma) but no luciferase expression in K562 (erythroid) or HeLa (fibroblastoid) cells. Consistent with its tissue-restricted pattern of expression, a polyclonal antipeptide antibody raised against novel sequence in exon 1B reacted with peptides of 220, 205, 45, and 40 kDa on immunoblots prepared from muscle and brain but not erythrocytes. In contrast to erythroid-specific exon 1E and tissue-restricted exon 1B, mRNAs containing exon 1A were detected in all 18 tissues examined. Like the 1E and 1B promoters, mapping identified an HS in the 5′ flanking genomic DNA/promoter region of exon 1A and another 6 kb downstream (chr8:41,873,229 and 41,866,593). An exon 1A anti-peptide antibody reacted with peptides of 205, 195, and 190 kDa on immunoblots prepared from numerous tissues, including erythrocytes ghosts. Regulation of ankyrin-1 expression by alternate promoters directing novel NH2-termini provides the basis for a complex pattern of tissue-specific ankyrin-1 isoform diversity. Characterization of the downstream alternate exon composition of the tissue-specific exon 1B and ubiquitous exon 1A-containing transcripts will allow a systematic evaluation of whether a specific spherocytosis-linked ankyrin-1 mutation could lead to a nonerythroid phenotype.