Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae is an important platform organism for synthesis of chemicals and fuels. However, the promoters used in most pathway engineering studies in S. cerevisiae have not been characterized and compared in parallel under multiple conditions that are routinely operated in laboratory and the number of known promoters is rather limited for the construction of large biochemical pathways. Here a total of 14 constitutive promoters from S. cerevisiae were cloned and characterized using a green fluorescent protein (GFP) as a reporter in a 2 µ vector pRS426, under varying glucose and oxygen concentrations. The strengths of these promoters varied no more than sixfold in the mean fluorescence intensity of GFP, with promoter TEF1p being the strongest and promoter PGI1p the weakest. As an example of application for these promoters in metabolic engineering, the genes involved in xylan degradation and zeaxanthin biosynthesis were subsequently cloned under the control of promoters with medium to high strength and assembled into a single pathway. The corresponding construct was transformed to a S. cerevisiae strain integrated with a D-xylose utilizing pathway. The resulting strain produced zeaxanthin with a titer of 0.74 ± 0.02 mg/L directly from birchwood xylan.

Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

“…The DNA assembler-based combinatorial approach requires an expression cassette of each individual gene, consisting of a unique promoter and terminator pair to avoid unwanted homologous recombination. ADH1, PGK1, and PYK1 are all medium-strength promoters and were selected in order not to limit the activity range of any one of the enzymes by using a set of promoters significantly different in transcription strength (26). More than 10 promoters are available for the expression of heterologous enzymes in S. cerevisiae (2,26,27).…”

“…ADH1, PGK1, and PYK1 are all medium-strength promoters and were selected in order not to limit the activity range of any one of the enzymes by using a set of promoters significantly different in transcription strength (26). More than 10 promoters are available for the expression of heterologous enzymes in S. cerevisiae (2,26,27). This requirement could be met by carefully designing the expression cassettes, taking into account the promoter strengths under the culture conditions.…”

Natural and Modified Promoters for Tailored Metabolic Engineering of the Yeast Saccharomyces cerevisiae

“…A more comprehensive characterization of several constitutive promoters has recently been carried out by Partow et al [ 30 ] and Sun et al [ 31 ]. Both studies constitute a very valuable source of knowledge for choosing promoters in yeast metabolic engineering.…”

“…These results substantiate our previous statement that not all promoters which are usually considered as constitutive can be expected to result in exactly the same level of expression under all experimental conditions or metabolic modes. Sun et al [ 31 ] cloned and characterized 14 different constitutive promoters including those tested by Partow et al [ 30 ]. All promoters were placed onto an episomal 2 μ plasmid and controlled the expression of the green fl uorescent protein (GFP), which was used here as a reporter.…”

Promoter and Terminator Discovery and Engineering

“…In similar fashion to other hosts, the sequencing of genomes (such as the CHO genome [12]) allowed for the discovery of novel, dynamic promoters such as pTXnip, which expresses proportionally to cell density [13]. Libraries of native promoters serve an important role as major synthetic parts and are among the most highly characterized [14,15]; however, they remain limited in their ability to sample complete gene expression ranges. Although multiple gene overexpression techniques have been used in E. coli [16][17][18] and S. cerevisiae [19][20][21][22], among other organisms, this approach can be limited and leads to the build-up of toxic intermediates that reduce productivity [23].…”