P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

Abstract

The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only. IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.KEYWORDS Aspergillus niger, low-pH-inducible promoter, itaconic acid, dynamic gene expression, metabolic engineering A spergillus niger is an excellent cell factory for the production of organic acids (1). The rational engineering of A. niger has attracted increasing attention, and great achievements have been made in this area (2, 3). Nevertheless, because heterologous pathways are controlled by constitutive promoters, e.g., the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA), most of this research has focused on static metabolic engineering; that is, the gene expression level is set without sensing changes in the pathway output or cellular environment (4). One disadvantage of static control is related to the trade-off between growth and the production of a desired compound; suboptimal productivity is obtained because these pathways can drain metabolites required for biomass synthesis (5). Metabolic engineering approaches are being developed to tackle this problem via a dynamic control system to compensate for changing conditions (6). In this system, the cell modulates its metabolic pathways dynamically to adjust fluxes such that the required metabolic intermediates are delivered at the appropriate levels and times to optimize growth (7).

Biomass-Derived Production of Itaconic Acid as a Building Block in Specialty Polymers

“…Using modified A. terreus , liquefied corn starch could be used more effectively for IA production compared to the wild-type strain [5]. At optimum conditions, using the most outstanding genetically engineered IA producer XH86-8, itaconate was produced at 77.6 g/L (after three days of fermentation) - which is approaching the industrial production yields [85,88].…”

“…Moreover, it is a promising strain in the production of itaconate as well. Through strain engineering and the application of a low-pH-induced-promoter that improves gene expression, Pgas in A. niger H915-1, 4.92 g/L IA was produced [88].…”

Inducible promoters and functional genomic approaches for the genetic engineering of filamentous fungi

“…Since constitutive expression of the CAD gene resulted in only a small yield of itaconate, the use of the P gas promoter led to a gradually increased expression level of CAD parallel to a decreasing pH value . Finally, a titer of 5 g/l was obtained, which was fivefold higher than comparable experiments in which the P gpdA promoter was used to drive expression of the CAD gene (Yin et al 2017 ).…”

Discovery and identification of medium‐chain fatty acid responsive promoters in Saccharomyces cerevisiae

“…For example, the sucrose‐inducible SUC2 promoter was applied in regulation of gene expression by using RNA interference , a low‐pH‐inducible promoter pGAS can promote gene expression at pH 2.0 , and with an ergosterol‐responsive promoter, metabolic flux could be diverted from the production of sterols to the end product amorphadiene . High‐throughput genomics technology can help in the search for promoters responsive to stress stimulus , and, by this method, an FPP‐responsive promoter was first found and then used to improve amorphadiene production by dynamic regulation in E. coli . Recently, a butanol‐responsive promoter was discovered in S. cerevisiae by using transcriptomic analysis .…”