title: GlnR and PhoP Directly Regulate the Transcription of Genes Encoding Starch-Degrading, Amylolytic Enzymes in Saccharopolyspora erythraea.
abstract: Starch-degrading enzymes hydrolyze starch- and starch-derived oligosaccharides to yield glucose. We investigated the transcriptional regulation of genes encoding starch-degrading enzymes in the industrial actinobacteriumSaccharopolyspora erythraeaWe observed that most genes encoding amylolytic enzymes (one α-amylase, one glucoamylase, and four α-glucosidases) were regulated by GlnR and PhoP, which are global regulators of nitrogen and phosphate metabolism, respectively. Electrophoretic mobility shift assays and reverse transcription-PCR (RT-PCR) analyses demonstrated that GlnR and PhoP directly interact with their promoter regions and collaboratively or competitively activate their transcription. Deletion ofglnRcaused poor growth on starch, maltodextrin, and maltose, whereas overexpression ofglnRandphoPincreased the total activity of α-glucosidase, resulting in enhanced carbohydrate utilization. Additionally, transcript levels of amylolytic genes and total glucosidase activity were induced in response to nitrogen and phosphate limitation. Furthermore, regulatory effects of GlnR and PhoP on starch-degrading enzymes were conserved inStreptomyces coelicolorA3(2). These results demonstrate that GlnR and PhoP are involved in polysaccharide degradation by mediating the interplay among carbon, nitrogen, and phosphate metabolism in response to cellular nutritional states. Our study reveals a novel regulatory mechanism underlying carbohydrate metabolism, and suggests new possibilities for designing genetic engineering approaches to improve the rate of utilization of starch in actinobacteria.IMPORTANCEThe development of efficient strategies for utilization of biomass-derived sugars, such as starch and cellulose, remains a major technical challenge due to the weak activity of associated enzymes. Here, we found that GlnR and PhoP directly regulate the transcription of genes encoding amylolytic enzymes and present insights into the regulatory mechanisms of degradation and utilization of starch in actinobacteria. Two nutrient-sensing regulators may play important roles in creating a direct association between nitrogen/phosphate metabolisms and carbohydrate utilization, as well as modulate the C:N:P balance in response to cellular nutritional states. These findings highlight the interesting possibilities for designing genetic engineering approaches and optimizing the fermentation process to improve the utilization efficiency of sugars in actinobacteria via overexpression of theglnRandphoPgenes and nutrient signal stimulation.
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Ya
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Cheng-Heng
authors.initials: CH
authors.lastname: Liao
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Yao
authors.affiliation: School of Electronics, Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Ye
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China bcye@ecust.edu.cn.
authors.forename: Bang-Ce
authors.initials: BC
authors.lastname: Ye