The stress-protectants and chemical chaperones ectoine and hydroxyectoine: enzymes, importer, exporter and transcriptional regulation

Changes within the external osmotic potential belong to the most ubiquitous stress factors that microbial cells encounter. A large group of Bacteria, but also a few Archaea and unicellular halophilic Eukarya possess the genetic information to produce the compatible solutes and chemical chaperones ectoine and its derivative 5-hydroxyectoine. These compounds are consequently amassed within the cytoplasm to counteract the reduction in turgor pressure resulting from the outflow of water during high extracellular osmolarity. Ectoines are not only major stress protective compounds for microorganisms but their physicochemical attributes and function-preserving features also entailed their industrial production and use in the fields of biotechnology, medicine and cosmetics. Ectoine biosynthesis from the precursor L-aspartate-β-semialdehyde is achieved in a three step enzymatic reaction involving the enzymes: EctB (L- 2,4-diaminobutyrate aminotransferase), EctA (L-2,4-diaminobutyrate acetyltransferase) and the key enzyme EctC (ectoine synthase). Furthermore, a hydroxyl group can be attached to the ectoine molecule by the ectoine hydroxylase (EctD) yielding 5-hydroxyectoine. The underlying biosynthetic genes are mostly encoded in a gene cluster [ectABC(D)] that is controlled by an osmotically responsive promoter. Besides de novo synthesis of the stress protective compounds, they can also be imported from environmental resources through specific osmotically controlled uptake systems. In the following paragraph the research subjects and major results of my PhD thesis will be briefly summarized: • Bioinformatical analysis focusing on the phylogenetic distribution of the genes for ectoine biosynthesis and investigation of their gene neighborhoods • Structural and functional analysis of ectoine/5-hydroxyectoine biosynthetic enzymes o Crystallographic, biochemical and functional analysis of the key enzyme for ectoine biosynthesis EctC led to the postulation of the performed reaction mechanism o Substrate ambiguity of the ectoine hydroxylase (EctD) was exploited for the stereo- and regioselective hydroxylation of the synthetic ectoine derivative homoectoine • Establishment of ectoine/5-hydroxyectoine-producing cell factories o Secretion of the products is independent of the mechanosensitive channels and ectoine import systems in the heterologous producer E. coli • Regulation of the expression of ectoine biosynthetic genes o Detailed molecular and functional analysis of the ect promoter from P. stutzeri A1501 led to the identification of an unusual sigma-70-type promoter and insights into the determinants involved in osmotic regulation of the ect genes o Bioinformatical, crystallographic and functional analysis of the MarR-type regulator EctR from Novosphingobium • Analysis and characterization of transporters present in the gene neighborhood of ectoine biosynthetic gene clusters o Co-transcriptionofnoveltransportersandmechanosensitivechannels o Functional characterization of two novel ectoine transporters with a broad substrate spectrum (EctI) and specificity for ectoines (EctU) o Functional analysis of mechanosensitive channels, that are encoded within ect gene clusters o Identification and characterization of an ectoine/5-hydroxyectoine specific exporter (EctE