Natural and Engineered Hydroxyectoine Production Based on the Pseudomonas stutzeri ectABCD-ask Gene Cluster

Abstract

We report on the presence of a functional hydroxyectoine biosynthesis gene cluster, ectABCD-ask, in Pseudomonas stutzeri DSM5190T and evaluate the suitability of P. stutzeri DSM5190 T for hydroxyectoine production. Furthermore, we present information on heterologous de novo production of the compatible solute hydroxyectoine in Escherichia coli. In this host, the P. stutzeri gene cluster remained under the control of its salt-induced native promoters. We also noted the absence of trehalose when hydroxyectoine genes were expressed, as well as a remarkable inhibitory effect of externally applied betaine on hydroxyectoine synthesis. The specific heterologous production rate in E. coli under the conditions employed exceeded that of the natural producer Pseudomonas stutzeri and, for the first time, enabled effective hydroxyectoine production at low salinity (2%), with the added advantage of simple product processing due to the absence of other cosolutes.As reviewed extensively elsewhere, halotolerant and halophilic microorganisms can adapt to extreme salinity by accumulation or biosynthesis of a range of osmolytes (8,14,21,32,36). Extreme halophiles like the archaeon Halobacterium salinarum usually recruit inorganic ions, while others, in particular, highly adaptive organisms, make use of a more flexible strategy and employ compatible solutes (also called extremolytes) to obtain osmotic equilibrium. Unlike inorganic ions and even at molar concentrations, these organic low-molecularweight osmolytes do not interfere negatively with normal cell metabolism, even at molar concentrations. In addition to their function as osmoprotectants for whole living cells, compatible solutes also protect biological macromolecules against physical stress in vitro. Therefore, they have found their way into a range of biochemical applications (25, 33) and even skin care products (5).The compatible solute ectoine (12) is often detected as the main solute in chemoheterotrophic eubacterial halophiles of broad salt tolerance.

A Specialized Aspartokinase Enhances the Biosynthesis of the Osmoprotectants Ectoine and Hydroxyectoine in Pseudomonas stutzeriA1501

“…The substantial increase in the hydroxyectoine and ectoine pool of osmotically stressed P. stutzeri A1501 cells is consistent with previous assessments of ectoine and hydroxyectoine production in response to osmotic stress in various microorganisms (8,9,32,33,58). However, the almost exclusive synthesis of hydroxyectoine by P. stutzeri A1501 is unusual, since most ectoine and hydroxyectoine producers typically contain a mixture of these two solutes with a considerable fraction of ectoine in exponential-phase cells (8,9,58). Our data, therefore, show that the ectABCD gene cluster of P. stutzeri A1501 is functional and allows the bacterium to amass considerable amounts of hydroxyectoine and moderate amounts of ectoine as osmostress protectants.…”

“…The type strain of P. stutzeri, DSM5190 T , is known to synthesize, in addition to large amounts of hydroxyectoine, minor quantities of trehalose and the peptide NAGGN in response to osmotic stress (58). Since these compatible solutes are also produced by many other Pseudomonas species (35), we assessed the presence of the corresponding structural genes in the genome sequence of P. stutzeri A1501 (67).…”

“…The genome sequences of P. stutzeri A1501 were analyzed for the presence of homologous enzymes involved in synthesis of the compatible solutes trehalose and N-␥-acetlyglutaminyl glutamine 1-amide (NAGGN) (3,35,58). The following protein sequences were used as search queries for BlastP: trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase of E. coli (NP_416410 and NP_416411), trehalose synthase of Streptomyces avermitilis (NP_823979), alpha amylase of Streptomyces coelicolor (NP_630189), maltooligosyltrehalose trehalohydrolase of Rhodopseudomonas palustris (NP_948984), trehalose synthase of Pyrococcus furiosus (NP_579471), trehalose phosphorylase of Propionibacterium acnes (YP_055814), and glutamine amidotransferase and NGG acetyltransferase of Pseudomonas aeruginosa (NP_252149).…”

“…T , whose genome has not yet been sequenced, an ectABCD_ ask_ect gene cluster is also present (58), and Seip et al observed that hydroxyectoine dominates the compatible solute pool of stationary-phase cells with minor amounts of ectoine and with more limited contributions of the disaccharide trehalose and the dipeptide NAGGN (58).…”

“…Lateral gene transfer is a major source of genetic and physiological innovation in the microbial world (59). The acquisition of the capacity to produce ectoine/ hydroxyectoine, in addition to the synthesis of the compatible solutes trehalose and the dipeptide NAGGN that are commonly found in pseudomonads (35,58), should enhance the ability of P. stutzeri A1501 to cope flexibly with osmotic stress in its varied habitats. In the P. stutzeri type strain DSM5190…”