Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium

Abstract

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter ؊1 ectoine with a space-time yield of 40 mg liter ؊1 h ؊1 , with the vast majority of the ectoine being excreted.Halophilic microorganisms live in highly saline environments. There are two major strategies of adaptation to these hostile conditions. Extreme halophiles such as Halobacterium salinarum accumulate salt in the cytosol to maintain the osmotic balance (the salt-in strategy). Other halophiles synthesize and accumulate small organic molecules as osmotic counterweights (the organic-osmolyte strategy). Unlike intracellular salt, the small organic compounds do not affect the metabolism of the organism and are thus called compatible solutes. The enzymes of organic-osmolyte strategists do not need to be haloadapted, allowing these organisms to cope with strong salinity fluctuations. Non-salt-tolerant bacteria like Escherichia coli are usually unable to synthesize large amounts of compatible solutes but may resist halostress to a certain extent by taking up and accumulating compatible solutes (8,9,12,27).Different classes of chemical compounds, including polyols, sugars, methylamines, and linear and cyclic amino acids and betaines, have been found to act as compatible solutes. Besides functioning as osmotic counterweights, compatible solutes were shown to protect biomolecules and whole cells against denaturation caused by heating, freezing, desiccation, or chemical agents (15,19,22). This property has attracted commercial attention. Compatible solutes can be used as chemical chaperones for protein folding, enhancers of PCR performance, cryoprotectants of microorganisms, cosmeceuticals, and pharmaceuticals (20, 32). A potentially promising future application could be the enhancement of drought tolerance or salt tolerance of transgenic plants (1, 37).The best-investigated compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), is biotechnologically produced with the halophilic bacterium Halomonas elongata (34). The physiology and genetics of ectoine biosynthesis in this bacterium have been studied in detail (7,23,24,25,26,29). The technical bioprocess for the synthesis of ectoine exploits the salt adaptation strategy of H. elongata and is called "bacterial milking" (34). When the medium has a high salt concentration, the bacterium s...

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

“…In order to avoid the problem of by-product separation, we aimed at establishing a heterologous production process to obtain almost pure hydroxyectoine. So far, de novo heterologous production has been reported only for ectoine (2,29,35,40) and not for hydroxyectoine. In all these studies, a coherent ectABC gene cluster was used.…”

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

“…Attempts are being made to establish production processes for ectoine and hydroxyectoine that are based on recombinant systems hyperexpressing the ect biosynthetic genes in easy-tomanipulate host strains, e.g., E. coli (5,40,49,56,58). Since E. coli does not naturally synthesize ectoine (28), there is no adequate metabolic and genetic adaptation of the aspartokinases and their structural genes in this host (14) to effectively supply the ectoine biosynthetic precursor aspartate-beta-semialdehyde under osmotic stress conditions.…”

Influence of initial glucose concentration on seed culture of sodium gluconate production by Aspergillus niger

“…This result was in accordance with the findings of Wucherpfennig et al (2011) that osmotic pressure could affect the spore germination and the high osmolality could increase time span. The interpretation was that the cells would produce compatible solutes in the early stage of seed culture to adapt to the environment and maintain the intercellular and external osmotic balance, which was more conducive to cell growth and metabolism (Schubert et al 2007). Notably, although the fermentation performances by 300 and 400 g/L initial glucose concentration were similar, the spore germination time with 300 g/L was obviously shorter than 400 g/L.…”

“…Similarly, the groups A, B, and E with low or high initial glucose concentrations had low glucose consumption rate and product yield, and more metabolic flux flowed to synthesize metabolites responding to environmental stresses for facilitating cell growth. In general, there was a relatively complete strategy for microorganism to respond to osmotic stress, containing the selfsynthesis or exogenous uptake of compatible solutes and osmoprotectants, metabolic flux redistribution, and membrane fluidity variations (Schubert et al 2007;Saum and Müller 2007;Simonin et al 2008;Varela et al 2003).…”