Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima

A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a VM of 2.9 Å3 Da-1 and 58% solvent content.

Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain

Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 °C and at pH 8.5, with a preference for esters with short acyl chains (C2–C10). Its 2.6-Å-resolution crystal structure revealed a classical α/β hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-Å-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA.

Monascus ruber as cell factory for lactic acid production at low pH

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175 g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190 g/l at pH 3.8 and 129 g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH