Background: As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. Results: The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L⁻¹ NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L⁻¹ ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L⁻¹), and also exceeds the production of Y2/ΔectD (2.21 g L⁻¹). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na⁺/H⁺ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L⁻¹ or 500 mg (g DCW) ⁻¹) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L⁻¹ NaCl stress, a maximum 10.5 g L⁻¹ ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)⁻¹ and a yield of 0.21 g g⁻¹ monosodium glutamate. Conclusion: The remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na⁺/H⁺ antiporters in their halophilic producers and thus lowering the medium salinity.

Background: Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.Results: A novelFlavobacteriaceae Sinomicrobiumα-amylase (FSA) was identified and characterized from genomic analysis of a novelFlavobacteriaceaespecies. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn²⁺- and Ca²⁺-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.Conclusions: The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.

Thermoacidophilic Alicyclobacillus strains attract great interests as the resource of thermostable or acidic enzymes. In this study, a putative gene encoding superoxide dismutase (AaSOD) was identified in a thermoacidophilic Alicyclobacillus strain. With a 16-fold activity observed, the AaSOD activity expressing in the medium of manganese enrichment was much higher than that in the iron medium. In addition, the purified AaSOD can be reconstituted exclusively with either Fe²⁺ or Mn²⁺, with its Mn-bound protein showing 25-fold activity than that of Fe-bound form. The optimal temperature for AaSOD reaction was 35°C, and was highly stable at any certain temperature up to 80°C. Of particular interest, the enzyme is found to be very stable across a wide pH range spanning from 2.0 to 10.0, which confers its robust stability in the acidic stomach environment and implies striking potentials as food additive and for medical use.

Whole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications. G. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ∆GOX1068∆GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ∆GOX1068∆GOX0854 as the biocatalyst, 61.6 g L−1 xylonate was produced from 58.4 g L−1 xylose at a yield of 1.05 g g−1. This process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol.

Mesophilic α-amylase from Flavobacteriaceae (FSA) is evolutionary closely related to thermophilic archaeal Pyrococcus furiosus α-amylase (PWA), but lacks the high thermostability, despite the conservation of most residues involved in the two-metal (Ca, Zn) binding center of PWA. In this study, a disulfide bond was introduced near the two-metal binding center of FSA (designated mutant EH-CC) and this modification resulted in a slight improvement in thermostability. As expected, E204G mutations in FSA and EH-CC led to the recovery of Ca²⁺-binding site. Interestingly, both Ca²⁺- and Zn²⁺-dependent thermostability were significantly enhanced; 153.1% or 50.8% activities was retained after a 30-min incubation period at 50 °C, in the presence of Ca²⁺ or Zn²⁺. The C214S mutation, which affects Zn²⁺-binding, also remarkably enhanced Zn²⁺- and Ca²⁺- dependent thermostability, indicating that Ca²⁺- and Zn²⁺-binding sites function cooperatively to maintain protein stability. Furthermore, an isothermal titration calorimetry (ITC) analysis revealed a novel Zn²⁺-binding site in mutant EH-CC-E204G. This metal ion cooperation provides a possible method for the generation of α-amylases with desired thermal properties by in silico rational design and systems engineering, to generate a Zn²⁺-binding site adjacent to the conserved Ca²⁺-binding site.