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Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five-to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.Trehalose (1-␣-glucopyranosyl-1-␣-glucopyranoside) is a nonreducing, particularly stable disaccharide formed by two glucose moieties (37). As a compatible osmolite (22) and protein stabilizer (39), trehalose shows a wide range of potential applications in biotechnology (increased stress tolerance of important crops, stability of recombinant proteins, etc.), as well as in the food industry (37).In the past, trehalose was produced by using Saccharomyces cerevisiae (32). The high cost of this system and the promising applications of the disaccharide led to the development of a new trehalose production process based on the enzymatic biotransformation of maltodextrins (25,26). The success of the enzymatic process has limited the interest in using microorganisms as alternative sources for trehalose synthesis. However, the recent development of metabolic engineering tools, allowing the rational design of microorganisms for metabolite production (27), prompted us to evaluate an alternative process for trehalose overproduction in the gram-positive bacterium Corynebacterium glutamicum (23). C. glutamicum was chosen for three major reasons: (i) it produces, and excretes, trehalose (15); (ii) it has a metabolic control architecture simpler than that of other microorganisms, maybe as a result of its comparatively small 3,500-kb genome size (10); and (iii) it is widely used in industrial biotechnological processes (10).Three pathways for trehalose synthesis have been characterized in C. glutamicum (45), similarly to that found in Mycobacterium species (7) (Fig. 1). The first is the TreS pathway, in which trehalose is formed by maltose isomerization (7, 31).The second is the TreY-TreZ pathway, le...

The prokaryotic oxidation of reduced inorganic sulfur compounds (RISCs) is a topic of utmost importance from a biogeochemical and industrial perspective. Despite sulfur oxidizing bacterial activity is largely known, no quantitative approaches to biological RISCs oxidation have been made, gathering all the complex abiotic and enzymatic stoichiometry involved. Even though in the case of neutrophilic bacteria such as Paracoccus and Beggiatoa species the RISCs oxidation systems are well described, there is a lack of knowledge for acidophilic microorganisms. Here, we present the first experimentally validated stoichiometric model able to assess RISCs oxidation quantitatively in Acidithiobacillus thiooxidans (strain DSM 17318), the archetype of the sulfur oxidizing acidophilic chemolithoautotrophs. This model was built based on literature and genomic analysis, considering a widespread mix of formerly proposed RISCs oxidation models combined and evaluated experimentally. Thiosulfate partial oxidation by the Sox system (SoxABXYZ) was placed as central step of sulfur oxidation model, along with abiotic reactions. This model was coupled with a detailed stoichiometry of biomass production, providing accurate bacterial growth predictions. In silico deletion/inactivation highlights the role of sulfur dioxygenase as the main catalyzer and a moderate function of tetrathionate hydrolase in elemental sulfur catabolism, demonstrating that this model constitutes an advanced instrument for the optimization of At. thiooxidans biomass production with potential use in biohydrometallurgical and environmental applications.

The production of bleached Kraft pulp generates inorganic and organic residues that are usually deposited on the soil surface or land-filled. Studies conducted to address the impact of these wastes on the environment are scarce. In this work, Monterey pine (Pinus radiata D. Don), an important tree for pulping, was evaluated for germination and development under greenhouse conditions in forest soils exposed to solid residues of the cellulose industry using the Kraft process. Soils exposed to 10 to 60% ashes, 10 to 70% fly ashes, or 10 to 30% dregs allowed substantial seed germination and seedling growth. In contrast, soils exposed to low proportions of brown rejects, grits, or a mixture of all these residues were detrimental for germination, plant growth, or both. The strongest negative effect (no germination) was observed with as low as 10% grits. The changes in pH and/or water content caused by solid wastes did not correlate with detrimental effects observed in various soil-residue combinations. No significant changes in the microbial community of soils exposed to these solid residues were observed by determination of culturable counts, or by terminal-restriction fragment length polymorphism analysis of the microbial community DNA. The presence of organic residues did not affect the ability of the soil microbial community to remove typical pulp bleaching chloroaromatics. However, inorganic wastes strongly decreased the removal of such compounds.

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.Trehalose (␣-glucopyranosyl-␣-glucopyranoside) is a stable, odor-free, nonreducing disaccharide that is widespread in nature (34). Its protein-stabilizing properties have suggested a wide range of commercial applications, ranging from cosmetics to the agricultural food sector (18). In the 1990s, the high cost of this disaccharide triggered the development of methods based on immobilized enzymes to obtain trehalose from maltodextrins (20,21). This achievement drastically reduced the commercial price of trehalose (34). A microbiological alternative for large-scale production of trehalose relying on Corynebacterium glutamicum (19), a gram-positive bacterium able to synthesize and excrete this sugar (37, 41), was recently proposed (27).Trehalose synthesis in C. glutamicum during growth on glucose proceeds through two major pathways ( Fig. 1): (i) from UDP-glucose and glucose-6-phosphate (OtsAB pathway) and (ii) from malto-oligosaccharides or ␣-1,4 glucans (TreYZ pathway) (36,42). Recently, a sixfold increase in OtsBA pathway activity in C. glutamicum (27) was achieved through heterologous expression of the Escherichia coli otsBA operon encoding the trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) (17). However, this approach resulted in only a twofold increase in the amount of excreted trehalose (27). These results suggested that further improvements in trehalose synthesis were hampered by the low activity of UDP-glucose pyrophosphorylase, which is responsible for UDP-glucose synthesis (17) according to the following reaction: glucose-1-P ϩ UTP 3 UDP-glucose ϩ pyrophosphate. This enzyme is critical in Lactococcus lactis and Streptococcus thermophilus during exopolysaccharide accumulation (3, ...