Beschreibung: Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MAT a/alpha, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP.

Beschreibung: Acremonium cellulolyticus is one of several fungi that offer promise as an alternative to Trichoderma reesei for use in industrial cellulase production. However, the mechanism of cellulase production has not been studied at the molecular level because adequate genetic engineering tools for use in A. cellulolyticus are lacking. In the present study, we developed a gene disruption method for A. cellulolyticus, which needs a longer homologous region length. We cloned a putative A. cellulolyticus creA gene that is highly similar to the creA genes derived from other filamentous fungi, and isolated a creA disruptant strain by using the disruption method. Growth of the creA disruptant on agar plates was slower than that of the control strain. In the wild-type strain, the CreA protein was localized in the nucleus, suggesting that the cloned gene encodes the CreA transcription factor. Cellulase and xylanase production by the creA disruptant were higher than that of the control strain at the enzyme and transcription levels. Furthermore, the creA disruptant produced cellulase and xylanase in the presence of glucose. These data suggest both that the CreA protein functions as a catabolite repressor protein, and that disruption of creA is effective for enhancing enzyme production by A. cellulolyticus.

Beschreibung: Variation in the growing environment can have significant impacts on the quantity and diversity of fungal secondary metabolites. In the industrial setting, optimization of growing conditions can lead to significantly increased production of a compound of interest. Such optimization becomes challenging in a drug-discovery screening situation, as the ideal conditions for one organism may induce poor metabolic diversity for a different organism. Here, the impact of different media types, including six liquid media and five solid media, on the secondary metabolite production of three fungal strains was examined in the context of the drug-discovery screening process. The relative production of marker compounds was used to evaluate the usefulness and reliability of each medium for the purpose of producing secondary metabolites.

Beschreibung: We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying alpha-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40[degree sign]C, which are higher than the optimal growth temperature of 30[degree sign]C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30[degree sign]C. However, alpha-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30[degree sign]C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30[degree sign]C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated.

Beschreibung: 5-Hydroxy-l-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified l-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, l-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the lamda red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of l-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4alpha-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

Beschreibung: Most whole cell biocatalysts have some problems with yields and productivities because of various metabolites produced as byproducts and limitations of substrate uptake. We propose a psychrophile-based simple biocatalyst for efficient bio-production using mesophilic enzymes expressed in psychrophilic Shewanella livingstonensis Ac10 cells whose basic metabolism was inactivated by heat treatment. The 45[degree sign]C heat-treated cells expressing lacZ showed maximum beta-galactosidase activity as well as chloroform/SDS-treated cells to increase membrane permeability. The fluorescent dye 5-cyano-2,3-ditolyl-tetrazolium chloride staining indicated that most basic metabolism of Ac10 was lost by heat treatment at 45 C for 10 min. The simple biocatalyst was applied for 3-HPA production by using Klebsiella pneumoniae dhaB genes. 3-HPA was stoichiometrically produced with the complete consumption of glycerol at a high production rate of 8.85 mmol 3-HPA/g dry cell/h. The amount of 3-HPA production increased by increasing the concentrations of biocatalyst and glycerol. Furthermore, it could convert biodiesel-derived crude glycerol to 3-HPA.

Beschreibung: Biosurfactants are the surface active compounds produced by micro-organisms. The eco-friendly and biodegradable nature of biosurfactants makes their usage more advantageous over chemical surfactants. Biosurfactants encompass the properties of dropping surface tension, stabilizing emulsions, promoting foaming and are usually non- toxic and biodegradable. Biosurfactants offer advantages over their synthetic counterparts in many applications ranging from environmental, food, and biomedical, cosmetic and pharmaceutical industries. The important environmental applications of biosurfactants include bioremediation and dispersion of oil spills, enhanced oil recovery and transfer of crude oil. The emphasis of present review shall be with reference to the commercial production, current developments and future perspectives of a variety of approaches of biosurfactant production from the micro-organisms isolated from various oil- contaminated sites and from the by-products of oleo-chemical industry wastes/ by-products viz. used edible oil, industrial residues, acid oil, deodorizer distillate, soap-stock etc.

Beschreibung: In this study, we demonstrate the one-step production of cadaverine (1,5-diaminopentane) from cellobiose using an Escherichia coli strain displaying beta-glucosidase (BGL) on its cell surface. L-lysine decarboxylase (CadA) derived from E. coli and BGL from Thermobifida fusca YX (Tfu0937) fused to the anchor protein Blc from E. coli were co-expressed using E. coli as a host. The expression of CadA was confirmed by Western blotting and BGL activity on the cell surface was evaluated using pNPG as a substrate. Growth on cellobiose as the sole carbon source was also achieved. The OD600 value of the BGL and CadA co-expressing strain was 8.0 after 48 h cultivation, which is higher than that obtained by growth on glucose (5.4 after 48 h cultivation). The engineered strain produced cadaverine from cellobiose more effectively than from glucose: 6.1 mM after 48 h from 28 g/L of consumed cellobiose, vs. 3.3 mM from 20 g/L of consumed glucose .

Beschreibung: Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.

Beschreibung: The mangroves are among the most productive and biologically important environments. The possible presence of cellulolytic enzymes and microorganisms useful for biomass degradation as well as taxonomic and functional aspects of two Brazilian mangroves were evaluated using cultivation and metagenomic approaches. From a total of 296 microorganisms with visual differences in colony morphology and growth (including bacteria, yeast and filamentous fungus), 179 (60.5%) and 117 (39.5%) were isolated from the Rio de Janeiro (RJ) and Bahia (BA) samples, respectively. RJ metagenome showed the higher number of microbial isolates, which is consistent with its most conserved state and higher diversity. The metagenomic sequencing data showed similar predominant bacterial phyla in the BA and RJ mangroves with an abundance of Proteobacteria (57.8% and 44.6%), Firmicutes (11% and 12.3%) and Actinobacteria (8.4% and 7.5%). A higher number of enzymes involved in the degradation of polycyclic aromatic compounds were found in the BA mangrove. Specific sequences involved in the cellulolytic degradation, belonging to cellulases, hemicellulases, carbohydrate binding domains, dockerins and cohesins were identified, and it was possible to isolate cultivable fungi and bacteria related to biomass decomposition and with potential applications for the production of biofuels. These results showed that the mangroves possess all fundamental molecular tools required for building the cellulosome, which is required for the efficient degradation of cellulose material and sugar release.