Improvement of shikimic acid production in Escherichia coli with growth phase-dependent regulation in the biosynthetic pathway from glycerol.

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33 g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.

High efficiency transformation by electroporation of Yarrowia lipolytica.

Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of F. lipolytica, we report here that pretreatment of F. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 10(10)/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform F. lipolytica that will be able to yield an efficiency of 2.1 × 10(4) transformants/ug for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for F. lipolytica transformation.

Microbial communities and bacterial diversity of spruce, hemlock and grassland soils of Tatachia Forest, Taiwan.

To evaluate the bacterial diversity of Tatachia Forest soils, 16S rDNA clone libraries of the spruce, hemlock and grassland soils were constructed. Further, the influence of physicochemical and biological properties of soil on microbial ecology, pH, moisture content, microbial population and biomass were also analyzed. The soil pH increased with the increasing of soil depth; whereas the microbial population, biomass, moisture content, total organic carbon and total nitrogen were reverse. Microbial populations were the highest in the summer season which also correlated with the highest moisture content. The phylogenetic analyses revealed that the clones from nine 16S rDNA clone libraries represented Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Gemmatimonadetes, Planctomycetes, Verrucomicrobia, candidate division TG1 and candidate division TM7. Members of Proteobacteria, Acidobacteria and Actinobacteria constituted 42.2, 35.1 and 7.8 % of the clone libraries, respectively; whereas the remaining bacterial divisions each comprised <3 %. The spruce site had the highest bacterial diversity among the tested sites, followed by the hemlock sites and the grassland sites with the least. The bacterial community is the more diverse in the organic layer than that in deeper horizons. Further, bacterial diversity through the gradient horizons was different, indicating that the bacterial diversity in the deeper horizons is not simply the diluted analogs of the surface soils and some microbes dominate only in the deeper horizons.

Seasonal variation of microbial populations and biomass in Tatachia grassland soils of Taiwan.

To investigate the seasonal variations of microbial ecology in grassland of Tatachia forest, soil properties, microbial populations, microbial biomass, and 16S rDNA clone library analysis were determined. The soil had temperatures 6.6-18.4 degrees C, pH 3.6-5.1, total organic carbon 1.11-10.68%, total nitrogen 0.18-0.78%, and C/N ratios 3.46-20.55. Each gram of dry soil contained bacteria, actinomycetes, fungi, cellulolytic, phosphate-solubilizing microbes, and nitrogen-fixing microbes 4.54 x 10(4) to 3.79 x 10(7), 3.43 x 10(2) to 2.17 x 10(5), 5.74 x 10(3) to 3.76 x 10(6), 1.97 x 10(3) to 1.34 x 10(6), 8.49 x 10(2) to 5.59 x 10(5), and 3.86 x 10(2) to 3.75 x 10(5) CFU, respectively. Each gram of soil contained 117-2,482 microg of microbial biomass carbon, 23-216 microg of microbial biomass nitrogen and 9-29 microg of DNA. The microbial populations, microbial biomass, and DNA decreased stepwise with the depth of soil, and they had low values in winter seasons. The microbial populations, microbial biomass carbon, microbial biomass nitrogen, and DNA at the BW2 horizon were 8.42-17.84, 19.26-64.40, 16.84-61.11, and 31.03-46.26% of those at the O horizon, respectively. When analyzing 16S rDNA library, members of Proteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, candidate division TMI, candidate division TM7, Gammatimonadetes, and Verrucomicrobia were identified. Members of Proteobacteria (44.4%) and Acidobacteria (33.3%) dominated the clone libraries. Within the phylum Proteobacteria, alpha-, beta-, and gamma-Proteobacteria were most numerous, followed by delta-Proteobacteria.

Seasonal variation of microbial ecology in hemlock soil of Tatachia Mountain, Taiwan.

BACKGROUND AND PURPOSE: Forest soil microorganisms and fauna decompose the organic materials, and thus strongly influence the nutrient cycling of the ecosystem. Soil microorganisms also contribute to soil structure and soil fertility. In Taiwan, the microbial distributions of soils have only been determined in acidic soil, inorganic acidic soil, upland soil, alkaline soil and power plant areas. There are few data on the microbial populations of forest soils. Tatachia Mountain is located in the central part of Taiwan and is a typical high altitude protected ecosystem area, designated as a National Park. This study investigated the role of microorganisms in the ecology and nutrient transformation of forest soil in Taiwan. METHODS: As part of long-term ecological research in Taiwan, the environmental conditions, seasons, microbial populations, biomass and organic acid contents of hemlock soil were investigated. We also studied the effect of depth on microbial populations and biomass. RESULTS: The soil temperatures were between 5.5 and 15.6 degrees C and the soil pH ranged from 3.3 to 4.4. Total organic carbon and total nitrogen contents ranged from 2.3 to 37.1% and from 0.3 to 1.7%, respectively. The carbon/nitrogen ratio was between 8.2 and 24.4. In topsoil, each gram of soil contained 10(5)-10(7) colony-forming units (CFU) culturable bacteria, 10(2)-10(5) CFU actinomycetes, 10(3)-10(5) CFU fungi, 10(4)-10(6) CFU cellulolytic microbes, 10(4)-10(6) CFU phosphate-solubilizing microbes, and 10(3)-10(6) CFU nitrogen-fixing microbes. Microbial populations were higher in topsoil compared with subsoil, but lower in topsoil than in organic layer. Microbial populations also decreased with the depth of soil. Microbial populations at 1E horizon were 0.6% to 9.4% of those at O horizon. The microbial biomass evaluated contained carbon 391-1013 mug, nitrogen 51-146 mug, malic acid 76-557 nM and succinic acid 37-527 nM per gram of soil. Summer season had higher microbial populations, biomass and organic content than winter season, but the differences were not significant. CONCLUSION: Heavy coverage of organic matter was found in hemlock and spruce soils and was associated with acidic pH. Microbial populations decreased with increasing soil depth. Microbes play a very important role in organic matter decomposition and nutrition transformation in hemlock soil.