Morphological, physiological, biochemical and molecular characterization of statin-producing Penicillium microfungi isolated from little-explored tropical ecosystems.

As hypercholesterolemia is a primary risk factor for coronary artery disease and stroke, there is now an increasing demand for cholesterol-lowering drugs. Statins are a group of extremely successful drugs that lower the cholesterol level in the blood. Natural statins are produced by fermentation using different species of microorganisms. The overall aim of the present study was to identify statin-producing microfungi, which were isolated from different types of little-explored mangrove and oil palm plantation soils. Isolated fungal cultures were characterized on the basis of morphological, physiological, biochemical, and molecular features. Morphological variability was detected amongst the fungal isolates in regard to colony morphology, conidiophores structures, and conidia coloration. Based on their physiological properties and enzyme assays, rapid differentiation of statin-producing isolates was achieved. Further molecular characterization allowed reliable identification of the selected Penicillium microfungi up to the species level. The identified Penicillium cintrinum ESF2M, Penicillium brefeldianum ESF21P, and Penicillium janthinellum ESF26P strains have a scientific interest as novel wild-type producers of natural statins.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2'-fucosyllactose in recombinant Escherichia coli.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and is important for infant nutrition and health. Because 2'-FL has potential as a functional ingredient in advanced infant formula and as a prebiotic in various foods, a cost-effective method for 2'-FL production is desirable. α1,2-Fucosyltransferase (α1,2-FT) is one of the key enzymes enabling the microbial biosynthesis of this complex sugar. However, the α1,2-FTs reported so far for the whole-cell biosynthesis of 2'-FL originate from pathogens, posing a potential hurdle for approval as a food production method depending on countries. In this study, 10 α1,2-FT genes from bacteria of biosafety level one were identified, and the main features of the deduced amino acid sequences were characterized. Four codon-optimized α1,2-FT genes were synthesized and introduced into Escherichia coli ΔL M15 strain containing the plasmid pBCGW encoding guanosine 5'-diphosphate-l-fucose biosynthetic enzymes. Among the four genes, 2'-FL was produced only by the α1,2-FT from Thermosynechococcus elongatus (Te2FT). Bifidobacterium thermacidophilum α1,2-FT (Bt2FT) showed high expression but was not active in E. coli ΔL M15. The other two α1,2-FTs were not expressed to a detectable level. During batch flask fermentation of Te2FT-expressing E. coli ΔL M15 cells, 0.49 g/L 2'-FL was obtained after 72 h of induction. This is comparable to the values previously reported for α1,2-FTs from Helicobacter pylori and Bacteroides fragilis.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.

A Simple Enzymatic Method for Quantitation of 2'-Fucosyllactose.

2'-Fucosyllactose (2'-FL) is one of the most important human milk oligosaccharides and has several health benefits for infants. The levels of 2'-FL in breast milk or samples from other sources can bequantified by high-performance liquid chromatography. However, this method cannot be used for simultaneous detection of the target compound in numerous samples. Here, we developed a simple method for quantifying 2'-FL in a microplate format. The method involves two steps: (1) release of L-fucose from 2'-FL by α-(1-2,3,4,6)-L-fucosidase and (2) measurement of NADPH formed during the oxidation of L-fucose by L-fucose dehydrogenase. This method enables measurement of up to 5 g/l 2'-FL in 50 min using a 96-well microplate. The efficiency and simplicity of the proposed method make it suitable for the analyses of a large number of samples simultaneously.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli.

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.