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Applied Microbiology and Biotechnology (v.61, #1)
Termite symbiotic systems: efficient bio-recycling of lignocellulose by M. Ohkuma (pp. 1-9).
Termites thrive in great abundance in terrestrial ecosystems and play important roles in biorecycling of lignocellulose. Together with their microbial symbionts, they efficiently decompose lignocellulose. In so-called lower termites, a dual decomposing system, consisting of the termite's own cellulases and those of its gut protists, was elucidated at the molecular level. Higher termites degrade cellulose apparently using only their own enzymes, because of the absence of symbiotic protists. Termite gut prokaryotes efficiently support lignocellulose degradation. However, culture-independent molecular studies have revealed that the majority of these gut symbionts have not yet been cultivated, and that the gut symbiotic community shows a highly structured spatial organization. In situ localization of individual populations and their functional interactions are important to understand the nature of symbioses in the gut. In contrast to cellulose, lignin degradation does not appear to be important in the gut of wood-feeding termites. Soil-feeding termites decompose humic substances in soil at least partly, but little is known about the decomposition. Fungus-growing termites are successful in the almost complete decomposition of lignocellulose in a sophisticated cooperation with basidiomycete fungi cultivated in their nest. A detailed understanding of efficient biorecycling systems, such as that for lignocellulose, and the symbioses that provide this efficiency will benefit applied microbiology and biotechnology.
Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production by R. de Vries (pp. 10-20).
The genus Aspergillus is widely used for the production of plant cell wall polysaccharide-degrading enzymes. The range of enzymes purified from these fungi covers nearly every function required for the complete degradation of cellulose, xyloglucan, xylan, galacto(gluco)mannan and pectin. This paper describes the Aspergillus enzymes involved in the degradation of these polysaccharides and discusses the regulatory systems involved in the expression of the genes encoding these proteins. The latter is of major importance in the large-scale production of these enzymes for industrial applications.
Biotin in microbes, the genes involved in its biosynthesis, its biochemical role and perspectives for biotechnological production by W. R. Streit; P. Entcheva (pp. 21-31).
Biotin (vitamin H) is one of the most fascinating cofactors involved in central pathways in pro- and eukaryotic cell metabolism. Since its original discovery in 1901, research has led to the discovery of the complete biotin biosynthesis pathways in many different microbes and much work has been done on the highly intriguing and complex biochemistry of biotin biosynthesis. While humans and animals require several hundred micrograms of biotin per day, most microbes, plants and fungi appear to be able to synthesize the cofactor themselves. Biotin is added to many food, feed and cosmetic products, creating a world market of 10–30 t/year. However, the majority of the biotin sold is synthesized in a chemical process. Since the chemical synthesis is linked with a high environmental burden, much effort has been put into the development of biotin-overproducing microbes. A summary of biotin biosynthesis and its biological role is presented; and current strategies for the improvement of microbial biotin production using modern biotechnological techniques are discussed.
Optimisation of cellobiose dehydrogenase production by the fungus Sclerotium (Athelia) rolfsii by R. Ludwig; D. Haltrich (pp. 32-39).
The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l−1 (15,000 units cytochrome c activity l−1) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.
Fed-batch cultivation of the docosahexaenoic-acid-producing marine alga Crypthecodinium cohnii on ethanol by M. de Swaaf; J. Pronk; L. Sijtsma (pp. 40-43).
The heterotrophic marine microalga Crypthecodinium cohnii produces docosahexaenoic acid (DHA), a polyunsaturated fatty acid with food and pharmaceutical applications. So far, DHA production has been studied with glucose and acetic acid as carbon sources. This study investigates the potential of ethanol as an alternative carbon source for DHA production by C. cohnii. In shake-flask cultures, the alga was able to grow on ethanol. The specific growth rate was optimal with 5 g l–1 ethanol and growth did not occur at 0 g l–1 and above 15 g l–1. By contrast, in fed-batch cultivations with a controlled feed of pure ethanol, cumulative ethanol addition could be much higher than 15 g l–1, thus enabling a high final cell density and DHA production. In a representative fed-batch cultivation of C. cohnii with pure ethanol as feed, 83 g dry biomass l–1, 35 g total lipid l–1 and 11.7 g DHA l–1 were produced in 220 h. The overall volumetric productivity of DHA was 53 mg l–1 h–1, which is the highest value reported so far for this alga.
Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in Terrabacter sp. strain DBF63 by H. Habe; M. Miyakoshi; J. Chung; K. Kasuga; T. Yoshida; H. Nojiri; T. Omori (pp. 44-54).
Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene (bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53–62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.
Cloning and characterization of two α-glucosidases from Bifidobacterium adolescentis DSM20083 by L. van den Broek; K. Struijs; J. Verdoes; G. Beldman; A. Voragen (pp. 55-60).
Two α-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both α-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-α-glucoside. The K m for pnp-α-glucoside was 1.05 and 0.47 mM and the V max was 228 and 113 U mg–1 for AglA and AglB, respectively. Using pnp-α-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37°C. For AglB, values of pH 6.8 and 47°C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant α-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.
Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum:implications for DNA microarray analysis by C. Glanemann; A. Loos; N. Gorret; L. B. Willis; X. M. O'Brien; P. A. Lessard; A. J. Sinskey (pp. 61-68).
The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum. Gene expression changes during C. glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR). The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling. Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data. Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C. glutamicum. Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes. Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.
Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects by B. S. Ferreira; C. R. C. Calado; F. van Keulen; L. P. Fonseca; J. M. S. Cabral; M. M. R. da Fonseca (pp. 69-76).
Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell—required for the induction of cutinase production—and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.
A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion by E. S. Gilbert; A. W. Walker; J. D. Keasling (pp. 77-81).
A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 μM parathion (146 mg l−1) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis–Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 μM p-nitrophenol (83 mg l−1) and P. putida KT2440 pSB337 inhibited by 1,000 μM p-nitrophenol (139 mg l−1). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.