Industrial Relevance of Trichoderma reesei as an Enzyme Producer.

Trichoderma reesei's potential as a rapid and efficient biomass degrader was first recognized in the 1950s when it was isolated from Army textiles during World War II. The microbe secreted cellulases that were degrading cotton-based tents and clothing of service members stationed on the Solomon Islands. In the 1970s, at the time of the first global oil crisis, research interest in T. reesei gained popularity as it was explored as part of the solution to the worlds growing dependence on fossil fuels. Much of this early work focused on classical mutagenesis and selection of hypercellulolytic strains. This early lineage was used as a starting point for both academic research with the goal of understanding secretion and regulation of expression of the complex mixture of enzymes required for cellulosic biomass decay as well as for its development as a host for industrial enzyme production. In 2001, at the onset of the second major oil crisis, the US Department of Energy supported research programs in microbial cellulases to produce ethanol from biomass which led to another surge in the study of T. reesei. This further accelerated the development of molecular biology and recombinant DNA tools in T. reesei. In addition to T. reesei's role in bio-ethanol production, it is used to produce industrial enzymes with a broad range of applications supporting the bio-based economy. To date there are around 243 commercially available enzyme products manufactured by fermentation of microorganisms; 30 of these are made using Trichoderma as a host, 21 of which are recombinant products sold for use in food, feed, and technical applications including textiles and pulp and paper.

Short communication: The effect of heat treatment of bovine colostrum on the concentration of oligosaccharides in colostrum and in the intestine of neonatal male Holstein calves.

The objective of this study was to determine the effect of the heat treatment (HT, 60°C for 60 min) on the concentration of bovine colostrum oligosaccharides (bCO) in pooled bovine colostrum and the intestine of neonatal male Holstein calves after feeding. First-milking colostrum was pooled from both primiparous and multiparous cows, and half of the pooled colostrum was heat-treated at 60°C for 60 min (HC), whereas the other half was not heat-treated and remained fresh (FC). At birth, 32 male Holstein calves were randomly assigned to 1 of 3 treatment groups: (1) control calves that did not receive colostrum for the duration of the experiment and were euthanized at 6 h (NC, n = 4) or 12 h (NC, n = 4), (2) calves fed fresh colostrum (FC) and were euthanized at 6 h (FC, n = 6) or 12 h (FC, n = 6), or (3) calves fed heat-treated colostrum (HC) and euthanized at 6 h (HC, n = 6) or 12 h (HC, n = 6). All calves were fed 2 L of colostrum within 1 h after birth. At dissection, digesta of the distal jejunum, ileum, and colon was collected and analyzed by liquid chromatography-mass spectrometry to determine the concentration of bCO within each intestinal region. The heat-treated colostrum displayed numerically higher concentrations of total bCO (3,511.6 µg/g) when compared with fresh colostrum (1,329.9 µg/g), with 3'-sialyllactose being the most abundant bCO in both fresh and HT colostrum. In contrast, calves fed HT colostrum displayed a lower amount of total bCO in the distal jejunum (221.91 ± 105.3 vs. 611.26 ± 265.1 µg/g), ileum (64.97 ± 48.39 vs. 344.04 ± 216.87 µg/g), and colon (25.60 ± 13.1 vs. 267.04 ± 125.81 µg/g) at 6 h of life when compared with calves fed fresh colostrum. No differences were observed in regard to the concentrations of total bCO in the intestine of FC and HC calves at 12 h of life. It is speculated that lower concentrations of bCO in the gastrointestinal tract of HC calves at 6 h of life could be due to the early establishment of beneficial bacteria, such as Bifidobacterium, in HC calves and their subsequent metabolism of bCO as a carbon source. These findings suggest that the heat treatment of colostrum increases the amount of free bCO, which may serve as prebiotics available to microbiota within the intestine of the neonatal calf.

Single-molecule dynamics of phytochrome-bound fluorophores probed by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) was used to investigate the hydrodynamic and photophysical properties of PR1 (phytofluor red 1), an intensely red fluorescent biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1Delta, which consists of the N-terminal 514 amino acids). Single-molecule diffusion measurements showed that PR1 has excellent fluorescence properties at the single-molecule level, making it an interesting candidate for red fluorescent protein fusions. FCS measurements for probing dimer formation in solution over a range of protein concentrations were enabled by addition of Cph1Delta apoprotein (apoCph1Delta) to nanomolar solutions of PR1. FCS brightness analysis showed that heterodimerization of PR1 with apoCph1Delta altered the chemical environment of the PR1 chromophore to further enhance its fluorescence emission. Fluorescence correlation measurements also revealed interactions between apoCph1Delta and the red fluorescent dyes Cy5.18 and Atto 655 but not Alexa Fluor 660. The concentration dependence of protein:dye complex formation indicated that Atto 655 interacted with, or influenced the formation of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for probing single-molecule dynamics in living cells in which the fluorescence signal can be controlled by the addition of various chromophores that have different structures and photophysical properties, thereby imparting different types of information, such as dimer formation or the presence of open binding faces on a protein.

Multiple roles of a conserved GAF domain tyrosine residue in cyanobacterial and plant phytochromes.

The phytochrome family of red/far-red photoreceptors has been optimized to support photochemical isomerization of a bound bilin chromophore, a process that triggers a conformational change and modulates biochemical output from the surrounding protein scaffold. Recent studies have established that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin-binding GAF domain of the cyanobacterial phytochrome Cph1 [Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome's glowing potential, Proc. Natl. Acad. Sci. U.S.A. 101, 17334-17339]. Here, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved. Saturation mutagenesis of Tyr176 in Cph1 establishes that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin-binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand-binding specificity of apophytochrome. On the basis of the hydrogen-bonding ability of Tyr176 substitutions that support the nonphotochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation, an interpretation consistent with site-directed mutagenesis of this conserved acidic residue.

Harnessing phytochrome's glowing potential.

Directed evolution of a cyanobacterial phytochrome was undertaken to elucidate the structural basis of its light sensory activity by remodeling the chemical environment of its linear tetrapyrrole prosthetic group. In addition to identifying a small region of the apoprotein critical for maintaining phytochrome's native spectroscopic properties, our studies revealed a tyrosine-to-histidine mutation that transformed phytochrome into an intensely red fluorescent biliprotein. This tyrosine is conserved in all members of the phytochrome superfamily, implicating direct participation in the primary photoprocess of phytochromes. Fluorescent phytochrome mutants also hold great promise to expand the present repertoire of genetically encoded fluorescent proteins into the near infrared.