Guanjun Chen

Thermomyces lanuginosus is the dominant fungus in maize straw composts.

The microbial community composition and function of three self-heating maize straw composts were compared by integrated meta-omics. The results revealed that the fungal communities were primarily dominated by the phylum Ascomycota (>90%) regardless of different nitrogen sources, which were exclusively composed of the Thermomyces, a genus of hemicellulose degraders. The bacterial community composition was affected by the addition of nitrogen sources, as the abundance of the Actinobacteria increased, while the Proteobacteria and Bacteroidetes decreased. Various hemicellulases and cellulases were detected in the composts, and the major xylanase secreted by Thermomyces lanuginosus was always present, revealing that it was the dominant fungus in hemicellulose hydrolysis and that bacteria and fungi might synergistically degrade lignocellulose. Thus, microbial communities in composts may develop a simple and stable structure of a dominant fungal species and limited numbers of bacterial species under the selective pressure of high temperature and maize straw as starting materials.

Dynamic changes of the dominant functioning microbial community in the compost of a 90-m3 aerobic solid state fermentor revealed by integrated meta-omics

The dynamic changes in the composition and function of both bacterial and fungal communities over time and at various depths in the compost of a 90-m(3) industrial-scale fermentor were explored using integrated meta-omics. The microbial communities in the middle layer (1.2m) of the compost developed a stable and simple structure over time, which was mainly composed of Thermobifida, Bacillus, Thermomyces and Aspergillus. According to the metaproteomic results, the bacterial community was more focused on cellulose degradation, characterized by 44% of the cellulases that were secreted by Thermobifida, while the fungal community was more likely to degrade hemicellulose, mainly via Thermomyces and Aspergillus. The results revealed that, under artificial control of the temperature and oxygen concentration, the efficiency of organic waste degradation was greatly increased and the fermentation cycle was shortened to 11 days.

Comparative secretome analysis of Aspergillus niger, Trichoderma reesei, and Penicillium oxalicum during solid-state fermentation.

Filamentous fungi such as Aspergillus spp., Trichoderma spp., and Penicillium spp. are frequently used to produce high concentrations of lignocellulosic enzymes. This study examined the discrepancies in the compositions and dynamic changes in the extracellular enzyme systems secreted by Aspergillus niger ATCC1015, Trichoderma reesei QM9414, and Penicillium oxalicum 114-2 cultured on corn stover and wheat bran. The results revealed different types and an abundance of monosaccharides and oligosaccharides were released during incubation, which induced the secretion of diverse glycoside hydrolases. Both the enzyme activities and isozyme numbers of the three fungal strains increased with time. A total of 279, 161, and 183 secretory proteins were detected in A. niger, T. reesei, and P. oxalicum secretomes, respectively. In the A. niger secretomes, more enzymes involved in the degradation of (galacto)mannan, xyloglucan, and the backbone of pectin distributed mostly in dicots were detected. In comparison, although P. oxalicum 114-2 hardly secreted any xyloglucanases, the diversities of enzymes involved in the degradation of xylan and β-(1,3;1,4)-d-glucan commonly found in monocots were higher. The cellulase system of P. oxalicum 114-2 was more balanced. The degradation preference provided a new perspective regarding the recomposition of lignocellulosic enzymes based on substrate types.

Wheat straw: An inefficient substrate for rapid natural lignocellulosic composting.

Composting is a promising method for the management of agricultural wastes. However, results for wheat straw composts with different carbon-to-nitrogen ratios revealed that wheat straw was only partly degraded after composting for 25days, with hemicellulose and cellulose content decreasing by 14% and 33%, respectively. No significant changes in community structure were found after composting according to 454-pyrosequencing. Bacterial communities were represented by Proteobacteria and Bacteroidetes throughout the composting process, including relatively high abundances of pathogenic microbes such as Pseudomonas and Flexibacter, suggesting that innocent treatment of the composts had not been achieved. Besides, the significant lignocellulose degrader Thermomyces was not the exclusively dominant fungus with relative abundance only accounting for 19% of fungal communities. These results indicated that comparing with maize straw, wheat straw was an inefficient substrate for rapid natural lignocellulose-based composting, which might be due to the recalcitrance of wheat straw.

Rce1, a novel transcriptional repressor, regulates cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei

Cellulase gene expression in the model cellulolytic fungus Trichoderma reesei is supposed to be controlled by an intricate regulatory network involving multiple transcription factors. Here, we identified a novel transcriptional repressor of cellulase gene expression, Rce1. Disruption of the rce1 gene not only facilitated the induced expression of cellulase genes but also led to a significant delay in terminating the induction process. However, Rce1 did not participate in Cre1‐mediated catabolite repression. Electrophoretic mobility shift (EMSA) and DNase I footprinting assays in combination with chromatin immunoprecipitation (ChIP) demonstrated that Rce1 could bind directly to a cbh1 (cellobiohydrolase 1‐encoding) gene promoter region containing a cluster of Xyr1 binding sites. Furthermore, competitive binding assays revealed that Rce1 antagonized Xyr1 from binding to the cbh1 promoter. These results indicate that intricate interactions exist between a variety of transcription factors to ensure tight and energy‐efficient regulation of cellulase gene expression in T. reesei. This study also provides important clues regarding increased cellulase production in T. reesei.

Subsite-specific contributions of different aromatic residues in the active site architecture of glycoside hydrolase family 12

The active site architecture of glycoside hydrolase (GH) is a contiguous subregion of the enzyme constituted by residues clustered in the three-dimensional space, recognizing the monomeric unit of ligand through hydrogen bonds and hydrophobic interactions. Mutations of the key residues in the active site architecture of the GH12 family exerted different impacts on catalytic efficiency. Binding affinities between the aromatic amino acids and carbohydrate rings were quantitatively determined by isothermal titration calorimetry (ITC) and the quantum mechanical (QM) method, showing that the binding capacity order of Tyr>Trp>His (and Phe) was determined by their side-chain properties. The results also revealed that the binding constant of a certain residue remained unchanged when altering its location, while the catalytic efficiency changed dramatically. Increased binding affinity at a relatively distant subsite, such as the mutant of W7Y at the −4 subsite, resulted in a marked increase in the intermediate product of cellotetraose and enhanced the reactivity of endoglucanase by 144%; while tighter binding near the catalytic center, i.e. W22Y at the −2 subsite, enabled the enzyme to bind and hydrolyze smaller oligosaccharides. Clarification of the specific roles of the aromatics at different subsites may pave the way for a more rational design of GHs.

An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization

ABSTRACT Cytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii.

Interruption of the denitrification pathway influences cell growth and magnetosome formation in Magnetospirillum magneticum AMB-1.

Aims:  Intracellular magnetosome synthesis in magnetotactic bacteria has been proposed to be a process involving functions of a variety of proteins. To learn more about the genetic control that is involved in magnetosome formation, nonmagnetic mutants are screened and characterized.

A copper-responsive promoter replacement system to investigate gene functions in Trichoderma reesei : a case study in characterizing SAGA genes

Trichoderma reesei represents an important workhorse for industrial production of cellulases as well as other proteins. The molecular mechanism underlying the regulation of cellulase production as well as other physiological processes in T. reesei is still insufficiently understood. We constructed a Ptcu1-based promoter substitution cassette that allowed one-step replacement of the endogenous promoter for controlling the target gene expression with copper. We then showed that copper repression of the histone acetyltransferase gene gcn5 phenocopied the gcn5 deletion strain. Using the same strategy, we further characterized the function of another putative Spt-Ada-Gcn5 acetyltransferase (SAGA) complex subunit encoding gene, ada2, in T. reesei. Similar to the repression of gcn5, the addition of copper to the Ptcu1-ada2 strain not only drastically reduced the vegetative growth and conidiation in T. reesei but also severely compromised the induced cellulase gene expression. The developed strategy will thus be potentially useful to probe the biological function of the large fraction of T. reesei genes with unknown functions including those essential genes in the genome to expand its extraordinary biotechnological potential.

Marinifilum albidiflavum sp. nov., isolated from coastal sediment

A novel Gram-stain-negative, facultatively anaerobic, filamentous, and yellowish-white-pigmented marine bacterium, designated strain FB208T, was isolated from marine sediment obtained off the coastal area of Weihai, China. Cells of strain FB208T were filamentous during exponential growth, fragmented to rods in the stationary growth phase and became spherical in aged cultures. It grew optimally at 33 °C, at pH 7.0-7.5 and in the presence of 2.0-3.0 % (w/v) NaCl. Based on the 16S rRNA gene sequence, strain FB208T was found to be closely related to Marinifilum flexuosum DSM 21950T (96.9 % similarity) and Marinifilum fragile JCM 15579T (96.4 %), with less than 90.0 % sequence similarity to other genera of the class Bacteroidia. Phylogenetic analysis, also based on 16S rRNA gene sequences, placed strain FB208T in the genus Marinifilum, family Marinifilaceae. The predominant isoprenoid quinone of strain FB208T was identified as menaquinone MK-7. The main cellular fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and iso-C17 : 1ω9c, and the major polar lipids were an unidentified lipid and aminophospholipid. The G+C content of the genomic DNA was 43.8 mol%. Based on these phylogenetic and phenotypic data, strain FB208T represents a novel species of the genus Marinifilum, for which the name Marinifilum albidiflavum sp. nov. is proposed. The type strain is FB208T (=KCTC 42591T=MCCC 1H00113T).