The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1.IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.
Cryostat microtome sections of birch wood degraded by white rot fungi were examined by light microscopy after treatment with two stains: astra-blue, which stains cellulose blue only in the absence of lignin, and safranin, which stains lignin regardless of whether cellulose is present. The method provided a simple and reliable screening procedure that distinguishes between fungi that cause decay by selectively removing lignin and those that degrade both cellulose and lignin simultaneously. Moreover, morphological characteristics specific to selective delignification were revealed.
Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a 14 C-labeled, polyethylene glycol-linked, high-molecular-weight -O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C ␣ and C  to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C ␣-C  cleavage of -O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), 14 C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 M to approximately 5 M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic -O-4 lignin model compounds between C ␣ and C  to release a benzylic fragment.
Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions.
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