丝状真菌中纤维素酶与半纤维素酶的合成调控简

综述了丝状真菌合成纤维素酶和半纤维素酶的相关调控研究进展。对最近研究文章分析发现：细胞外信号分子（C源、光信号）,转录因子以及染色质重建等对丝状真菌合成调控纤维素酶及半纤维素酶有重要影响。同时解析丝状真菌合成纤维素酶和半纤维素酶调控网络,以期为利用基因工程改造纤维素酶和半纤维素酶生产工业菌株提供理论指导。     

Metabolic engineering strategies for the improvement of cellulase production by Hypocrea jecorina

Hypocrea jecorina (= Trichoderma reesei) is the main industrial source of cellulases and hemicellulases used to depolymerise plant biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. Cellulases are formed adaptively, and several positive (XYR1, ACE2, HAP2/3/5) and negative (ACE1, CRE1) components involved in this regulation are now known. In addition, its complete genome sequence has been recently published, thus making the organism susceptible to targeted improvement by metabolic engineering. In this review, we summarise current knowledge about how cellulase biosynthesis is regulated, and outline recent approaches and suitable strategies for facilitating the targeted improvement of cellulase production by genetic engineering.     

The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.     

A complete protein pattern of cellulase and hemicellulase genes in the filamentous fungus Trichoderma reesei

The complete protein pattern of cellulase and hemicellulase genes was studied through the Genome-wide analysis in Trichoderma reesei. The genome database revealed the presence of 39 ORFs encoding related proteins, including 32 enzymes with a catalysis domain related to cellulases and hemicellulases and 7 related proteins with a cellulose-binding module (CBM). Ten of these encoded yet undescribed enzymes, including six novel beta-glucosidases or xylosidases, two putative xylanases and two undescribed mannases. To better illustrate the relation of these 39 related proteins, four groups were created and analyzed by phylogenetic analysis: group A corresponding to xylanases, group B belonging to mannases and acting to degrade mannan; group C containing all known and putative cellulose-degrading proteins that have highly conserved CBMs; and group D containing beta-glucosidase and beta-xylosidase. Group D was the largest group, in which 8 beta-glucosidases appeared to be non-secreted proteins.     

Biochemistry and genetics of actinomycete cellulases.

The order Actinomycetales includes a number of genera that contain species that actively degrade cellulose and these include both mesophilic and facultative thermophilic species. Cellulases produced by strains from two of the genera containing thermophilic organisms have been studied extensively: Microbispora bispora and Thermomonospora fusca. Fractionation of M. bispora cellulases has identified six different enzymes, all of which were purified to near homogeneity and partially characterized. Two of these enzymes appear to be exocellulases and gave synergism with each other and with the endocellulases. The structural genes of five M. bispora cellulases have been cloned and one was sequenced. Fractionation of T. fusca cellulases has identified five different enzymes, all of which were purified to near homogeneity and partially characterized. One of the T. fusca enzymes gives synergism in the hydrolysis of crystalline cellulose with several T. fusca endocellulases and with Trichoderma reesei CBHI but not with T. reesei CBHII. Each T. fusca cellulase contains distinct catalytic and cellulose binding domains. The structural genes of four of the T. fusca endoglucanases have been cloned and sequenced, while three cellulase genes have been cloned from "T. curvata". The T. fusca cellulase genes are expressed at a low level in Escherichia soli, but at a high level in Streptomyces lividans. Sequence comparisons have shown that there are no significant amino acid homologies between any of the catalytic domains of the four T. fusca cellulases, but each of them shows extensive homology to several other cellulases and fits in one of the five existing cellulase gene families. There have been extensive studies of the regulation of the synthesis of these cellulases and a number of regulatory mutants have been isolated. This work has shown that the different T. fusca cellulases are coordinately regulated over a 100-fold range by two independent controls; induction by cellobiose and repression by any good carbon source.     

Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina)

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.     

丝状真菌红褐肉座菌(Hypocrea jecorina)纤维素酶基因的转录调控研究进展

红褐肉座菌(Hypocrea jecorina,即Trichoderma reesei的有性型)是工业上重要的纤维素酶生产菌株,也是用于研究纤维素酶和半纤维素酶基因转录调控机制的模式菌株。在诱导物存在的条件下,H.jecorina可以迅速启动这些糖苷水解酶基因的转录表达,但不同的诱导物对纤维素酶和半纤维素酶基因的诱导表达模式存在一定差异。目前对不溶性诱导物如结晶纤维素如何诱导这些基因的起始转录问题有3种假设;并且已发现某些参与调控纤维素酶基因转录的正调控因子(Xyr1、Ace2、Hap2/3/5)和负调控因子(Ace1、Cre1),这些调控因子可在纤维素酶基因启动子上结合且彼此间可能发生相互作用。本文系统综述了红褐肉座菌纤维素酶基因转录表达调控中的关键因素及其相互作用的相关研究进展。     

Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans

The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions. A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars. The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose. Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied. Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source. Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells. The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively. Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose. These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.     

2种纤维素酶水解效果比较及酶蛋白组分分析

比较了自产纤维素酶和商品纤维素酶的水解效果,并采用超滤、层析、SDS-PAGE相结合的方法分析2种纤维素酶蛋白组分的差异。里氏木霉以纸浆为C源合成的自产纤维素酶的水解得率高于商品纤维素酶,自产纤维素酶水解48h的得率为66.24%,商品纤维素酶的得率为52.19%。自产纤维素酶中存在着Cel6A酶组分和XYNⅡ酶组分,而商品纤维素酶中没有检测到这2种酶组分。自产纤维素酶和商品纤维素酶的Cel1A酶组分和Cel7A酶组分间存在着分布和含量上的差异。自产纤维素酶在相对分子质量(2.5～3.5)×104范围内存在着几条蛋白条带,而商品纤维素酶则是在相对分子质量3.5×104附近存在着几条蛋白条带。