Enzymatic degradation of plant polysaccharides has many industrial applications, such as within the paper, food, and feed industry and for sustainable production of fuels and chemicals. Cellulose, ...hemicelluloses, and pectins are the main components of plant cell wall polysaccharides. These polysaccharides are often tightly packed, contain many different sugar residues, and are branched with a diversity of structures. To enable efficient degradation of these polysaccharides, fungi produce an extensive set of carbohydrate-active enzymes. The variety of the enzyme set differs between fungi and often corresponds to the requirements of its habitat. Carbohydrate-active enzymes can be organized in different families based on the amino acid sequence of the structurally related catalytic modules. Fungal enzymes involved in plant polysaccharide degradation are assigned to at least 35 glycoside hydrolase families, three carbohydrate esterase families and six polysaccharide lyase families. This mini-review will discuss the enzymes needed for complete degradation of plant polysaccharides and will give an overview of the latest developments concerning fungal carbohydrate-active enzymes and their corresponding families.
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CEKLJ, DOBA, EMUNI, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK
Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to ...alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose. L: -Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses.
Carbohydrate Active enzyme ...(CAZy) annotation of the R. oryzae identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs) and a high number of glycosyl transferases (GTs) and carbohydrate esterases (CEs). A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars), chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of R. oryzae to its environment.
CAZy analyses of the genome of the zygomycete fungus R. oryzae and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Trichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further ...improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes.In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus.
The modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis.
This study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in T. reesei.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The ability of baker's yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many ...of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase in the glycolytic enzyme capacities (Vmax) and/or by changes in the concentrations of low-molecular-weight substrates, products, and effectors. The goal of the present study was to understand the time-dependent, multilevel regulation of glycolytic enzymes during a switch from fully respiratory conditions to fully fermentative conditions. The switch from glucose-limited aerobic chemostat growth to full anaerobiosis and glucose excess resulted in rapid acceleration of fermentative metabolism. Although the capacities (Vmax) of the glycolytic enzymes did not change until 45 min after the switch, the intracellular levels of several substrates, products, and effectors involved in the regulation of glycolysis did change substantially during the initial 45 min (e.g., there was a buildup of the phosphofructokinase activator fructose-2,6-bisphosphate). This study revealed two distinct phases in the upregulation of glycolysis upon a switch to fermentative conditions: (i) an initial phase, in which regulation occurs completely through changes in metabolite levels; and (ii) a second phase, in which regulation is achieved through a combination of changes in Vmax and metabolite concentrations. This multilevel regulation study qualitatively explains the increase in flux through the glycolytic enzymes upon a switch of S. cerevisiae to fermentative conditions and provides a better understanding of the roles of different regulatory mechanisms that influence the dynamics of yeast glycolysis.
When faced with environmental changes, microbes often enter a temporary growth arrest during which they reprogram the expression of specific genes to adapt to the new conditions. A prime example of ...such a lag phase occurs when microbes need to switch from glucose to other, less-preferred carbon sources. Despite its industrial relevance, the genetic network that determines the duration of the lag phase has not been studied in much detail. Here, we performed a genome-wide Bar-Seq screen to identify genetic determinants of the
glucose-to-galactose lag phase. The results show that genes involved in respiration, and specifically those encoding complexes III and IV of the electron transport chain, are needed for efficient growth resumption after the lag phase. Anaerobic growth experiments confirmed the importance of respiratory energy conversion in determining the lag phase duration. Moreover, overexpression of the central regulator of respiration,
, leads to significantly shorter lag phases. Together, these results suggest that the glucose-induced repression of respiration, known as the Crabtree effect, is a major determinant of microbial fitness in fluctuating carbon environments.
The lag phase is arguably one of the prime characteristics of microbial growth. Longer lag phases result in lower competitive fitness in variable environments, and the duration of the lag phase is also important in many industrial processes where long lag phases lead to sluggish, less efficient fermentations. Despite the immense importance of the lag phase, surprisingly little is known about the exact molecular processes that determine its duration. Our study uses the molecular toolbox of
combined with detailed growth experiments to reveal how the transition from fermentative to respirative metabolism is a key bottleneck for cells to overcome the lag phase. Together, our findings not only yield insight into the key molecular processes and genes that influence lag duration but also open routes to increase the efficiency of industrial fermentations and offer an experimental framework to study other types of lag behavior.
Thermophilic fungi have the potential to produce industrial-relevant thermostable enzymes, in particular for the degradation of plant biomass. Sordariales is one of the few fungal orders containing ...several thermophilic taxa, of which many have been associated with the production of thermostable enzymes. The evolutionary affiliation of Sordariales fungi, especially between thermophiles and non-thermophilic relatives, is however poorly understood. Phylogenetic analysis within the current study was based on sequence data, derived from a traditional Sanger and highly multiplexed targeted next generation sequencing approach of 45 isolates. The inferred phylogeny and detailed growth analysis rendered the trait ‘thermophily’ as polyphyletic within Chaetomiaceae (Sordariales, Sordariomycetes), and characteristic to: Myceliophthora spp., Thielavia terrestris, Chaetomium thermophilum, and Mycothermus thermophilus. Compared to mesophiles, the isolates within thermophilic taxa produced enzyme mixtures with the highest thermostability of known cellulase activities. Temperature profiles of the enzyme activities correlated strongly with the optimal growth temperatures of the isolates but not with their phylogenetic relationships. This strong correlation between growth and enzyme characteristics indicated that detailed analysis of growth does give predictive information on enzyme physiology. The variation in growth and enzyme characteristics reveals these fungi as an excellent platform to better understand fungal thermophily and enzyme thermostability.
•RPB2 (>3 kb) of Sordariales isolates were sequenced using next generation sequencing.•Complete RPB2 sequences are more informative than ITS, LSU, EF1, and EF3 sequences.•The trait thermophily was rendered polyphyletic in Chaetomiaceae.•Thermophiles compared to mesophiles produce the most thermostable cellulases.•Optimal growth temperatures give predictive information on enzyme thermostability.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Species of the genus
Myceliophthora
and its teleomorph
Corynascus
have attracted increasing interest due to their potential to produce thermostable enzymes. This study re-assessed the phylogenetic ...relationship of 49 isolates of nine species belonging to
Myceliophthora
and
Corynascus
. One species,
M. vellerea
, was shown not to belong to the genus
Myceliophthora
and should be placed in the genus
Ctenomyces
. The other species belonged to two phylogenetic clusters: mesophilic fungi with the type species
M. lutea
and
C. sepedonium
, and thermophilic fungi with
M. thermophila, M. hinnulea
and
C. thermophilus
. The phylogenetic data provides no clear separation of the two genera
Corynascus
and
Myceliophthora
. To avoid confusion in future taxonomic studies, it is proposed that all existing
Corynascus
species be renamed to
Myceliophthora
, which is the old name and the one more frequently used. Furthermore, this study identified two groups within the isolates listed as
M. thermophila
and assigned one group (five isolates) to
M. heterothallica
based on AFLP analysis and mating behavior. This study provides new insights into the genetic differences within the genus
Myceliophthora
and will therefore be essential for the interpretation of future genomic and physiological studies of these species.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with ...emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.
Fungal enzymes efficiently release fermentable sugars from plant biomass. This study shows that combining enzyme mixtures of Aspergillus niger and Trichoderma reesei improves the release of fermentable sugars. It also shows that the fungal growth conditions strongly effect the produced enzyme sets and their saccharification capacity. Overall, this study provides potential leads to improve industrial enzyme productions for plant biomass saccharification.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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