Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria 1 2 Iain Anderson *, Birte Abt

38 Pages·2012·1.73 MB·English

Lawrence Berkeley National Laboratory
Lawrence Berkeley National Laboratory
Title
Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria
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https://escholarship.org/uc/item/5dq988t1
Author
Anderson, Iain
Publication Date
2012-12-01
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University of California
1 Genomics of Aerobic Cellulose Utilization Systems in Actinobacteria
2
3 Iain Anderson1*, Birte Abt2, Athanasios Lykidis1, Hans-Peter Klenk2, Nikos Kyrpides1,
4 Natalia Ivanova1
5
6 1DOE Joint Genome Institute, Walnut Creek, California, United States of America
7 2Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures,
8 Braunschweig, Germany
9
10 *Corresponding author: [email protected]
Abstract
11
12 Cellulose degrading enzymes have important functions in the biotechnology industry,
13 including the production of biofuels from lignocellulosic biomass. Anaerobes including
14 Clostridium species organize cellulases and other glycosyl hydrolases into large
15 complexes known as cellulosomes. In contrast, aerobic actinobacteria utilize systems
16 comprised of independently acting enzymes, often with carbohydrate binding domains.
17 Numerous actinobacterial genomes have become available through the Genomic
18 Encyclopedia of Bacteria and Archaea (GEBA) project. We identified putative cellulose-
19 degrading enzymes belonging to families GH5, GH6, GH8, GH9, GH12, GH48, and
20 GH51 in the genomes of eleven members of the actinobacteria. The eleven organisms
21 were tested in several assays for cellulose degradation, and eight of the organisms
22 showed evidence of cellulase activity. The three with the highest cellulase activity were
23 Actinosynnema mirum, Cellulomonas flavigena, and Xylanimonas cellulosilytica.
24 Cellobiose is known to induce cellulolytic enzymes in the model organism Thermobifida
25 fusca, but only Nocardiopsis dassonvillei showed higher cellulolytic activity in the
26 presence of cellobiose. In T. fusca, cellulases and a putative cellobiose ABC transporter
27 are regulated by the transcriptional regulator CelR. Nine organisms appear to use the
28 CelR site or a closely related binding site to regulate an ABC transporter. In some, CelR
29 also regulates cellulases, while cellulases are controlled by different regulatory sites in
30 three organisms. Mining of genome data for cellulose degradative enzymes followed by
31 experimental verification successfully identified several actinobacteria species which
32 were not previously known to degrade cellulose as cellulolytic organisms.
Introduction
33
34 Aerobic cellulolytic actinobacteria and aerobic fungi have been shown to use a system
35 for cellulose degradation consisting of sets of soluble cellulases and hemicellulases.
36 Most of these independent cellulolytic enzymes contain one or more carbohydrate
37 binding domains. This is in contrast to the system found in many anaerobic bacteria and
38 fungi which consists of multienzyme assemblies attached to the outer surface of the cell,
39 the cellulosomes (reviewed in [1]). Cellulosomes are usually anchored to the surface of
40 the cell through protein-protein interactions and to the carbohydrate substrate through
41 carbohydrate binding domains on the scaffolding protein or on the catalytic enzymes [1].
42 Previous work on cellulose degradation in actinobacteria has focused on two model
43 organisms, Thermobifida fusca and Cellulomonas fimi (reviewed in [2]). The system of T.
44 fusca is composed of three non-processive endocellulases (E1/Cel9B, E2/Cel6A,
45 E5/Cel5A), which cleave cellulose at random sites along cellulose chains, two
46 exocellulases (E3/Cel6B and E6/Cel48A), which cleave cellobiose units from the ends of
47 cellulose chains in a processive manner, and one processive endocellulase (E4/Cel9A).
48 The latter enzyme combines features of both endo- and exo-type enzymes: it makes an
49 initial endocellulolytic cleavage followed by release of cellotetraose units from the
50 cleaved substrate [2]. Exocellulase E6/Cel48A and processive endocellulase E4/Cel9A
51 remove cellooligosaccharides from the reducing end, while exocellulase E3/Cel6B acts
52 on the nonreducing end [3]. Synergism is observed between exo- and endocellulases
53 (endo/exo synergism) or when different classes of exocellulases are combined (exo/exo
54 synergism); processive endocellulase displays synergism with both exo- and
55 endocellulases. A transcription factor regulating the expression of T. fusca cellulases
56 (CelR) has been identified, and in vitro experiments indicate that cellobiose acts as an
57 effector causing dissociation of the CelR-DNA complex [4]. The set of cellulases in C.
58 fimi is also comprised of three endocellulases (CenA, CenB and CenD), two
59 exocellulases (CbhA and CbhB), and a processive endocellulase CenC [2]. While these
60 belong to the same glycosyl hydrolase families as the corresponding T. fusca enzymes,
61 the sequences are not closely related in most cases.
62 The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project has generated
63 genome sequences for a number of actinobacteria [5]. Four of these organisms are
64 known to degrade cellulose: Cellulomonas flavigena 134, DSM 20109 [6],
65 Thermobispora (formerly Microbispora) bispora R51, DSM 43833 [7],
66 Thermomonospora curvata DSM 43183 [8], and Xylanimonas cellulosilytica XIL07,
67 DSM 15894 [9]. During analysis of the other actinobacterial genomes, we observed that
68 many contained glycosyl hydrolases similar to endo- and exocellulases of T. fusca and C.
69 fimi, although the organisms were not known to be cellulolytic. These organisms are
70 Actinospica robiniae GE134769, DSM 44927, Actinosynnema mirum 101, DSM 43827,
71 Catenulispora acidiphila ID139908, DSM 44928, Jonesia denitrificans 55134, DSM
72 20603, Nocardiopsis dassonvillei IMRU 509, DSM 43111, Stackebrandtia nassauensis
73 LLR-40K-21, DSM 44728, and Streptosporangium roseum NI 9100, DSM 43021. We
74 present here an analysis of the cellulolytic enzymes of these actinobacteria and
75 comparison with the genomes of known cellulolytic actinobacteria as well as
76 experimental demonstration of cellulose degradation by these bacteria.
77
Results
78
79 Cellulose degradation: computational analysis of glycosyl hydrolases.
80 Experimentally characterized endocellulases are from glycosyl hydrolase families GH5,
81 GH6, GH8, GH9, GH12, and GH51, while predicted exocellulases belong to families
82 GH6 and GH48 according to CAZy classification [10]. While all experimentally
83 characterized actinobacterial GH48 family enzymes are reducing-end exocellulases, GH6
84 family enzymes may have either endo- or exocellulase activity. Similarly, GH9 family
85 enzymes may have either processive or non-processive endocellulase activity. In order to
86 distinguish between different enzymatic activities within the same GH family we
87 performed phylogenetic analysis of catalytic domains of proteins assigned to GH9 and
88 GH6 families (Figure 1a and 1b, respectively).
89 GH9 proteins have been assigned to different “themes” based on their sequence
90 similarity and possession of domains other than the glycosyl hydrolase domain [11]. The
91 processive endocellulases found in actinobacteria belong to Theme B, and they are
92 generally composed of a GH9 catalytic domain followed by a carbohydrate-binding
93 CBM3 domain, a fibronectin type 3 (fn3) domain, and a CBM2 or a second CBM3
94 domain. A processive endocellulase, very similar in sequence to the T. fusca enzyme
95 (Tfu_2176, Cel9A), has been identified in C. flavigena and is named CBP105 [12]. Four
96 of the other actinobacteria included in the study are likely to have processive
97 endocellulases, based on clustering with the T. fusca and C. flavigena enzymes in
98 phylogenetic analysis (Figure 1a) and similar domain architecture. Cfla_0139 and
99 Acel_0970 also belong to Theme B, and therefore may be processive endocellulases. If
100 they are, then C. flavigena and A. cellulolyticus would each have two processive
101 endocellulases. Catalytic domains of GH6 family proteins can be divided into
102 exocellulase subfamily clustering with Tfu_0620 (E3/Cel6B) and endocellulase
103 subfamily clustering with Tfu_1074 (E2/Cel6A) (Figure 1b).
104 Table 1 summarizes the distribution of putative endo- and exocellulases in the eleven
105 actinobacterial genomes that we studied, along with the cellulases from the previously
106 published genomes of T. fusca and Acidothermus cellulolyticus. Most genomes included
107 in this study encode genes for predicted endocellulases and two exocellulases, one acting
108 on the non-reducing end of cellulose polymers (GH6 family enzyme) and the other acting
109 on the reducing end (GH48 family enzyme). Two exceptions are S. nassauensis, which
110 lacks a reducing end exocellulase of GH48 family, and T. curvata, in which no
111 exocellulases were identified. Two pseudogenes with similarity to the reducing- and non-
112 reducing end exocellulases, Tcur_4566 and Tcur_4570 (Table 1), were found in the latter
113 genome suggesting recent loss of cellulose-degrading capability by this T. curvata strain.
114 With the exception of GH6-family exocellulase from S. nassauensis, all other
115 exocellulases found in actinobacterial genomes included in this study have at least one
116 non-catalytic carbohydrate-binding module (CBM), which play an important role in
117 hydrolysis of insoluble cellulosic substrates [13]. Analysis of exocellulase domain
118 architecture revealed common themes of catalytic and CBM domain arrangement.
119 Reducing end exocellulases of GH48 family can be grouped into 2 types: those with N-
120 terminal CBM (type I or Thermobifida-like) and those with C-terminal CBM (type II or
121 Cellulomonas-like) (Figure S1a). Similarly non-reducing-end exocellulases of GH6
122 family can be grouped into Thermobifida-like type I with N-terminal CBM and
123 Cellulomonas-like type II with C-terminal CBM (Figure S1b). The domain arrangement
124 of non-processive endocellulases of GH6 family appears to be the reverse of that of GH6
125 family exocellulase with Thermobifida-like type I having C-terminal CBM and
126 Cellulomonas-like type II having N-terminal CBM (Figure S1c). Considering that both
127 reducing and non-reducing end exocellulases are present in 10 out of 12 genomes
128 included in the study, one would expect to find many combinations of different domain
129 architectures. Instead, a remarkable conservation is observed: those organisms possessing
130 type I reducing-end exocellulase also have type I non-reducing end exocellulase and type
131 I non-processive endocellulase of GH6 family (Thermobifida type, Table S1). Likewise,
132 actinobacteria with type II reducing-end exocellulase have type II non-reducing end
133 exocellulase and mostly type II non-processive endocellulase (Cellulomonas type, Table
134 S1). We hypothesize that this non-random distribution of enzymes with different domain
135 architectures may reflect optimization of actinobacterial cellulase system to achieve
136 maximal synergy between endo- and exocellulases. Two exceptions from this conserved
137 domain architecture are C. acidiphila and A. robiniae, which contain enzymes with both
138 types of domain arrangements. Both genomes have the largest and the most diverse sets
139 of glycosyl hydrolases as compared to other actinobacteria included in this study (see
140 below). The catalytic activity and expression of the enzymes in these organisms require
141 further experimental investigation.
142 Cellulose degradation: computational analysis of auxiliary genes. In addition to
143 predicted cellulases, we identified genes in these actinobacteria for beta-glucosidases,
144 beta-glucan glucohydrolases and cellobiose phosphorylases, enzymes required for
145 cellobiose utilization within the cell (Table S2). All of the organisms have at least one
146 beta-glucosidase, most have beta-glucan glucohydrolases, and two (C. flavigena and X.
147 cellulosilytica) have cellobiose phosphorylases. The beta-glucosidases belong to families
148 GH1 and GH3, the beta-glucan glucohydrolases belong to family GH3, and the cellobiose
149 phosphorylases belong to family GH94.
150 Another component required for cellulose degradation is a transporter for cellobiose.
151 All of the actinobacteria considered here, except A. robiniae and N. dassonvillei, have an
152 ABC transporter whose binding protein has at least 48% similarity to that of a
153 characterized cellobiose/cellotriose ABC transporter from Streptomyces reticuli [14]. In
154 most cases this ABC transporter is adjacent to a beta-glucosidase and a LacI family
155 transcriptional regulator (Figure 2), although in C. flavigena a beta-glucosidase is not
156 present. The beta-glucosidases generally lack signal peptides and thus are predicted to be
157 intracellular, with the possible exception of Xcel_2614, which has a signal peptide
158 probability of 0.465. For most of the ABC transporters, only the substrate binding
159 protein and membrane proteins are found together; however, in T. curvata an ABC
160 transporter ATPase protein (Tcur_1737) is adjacent to the other subunits (Figure 2). S.
161 roseum and T. bispora have two copies of the ABC transporter operon; however, the
162 proteins making up the operons are not closely related and therefore do not appear to
163 result from recent duplications.
164 Cellulose degradation: experimental verification. Based on the above predictions of
165 the presence of cellulases, we performed experiments to determine whether these
166 organisms actually secrete active cellulolytic systems. Table 2 shows the results of
167 several cellulase assays performed on the eleven actinobacteria. In the clearing test,
168 which was performed with quite recalcitrant microcrystalline cellulose, only A. mirum
169 gave a positive result, while in the filter paper test, A. mirum, C. flavigena, and X.
170 cellulosilytica showed cellulolytic activity. We also tested these actinobacteria on
171 azurine cross-linked hydroxyethylcellulose (AZCL-HEC) plates, where a blue color
172 indicates cellulolytic activity. A. mirum and C. flavigena gave the strongest response
173 (Figure 3), and altogether eight of the actinobacteria were positive in this assay (Table 2,
174 Figure 3). C. acidiphila gave a positive result on AZCL-HEC plates only when grown at
175 its optimal pH of 5.5. Two of the organisms showed no reproducible cellulolytic activity
176 in AZCL-HEC test: T. bispora, and T. curvata, and for one organism (S. roseum) the
177 results were inconsistent: while in the initial testing it displayed some cellulolytic
178 activity, this observation could not be confirmed in later tests. Since cellobiose is known
179 to induce cellulases in T. fusca, we tested whether addition of 0.01% cellobiose would
180 induce cellulolytic activity. N. dassonvillei showed a stronger response on AZCL-HEC
181 plates when cellobiose was present (Table 2), but there was no effect on the other
182 organisms.
183 Transcriptional regulation. In T. fusca, cellulase production is regulated by
184 cellobiose through the LacI family transcriptional regulator CelR [4]. We checked to see
185 if the actinobacteria studied here have homologs of T. fusca CelR. A phylogenetic tree
186 was constructed, composed of proteins that have at least 1e-50 BLASTp score to T. fusca
187 CelR (Figure S2). All of the actinobacteria included in the study have at least one CelR-
188 related transcriptional regulator within the specified cutoff, and some of them have
189 several close homologs of CelR, the highest number being four in A. robiniae and S.
190 roseum.
191 A phylogenetic cluster is formed by T. fusca CelR, Ndas_0809, Sros_3304,
192 Tbis_1895, Tcur_1732, and Snas_6278. With the exception of Ndas_0809, these
193 transcriptional regulators are found in operons with putative cellobiose ABC transporters