Accepted Manuscript

Expression of a recombinant Lentinula edodes cellobiohydrolaseby Pichia pastoris and its effects on in vitro ruminal fermentationof agricultural straws

Lizhi Li, Mingren Qu, Chanjuan Liu, Ke Pan, Lanjiao Xu, KehuiOuYang, Xiaozhen Song, Yanjiao Li, Xianghui Zhao

PII: S0141-8130(19)30672-5DOI: https://doi.org/10.1016/j.ijbiomac.2019.05.043Reference: BIOMAC 12328

To appear in: International Journal of Biological Macromolecules

Received date: 27 January 2019Revised date: 4 April 2019Accepted date: 6 May 2019

Please cite this article as: L. Li, M. Qu, C. Liu, et al., Expression of a recombinantLentinula edodes cellobiohydrolase by Pichia pastoris and its effects on in vitro ruminalfermentation of agricultural straws, International Journal of Biological Macromolecules,https://doi.org/10.1016/j.ijbiomac.2019.05.043

This is a PDF file of an unedited manuscript that has been accepted for publication. Asa service to our customers we are providing this early version of the manuscript. Themanuscript will undergo copyediting, typesetting, and review of the resulting proof beforeit is published in its final form. Please note that during the production process errors maybe discovered which could affect the content, and all legal disclaimers that apply to thejournal pertain.

ACC

EPTE

D M

ANU

SCR

IPT

1

Expression of a recombinant Lentinula edodes cellobiohydrolase by Pichia

pastoris and its effects on in vitro ruminal fermentation of agricultural straws

Lizhi Li§, Mingren Qu§, Chanjuan Liu, Ke Pan, Lanjiao Xu, Kehui OuYang, Xiaozhen

Song, Yanjiao Li, Xianghui Zhao

Jiangxi Province Key Laboratory of Animal Nutrition/Engineering Research Center of

Feed Development, Jiangxi Agricultural University, Nanchang Jiangxi 330045, China

Corresponding author at: Jiangxi Province Key Laboratory of Animal Nutrition/Engineering Research Center

of Feed Development, Jiangxi Agricultural University, Nanchang Jiangxi, China.

E-mail: zhaoxh001@163.com.

§These authors contributed equally to this work.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

2

Abstract

An experiment was conducted to determine the effects of recombinant

cellobiohydrolase on the hydrolysis and in vitro rumen microbial fermentation of

agricultural straws including rice straw, wheat straw, and corn straw. The

cellobiohydrolase from Lentinula edodes (LeCel7A) was produced in Pichia pastoris.

The optimal temperature and pH for LeCel7A were 60°C and 5.0, respectively. The

recombinant protein enhanced the hydrolysis of three straws. During in vitro rumen

fermentation of three straws, the fiber digestibility, concentration of acetate and total

volatile fatty acids, and fermentation liquid microbial protein were increased by

LeCel7A. High throughput sequencing and real-time PCR data showed that the effects

of LeCel7A on ruminal microbial community depended on the fermentation substrates.

The relative abundances of Prevotellaceae_UCG_003 and Saccharofermentans were

increased by LeCel7A regardless of agricultural straws. With rice straw, LeCel7A

increased the relative abundances of Desulfovibrio, Ruminococcaceae and its some

genus. With wheat straw, LeCel7A increased the relative abundances of

Succiniclasticum, Ruminococcus flavefaciens, and Ruminococcus albus. With corn

straw, Succiniclasticum, Christensenellaceae_R_7_group and Desulfovibrio were

increased by LeCel7A. This study demonstrates that LeCel7A could enhance the

hydrolysis and in vitro ruminal fermentation of agricultural straws, showing the

potential of LeCel7A for improving the utilization of agricultural straws in ruminants.

Key words: Recombinant Lentinula edodes cellobiohydrolase; agricultural straw; in

vitro ruminal fermentation

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

3

1. Introduction

Agricultural by-products, for instance rice straw, wheat straw, and maize straw,

are generated in billions of tons per year all around the world [1]. These fibrous

materials contain considerable quantities of cellulose and hemicellulose and have the

potential to be a valuable feed source for ruminants. However, though these straws

could be utilized by ruminants through the ruminal microorganisms [2], their nutritive

feeding value is limited by their high-order molecular packing of lignocelluloses [3].

Consequently, the majority of these agricultural straws are as wastes either left in the

field for natural decay or burnt adding to environmental pollution [4]. In past years,

some physical and chemical technologies such as steaming, alkaline, and acidic

treatments have been investigated to improve the nutritional value of agricultural

straws [5, 6]. However, the application of these methods presents many disadvantages,

including high energy consumption [7] and high risk to the animal and environment,

especially when alkali used [8]. Biological treatments, including the use of white rot

fungi and their enzyme, have the potential to eliminate/reduce the problems associated

with physicochemical methods and appear to be the most promising in improvement

of straws digestibility [4, 9].

As one of white-rot fungi, Lentinula edodes, commonly referred to as the

Shiitake mushroom, produces all the core enzymes essential to the complete

enzymatic hydrolysis of lignocellulose [10]. L. edodes can use its enzymatic

machineries to break down lignocellulose and improve nutritive value of low quality

feeds, such as rape straw, wheat straw, rice straw, corn stover, and sugarcane bagasse,

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

4

but also resulted in the great losses of cellulose and hemicellulose during the

degradation, which limits its practical use [4, 11, 12]. In addition, considering that the

long incubation time of L. edodes and its cultivation products may contain toxic

substances [6], crude enzyme extracts of L. edodes as feed additive may be also not an

attractive option for treatment of straws. However, at least it means that L. edodes is a

good source for fibrolytic enzymes gene pool. Using heterologous expression

technology to obtain the enormous amount of interest enzyme protein within a short

time may be an easy way worthy of consideration.

From the perspective of the best-studied enzyme systems, the heart of

depolymerization of cellulose to glucose comprises cellobiohydrolases,

endoglucanases and β-glucosidase [13]. Cellobiohydrolases (also called

exoglucanases) hydrolyze the crystalline parts of the substrate by initiating their

action from the reducing or non-reducing ends of the cellulose chains, producing

primarily cellobiose and decreasing the substrate polymerization degree very slowly

[14, 15]. Cellobiohydrolases are key components in the multi-enzyme cellulose

complexes. Many studies have been conducted to investigate the functional

characterization of cellobiohydrolases and its potential synergistic role in the

enzymatic hydrolysis of biomass. Some results showed that cellobiohydrolases from

Trichoderma reesei displayed a high synergistic effect with cellulase and xylanase and

enhanced the enzymatic hydrolysis of corn stover, rice straw, and wheat straw [16, 17].

Within the rumen of ruminants, a complex group of anaerobic microorganisms,

including bacteria, archaea and eukaryotes, produces a vast array of lingo-cellulolytic

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

5

enzymes, which means that the rumen can provide an enzyme-containing

environment for the synergistic action and boosting activity of cellobiohydrolases.

Lee et al. (2001) cloned and characterized two cellobiohydrolases genes from L.

edodes, one of which belonged to glycosyl hydrolase family 7 (LeCel7A) [10]. Based

on the effects of L. edodes on agricultural straws degradation aforementioned and

characteristics of rumen, we hypothesize that supplementation of LeCel7A in the

rumen could enhance the hydrolysis and ruminal fermentation of agricultural straws,

however, little information is available. Therefore, this study recombined, expressed

and purified the LeCel7A by P. pastoris, investigated the effects of recombinant

LeCel7A on the hydrolysis of agricultural straws, and evaluated the effects of

recombinant LeCel7A on in vitro ruminal fermentation and microbial community of

agricultural straws and consequently the application possibility of LeCel7A in the

utilization of agricultural straws by ruminants.

2. Materials and Methods

This study was approved by the Animal Care and Use Committee of the College of

Animal Science and Technology of the Jiangxi Agricultural University.

2.1 Strains and reagents

P. pastoris strain X-33, expression vector pPICZαA, and antibiotic zeocin were

purchased from Invitrogen Corporation (Invitrogen, Carlsbad, CA). The competent

Escherichia coli DH5α cells and TIANprep Mini Plasmid Kit were purchased from

Tiangen Biotech (BEIJING) Co., Ltd (Beijing, China). Primers synthesis and DNA

sequencing were performed in Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China).

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

6

The N-glycosidase F (PNGase F), O-glycosidase, and α2-3,6,8,9 neuraminidase were

purchased from NEB Biolabs (Beijing, China). Other reagents were obtained from

standard commercial sources.

2.2 Synthesis of LeCel7A gene and construction of expression vector

The coding sequence of LeCel7A (GenBank accession AF411250), was

synthesized by GenScript Nanjing Co., Ltd. (Nanjing, China) with the following

modifications: Gene sequence was optimized for the preferred codon usage in P.

pastoris without altering the encoded amino acid sequence; the signal sequences

containing 18 codons and the stop codon were removed from the synthesized gene

sequence; and the unique restriction sites including EcoR I and Xba I were used for

plasmid construction, meanwhile, two base pairs (TT/AA) were introduced in the

protein C-terminal domains before the Xba I site to avoiding a translation frame shift

when the vector pPICZαA was used in this study.

The synthetic DNA was ligated into pPICZαA at the EcoR I and Xba I restriction

sites. The resulting plasmid was designated pPICZαA-LeCel7A and transformed into

E. coli DH5α. The pPICZαA-LeCel7A plasmids were extracted from transformed E.

coli and verified by PCR amplification using the α-Factor sequencing primer

(5´-TACTATTGCCAGCATTGCTGC-3´) and 3´ AOX1 sequencing primer

(5´-GCAAATGGCATTCTGACATCC-3´), restriction analysis and sequencing.

2.3. Transformation of P. pastoris and screening of LeCel7A expression stain

Recombinant plasmid pPICZαA-LeCel7A was linearized by restriction digestion

with Sac I and transformed into competent P. pastoris X33 by electroporation

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

7

(MicroPulser; Bio-Rad, CA, USA). The transformants were preliminarily screened on

the YPD agar plates containing zeocin and incubated at 30oC for three days. The

positive colonies were certified for integration of LeCel7A gene into the Pichia

genome by PCR using the primers of α-Factor and 3′AOX1 primers. For their enzyme

activities, the confirmed transformants were further grown in 2 ml of BMGY medium

and shaken overnight at 30°C. The cultures were centrifuged for 5 min at 2,000 rpm.

The pellets were then inoculated into 10 ml of BMMY medium in 100-ml flasks and

shaken at 30°C to induce expression. During the following 1-3 days, methanol was

supplemented every 24 h to the culture to a final concentration of 1.0% (v/v) for

maintaining the induction. On the final day, yeast cultures were collected and

centrifuged at 10,000 rpm for 5 min at 4°C. Supernatants were assayed for the

LeCel7A activity using Avicel (Avicel PH-101, catalog no. 11365; Sigma-Aldrich) as

substrates. The reaction mixture contained supernatants, 1% (w/v) Avicel, and 0.1 M

citrate/disodium hydrogen phosphate buffer. The assay was carried out at 40 °C for 1

h with gentle mixing by measuring the released reducing sugars. Supernatant with the

highest activity was further confirmed by sodium dodecyl sulphate polyacrylamide gel

electrophoresis (SDS-PAGE) staining with Coomassie Blue. The recombinant strain

with the highest activity was selected and kept for further incubation (Data are

included in the Supplemental Material).

2.4. Laboratory scale production of LeCel7A by P. pastoris

According to the Pichia fermentation process guidelines of Invitrogen, a seed

culture of recombinant LeCel7A strain screened was prepared in BMGY medium and

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

8

then inoculated into the batch medium. The production of LeCel7A was carried out in

a 4.0-liter working volume vertical glass bioreactor (Minifors 2, Infors HT,

Switzerland). Culture temperature was controlled at 30°C, pH was controlled at 6.0

with addition of 25% ammonium hydroxide, and the dissolved-oxygen concentration

was maintained above 20% saturation by controlling the stirrer speed or

supplementing pure oxygen, whereas the airflow was kept constant at 4 liters h-1. The

following glycerol fed-batch phase and methanol fed-batch phase were performed

according to the Pichia fermentation process guidelines of Invitrogen. Sample was

taken every 12 hours during the methanol fed-batch phase and the activity was

determined. When the activity no longer increased, the culture was finished.

The yeast culture was collected and centrifuged at 10,000 rpm for 5 min at 4°C.

Supernatants was concentrated and buffer exchanged in a binding buffer (pH 8.0)

consisting of 0.05 M sodium dihydrogen phosphate and 0.3 M sodium chloride using

a membrane separation system (Jinan Bona Biological Technology Co., Ltd., Jinan,

China) and loaded on a 5 ml Bio-Scale Mini Nuvia IMAC Ni-Charged column

(Bio-Rad, CA, USA) connected to a low-pressure chromatographic system (Biologic

LP; Bio-Rad, CA, USA) at 1.0 ml/min. The bound LeCel7A was first washed with the

aforementioned binding buffer containing 0.04 M imidazole (pH 8.0) and then eluted

with the binding buffer containing 0.25 M imidazole (pH 8.0). The flow-through,

which contained LeCel7A, was collected and changed to citrate/disodium hydrogen

phosphate buffer through Amicon Ultra-15 centrifugal filter unit. The protein sample

was verified by Western blot and quantified with Bradford Protein Assay Kit (Sangon

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

9

Biotech, Shanghai, China). SDS-PAGE was carried out to confirm the LeCel7A

protein purity. The N-glycosidase F, O-glycosidase, and α2-3,6,8,9 neuraminidase

were used to investigate the glycosylation of purified LeCel7A according to the

manufacturer's instructions.

2.5 Characteristics of recombinant LeCel7A

The activity of recombinant LeCel7A was measured according to the

aforementioned description. Determining the effects of pH on recombinant LeCel7A

activity was performed by the reaction mixtures containing purified enzyme, 1% (w/v)

Avicel, and 100 mM citrate/disodium hydrogen phosphate buffer (pH 3.0−7.0) at

40°C. The temperature dependency for recombinant LeCel7A activity was determined

by incubating reaction mixtures at 20−80 °C at the optimum pH.

2.6 Enzymatic hydrolysis of agricultural straws

Three agricultural straws including rice straw, wheat straw, and corn straw

(air-dry basis) were used in this study. The hydrolysis was performed using 8 µg

purified recombinant LeCel7A and 20 mg straw substrates in 2 ml 0.1 M

citrate/disodium hydrogen phosphate buffer (pH 5.0). The mixtures were incubated in

10 mL centrifuge tubes and shaken (200 rpm) for 24 h at 60°C as triplicates. The

blanks without recombinant LeCel7A were incubated similarly. The reducing sugars

released in samples were determined using alkaline 3,5-dinitrosalicylic acid reagent.

2.7 In vitro ruminal fermentation

An in vitro study was carried out to investigate the effects of LeCel7A on ruminal

fermentation. Rumen fluid was obtained from three ruminally fistulated beef cattle fed

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

10

a diet consisting of 700 g/kg rice straw and 300 g/kg concentrates before morning

feeding. The rumen liquid collected was filtered through four lays of cheesecloth and

mixed (1:2 v/v) with anaerobic buffer [18]. All manipulations were done under

continuous flushing with CO2. Fermentation was conducted in 120-mL serum bottles

to which 500 mg of agricultural straws and 60 mL of buffered rumen fluid were added.

Two hundred microgram purified LeCel7A was added into the incubation bottles as

LeCel7A treatments, and the same amount of citrate/disodium hydrogen phosphate

buffer instead of LeCel7A were incubated similarly as controls. Bottles were closed

and incubated in a shaking water bath at 39°C for 48 h. All samples were incubated in

triplicate. A blank (rumen fluid without substrate) was incubated in duplicate for

correction of residual dry matter (DM) in samples. The gas produced during

fermentation was expelled per 12 h. Fermentation was terminated by placing the

bottles on the ice, and the residue was filtered using pre-weighted nylon bag (37 µm

pore size) for the determination of in vitro NDF digestibility (IVNDFD). Samples of

filtrate were determined the ruminal pH immediately. One milliliter of ruminal fluid

was preserved by adding 1 ml of deproteinizing solution (100 g/L metaphosphoric

acid and 0.6 g/L crotonic acid) to determine volatile fatty acids (VFA). Ten milliliter

of filtrate was preserved to determine ammonia-N concentration and microbial protein

synthesis. The filtrate samples were also collected and stored at –80°C for DNA

extraction and microbial community determination.

2.6. Analytical procedures

The samples were analyzed for DM by drying at 65°C for 72 h. The NDF content

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

11

in samples was analyzed according to Van Soest et al. (1991) [19]. Ammonia-N in the

samples was analysed according to Weatherburn (1967) [20]. The VFA concentrations

in the filtered samples were determined by a gas chromatography (GC-2014;

Shimadzu Corp., Kyoto, Japan) equipped with a flame ionization detector, fitted with

a 30 m × 0.32 mm I.D. × 0.25 μm thickness film Stabilwax-DA column (Restek Corp.,

Bellefonte, PA, United States). Crotonic acid was used as an internal standard.

Fermentation liquid microbial protein (FLMCP) synthesis was determined according

to Makkar et al. (1982) [21].

The ruminal microbiota was determined by the high-throughput sequencing at Gene

Denovo Co. (Guangzhou, China) using an Illumina Hiseq2500 platform. The raw

Fastq data obtained was processed and analyzed according to our previous report [22].

Based on the operational taxonomic units (OTUs), bacterial richness indices and

bacterial diversity indices were determined. Principal coordinate analysis (PCoA) was

applied on the Bray-Curtis distances to generate two-dimensional plots. Linear

discriminant analysis (LDA) effect size (LEfSe) method was used to identify the most

differentially abundant taxons between groups and LDA ≥ 2.5 was chosen to indicate

significant difference during the analysis of high-throughput sequencing data [23].

The 16S ribosomal deoxyribonucleic acid (rDNA) copy numbers of total bacteria and

three fibrolytic bacterial species (Fibrobacter succinogenes, Ruminococcus

flavefaciens, and Ruminococcus albus) were determined according to our previous

report [22].

2.7 Statistical Analyses

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

12

Statistical analysis was performed using a One-way ANOVA in IBM SPSS

statistics version 20. Significance was declared at P ≤ 0.05.

3. Results and Discussion

3.1 Production and analysis of LeCel7A

For the production of LeCel7A, the LeCel7A gene from L. edodes was optimized

for the preferred codon usage and expressed in P. pastoris in the present study. The

native LeCel7A includes more than 19 % codons, such as GGC (Gly), UGU (Cys),

and AGC (Ser), which share <10 ‰ usage in P. pastoris. These rare codons were

replaced by preferred ones which were more frequently used in P. pastoris. In

addition, an algorithm (GenScript Nanjing Co., Ltd., Nanjing, China) with parameter

settings was used to optimize the mRNA secondary structure and repeat sequences of

LeCel7A, and the G+C content was adjusted from 52.8% in original gene to 40.8% in

optimized gene, which is much closer to the G+C content of P. pastoris (42.7%). The

codon-optimized gene shares 77.1% identity with the original LeCel7A gene.

The LeCel7A expressed and purified was verified by SDS-PAGE and Western blot

(Fig. 1A). Two intense protein bands at about 83 kDa and 47 kDa were observed in

the supernatant of the recombinant strain compared with the wild type. Similarly,

Taipakova et al. (2011) also observed two protein bands during the heterogenous

expression of LeCel7A using Escherichia coli [24]. One protein band with the

molecular weight of 83 kDa was larger than the predicted size (51 kDa) based on the

primary sequence of LeCel7A in the present study, which maybe resulted partly from

the increased glycosylation of LeCel7A when expressed in P. pastoris. One potential

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

13

N-glycosylation sites and 40 potential O-β-GlcNAc attachment sites were found in the

LeCel7A sequence using the NetNGlyc 1.0

(http://www.cbs.dtu.dk/services/NetNGlyc/) and YinOYang 1.2 tools

(http://www.cbs.dtu.dk/services/YinOYang/). The glycosylation of purified LeCel7A

was further verified using N-glycosidase and O-glycosidase. However, the

glycosidase treatment did not cause a significant shift in migration (Fig. 1B and Fig.

1C). The results are confusing and need to be elucidated in further studies.

3.2 Characteristics of recombinant LeCel7A

The recombinant LeCel7A exhibited high hydrolytic activity ranging from 50-60℃

and the optimum temperature for enzyme activity was at 60℃ (Fig. 2A). Though the

activity decreased dramatically at 70-80°C, it still kept the 60% of its optimum

activity, which suggested that the LeCel7A in the present study featured resistance to

high temperature. The recombinant LeCel7A showed maximum activity at pH 5.0 and

showed broad pH adaptability (>60% of the maximum activity at pH 3.0-7.0) (Fig.

2B). Current results were partly similar to the report by Bissenbaev et al. (2014), in

which the activity for recombinant LeCel7A by Saccharomyces cerevisiae was shifted

to high values at 60-70°C [25], but were different from the report by Taipakova et al.

(2011), in which the activity for recombinant LeCel7A by E. coli was maximum at

50°C and pH 7.0 [24]. Different expression system used in these studies may be

responsible for the discrepant temperature and pH dependency of LeCel7A [26, 27].

3.3 Enzymatic hydrolysis of agricultural straws

Abilities of LeCel7A to hydrolyze different agricultural straws were analyzed

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

14

(Fig. 3). The results showed that LeCel7A could significantly accelerated the

hydrolysis of all agricultural straws. After 24 h of incubation, the reducing sugars

content was increased by 5.7%, 23.1%, and 24.0% during the enzymolysis of rice

straw, wheat straw, and corn straw, respectively, relative to the control group.

Similarly, previous studies also found that Cel7A from some fungus exhibited high

hydrolytic efficiency toward agricultural straws [16, 17]. In addition, the dramatical

ability of LeCel7A to hydrolyze agricultural straws may help to explain why

significant cellulose losses were observed in L. edodes cultivation using agricultural

straws [4, 11, 12].

3.4 Effects of LeCel7A on in vitro ruminal fermentation of agricultural straws

Effects of LeCel7A on ruminal fermentation and microbial community in in vitro

cultures of agricultural straws were investigated (Table 1). The results showed that

LeCel7A increased IVNDFD for rice straw, wheat straw, and corn straw by 8.4%,

5.1%, and 5.5%, respectively, compared with controls. After incubation of 48 h,

regardless of agricultural straws, LeCel7A did not affect the ruminal pH, averaging

6.76 (data not shown). Supplemental LeCel7A increased the total VFA concentration

by 3.9%, 5.5%, 9.3% for rice straw, wheat straw, and corn straw, respectively, relative

to controls. The acetate and propionate concentrations were also greater in

LeCel7A-added agricultural straws than in the controls, which resulted in greater total

VFA production observed in the treatments. Regardless of agricultural straws, the

ammonia-N concentration was increased by added LeCel7A. Supplemental LeCel7A

increased FLMCP synthesis for rice straw, wheat straw, and corn straw by 10.5%,

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

15

18.7%, and 22.9%, respectively.

The high-throughput sequencing technology was used to investigate the effects of

LeCel7A on microbial community in in vitro ruminal incubation of agricultural straws.

To explore the dissimilarities in microbial composition between LeCel7A and control

groups, PCoA analysis based on Bray-Curtis metric was performed (Fig. 4). The

results showed that the microbiota clustered separately and axes accounted for 78.2%,

67.8%, and 74.7% of the total variation detected for rice straw, wheat straw, and corn

straw, respectively, suggesting that certain bacterial species may be affected by

LeCel7A.

Based on the Silva taxonomic database and using the analysis program Uparse, the

bacterial OTUs were classified and confirmed to 31 phyla and 296 genera in this

research. The dominant bacterial phylum was Bacteroidetes (47.7-53.9%), followed

by Firmicutes (27.9%-31.9%) and Verrucomicrobia (9.2-10.3%) in all groups (Fig.

5A). At the genus level, Prevotella_1 (9.8-17.9%) and Rikenellaceae_RC9_gut_group

(9.8-12.8%) were the dominant bacteria (Fig. 5B). To identify the taxon had the great

impact on microbial community, LEfSe analysis was performed and biomarkers were

found in both LeCel7A and control groups for three straws (Fig. 6). There were 36, 51

and 94 taxa found as biomarker for rice straw, wheat straw, and corn straw,

respectively. With rice straw, 16 taxa were increased by LeCel7A, among which

Saccharofermentans, Desulfovibrio, Prevotellaceae_UCG_003 and some genus

belonging to Ruminococcaceae, such as Ruminococcaceae_UCG_010,

Ruminococcaceae_UCG_013 and so on, were found to be predominant bacteria; 20

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

16

taxa were decreased by LeCel7A, among which Bacteroidetes,

Lachnospiraceae_AC2044_group, and Fibrobacter were found to be predominant

bacteria. With wheat straw, 23 taxa were increased by LeCel7A, among which

Prevotellaceae_UCG_003, Saccharofermentans, and Succiniclasticum were

predominant bacteria; 28 taxa were decreased by LeCel7A, among which

Fibrobacteres, Lentisphaerae, and Lachnospiraceae_AC2044_group were

predominant bacteria. With corn straw, 53 taxa were increased by LeCel7A, among

which Prevotellaceae_UCG_003, Saccharofermentans, and

Christensenellaceae_R_7_group were found to be predominant bacteria; 41 taxa were

decreased by LeCel7A, among which Bacteroidetes,

Ruminococcaceae_NK4A214_group, and Lachnospiraceae_AC2044_group were

predominant bacteria.

Besides the high-throughput sequencing technology, the quantity of total bacteria

and relative abundance of three representative fibrolytic bacteria species in the

fermentation liquid was determined using real-time PCR (Fig. 7). With rice straw,

LeCel7A tended to increase the relative abundance of R. flavefaciens and R. albus but

significantly decreased the relative abundance of F. succinogenes. With wheat straw,

LeCel7A significantly increased the relative abundance of R. flavefaciens and R. albus.

With corn straw, the relative abundance of R. flavefaciens was significantly increased

by supplemental LeCel7A.

Previous studies have shown that exogenous fibrolytic enzymes could enhance the

in vitro rumen fermentation of agricultural straws, including increased VFA

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

17

production and fiber digestibility [28-30]. Exogenous fibrolytic enzyme increased the

ruminal microorganism colonization of substrate, which accelerated the hydrolysis of

lignocellulose substrate [31]. However, the fibrolytic enzymes used in these studies

was mostly composite enzymes, and little information is available for the effects of

purified cellobiohydrolases on the ruminal fermentation. In current study, LeCel7A

significantly increased the IVNDFD during the incubations of three agricultural

straws, which might be due to the accelerated enzymatic hydrolysis of fibrous

material in the three straws caused by LeCel7A. Accordingly the acetate concentration,

total VFA concentration, and fibrolytic bacteria were significantly increased by

supplemental LeCel7A regardless of straws. In the rumen, VFAs are produced as

end-products of microbial fermentation and dietary carbohydrates including cellulose,

hemicellulose and starch, are the main fermentation substrates [32]. The relative

concentrations of the individual VFAs are determined by the type of dietary

carbohydrate [33]. Fermentation of structural carbohydrates (i.e. cellulose and

hemicellulose), compared to fermentation of starch, encourages the growth of acetate

producing bacterial species and consequently yielded high amount of acetate [32].

The relative abundance of genus Prevotellaceae_UCG_003 and Saccharofermentans

were increased by LeCel7A regardless of straws in the current study. Similarly, the

Prevotellaceae_UCG_003 abundance was increased significantly with increased

dietary NDF content or fiber digestibility of straws in previous studies [22, 34-36].

The genus Prevotellaceae_UCG_003 belongs to the family Prevotellaceae, which has

long been recognized as one of the predominant bacterial group inhabiting the rumen

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

18

[37]. Prevotellaceae could utilize various sugars and produce acetic and succinic acids

as major fermentation end products [38]. Among the family Prevotellacaea, some

genus or species contain endocellulase, β-glucosidase, and endoxylanase and are

involved in utilization of starch and plant cell-wall components digestion [39-41]. As

an uncultured genus, Prevotellaceae_UCG-003 maybe has the similar hydrolysis

activity, which contributes to the increase of IVNDFD for straws, but requires further

study. Saccharofermentans is known to play an important role in fiber degradation and

produces acetate as main end-products [42, 43]. Zhang et al. (2017) found that

Saccharofermentans was linearly increased with increased dietary forage content and

was positively associated with ruminal acetate concentration, which was consistent

with the current study [43]. The relative abundance of genus Desulfovibrio was

increased by LeCel7A for rice straw and corn straw. Desulfovibrio belongs to

sulfate-reducing bacteria and could metabolize lactate and pyruvate into acetate and

CO2 when the latter serves as an electron donor for sulfate reduction [44]. Therefore,

it was understandable that the increased acetate concentration associated with

increased relative abundance of genus Desulfovibrio. Some genus of

Ruminococcaceae, i.e. Ruminococcaceae_UCG_010, Ruminococcaceae_UCG_011,

and Ruminococcaceae_UCG_013 for rice straw, Ruminococcus_1 for wheat straw,

and Ruminococcaceae_UCG_010 and Ruminococcaceae_UCG_005 for corn straw,

were increased by LeCel7A in the present study. The family Ruminococcaceae

belongs to the phylum Firmicutes, which mainly digest cellulose, hemicellulose, and

some simple sugars widely existing dietary forage resulting in increased acetate

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

19

concentration [45, 46]. Though the metabolic characters of members from

Ruminococcaceae were variable, most of its genus are acetate producing bacteria and

could ferment carbohydrates to acetate as the major products [47]. As the

representative fibrolytic bacteria, R. flavefaciens, F. succinogenes, and R. albus were

all the important members of Ruminococcaceae and contribute significantly to fiber

metabolism and acetate production [48]. Previous in vitro studies showed that

proliferation of the three fibrolytic bacteria was often accompanied by the increase in

fiber digestion and acetate production [49, 50]. The IVNDFD, acetate concentration,

and part of the three representative fibrolytic bacteria were increased by LeCel7A in

the present study, which were in agreement with these reports. The relative abundance

of genus Succiniclasticum and Bacteroides were increased by LeCel7A for wheat

straw and corn straw. The genus Succiniclasticum belonging to the family

Acidaminococcaceae could convert succinate to propionate, the most important

precursor of glucose in ruminants [51, 52]. Therefore, the increased Succiniclasticum

may be relative to the increased propionate concentration by LeCel7A for wheat straw

and corn straw. The Bacteroides belongs to the family Bacteroidaceae. Many studies

reported that some Bacteroides species could ferment plant fiber with acetic acid and

succinic acid as major metabolic end products [53, 54], which means that the

Bacteroides maybe plays an important role in ruminal fiber digestibility. Besides the

increased taxa, some taxa including Bacteroidetes, Fibrobacteres, Lentisphaerae, and

so on, were reduced by supplemental LeCel7A. The microbial community was

dominated by Bacteroidetes and Firmicutes regardless of groups in the current study,

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

20

which is consistent with the study by de Oliveira et al. (2013) [55]. The reduction of

Bacteroidetes and other taxa may be due to the competition of faster growing

cellulolytic bacteria belonging to Firmicutes in obtaining nutrients or energy [56, 57].

Supplemental LeCel7A significant increased the ammonia-N concentration for

three straws in the present study. The ammonia-N concentration during fermentation

depends on the extent of dietary nitrogen degradation and nitrogen uptake by ruminal

bacteria. The higher production of ammonia-N for LeCel7A treatments may be related

to higher dietary nitrogen degradation compared with control treatments, and

increased protein or amino-acid degrading bacteria. The genus Desulfovibrio,

Bacteroides, and phylum Synergistetes increased by LeCel7A could degrade peptides

or amino acids and played important role in dietary nitrogen degradation [58-60],

which supported the deduction. The concentrations of ammonia-N exceeded 5 mg/dl

in all groups, which suggests that microbial growth may not be limited by available

ammonia-N in the present study [61]. Increased microbial protein production with

supplemental LeCel7A may relate to the synchrony between protein and carbohydrate

digestion during fermentation. Microbial protein synthesis depends largely on the

available amount and fermentation rate of carbohydrates and N in the rumen. With

enough available ammonia-N, increased NDF digestion by LeCel7A may stimulate

the growth of more ruminal bacteria, which was consistent with the study by Sommart

et al. (2000) [62].

4. Conclusions

The LeCel7A was expressed and produced using P. pastoris and shown

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

21

maximum activity at 60°C and pH 5.0. The LeCel7A could improve the hydrolysis

and in vitro ruminal fermentation of agricultural straws by increasing the digestion of

fiber, changing the microbial community, stimulating the growth of fibrolytic bacteria

and production of VFA, as well as increased rumen microbial protein synthesis. The

LeCel7A might be useful for improving the utilization of agricultural straws as a feed

additive for ruminants.

Funding

This work was supported by National Key R&D Program of China

(2018YFD0501804), the National Natural Science Foundation of China (31760687),

and the National Beef Cattle Industry Technology & System (CARS-Beef Cattle

System: CARS-38).

Notes

The authors declare no conflict of interest.

References

[1] M. Sain, S. Panthapulakkal, Bioprocess preparation of wheat straw fibers and their characterization,

Ind. Crop. Prod. 23 (2006) 1-8.

[2] I.K. Cann, Y. Kobayashi, M. Wakita, S. Hoshino, Effects of 3 chemical treatments on in vitro

fermentation of rice straw by mixed rumen microbes in the presence or absence of anaerobic rumen

fungi, Reprod. Nutr. Dev. 34 (1994) 47-56.

[3] Alborés, S. Pianzzola, M.J. Soubes, M. Cerdeiras, M. Pía, Biodegradation of agroindustrial wastes

by Pleurotus spp for its use as ruminant feed, Electron. J. Biotechn. 9 (2006) 215-220.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

22

[4] X. Zhao, J. Gong, Z. Shan, K. Ouyang, X. Song, C. Fu, L. Xu, M. Qu, Effect of Fungal Treatments

of Rape Straw on Chemical Composition and in vitro Rumen Fermentation Characteristics,

BioResources 10 (2015) 622-637.

[5] B.W. Alexander, A.H. Gordon, J.A. Lomax, A. Chesson, Composition and rumen degradability of

straw from three varieties of oilseed rape before and after alkali, hydrothermal and oxidative treatment,

J. Sci. Food Agric. 41 (1987) 1-15.

[6] C. Sarnklong, J.W. Cone, W. Pellikaan, W.H. Hendriks, Utilization of Rice Straw and Different

Treatments to Improve Its Feed Value for Ruminants: A Review, Asian-Aust. J. Anim. Sci. 23 (2010)

680-692.

[7] J.X. Liu, E.R. Orskov, X.B. Chen, Optimization of steam treatment as a method for upgrading rice

straw as feeds, Anim. Feed Sci. Tech. 76 (1999) 345-357.

[8] P.J.V. Soest, Rice straw, the role of silica and treatments to improve quality, Anim. Feed Sci.

Tech.130 (2006) 137-171.

[9] O. Loera, Efficiency of lignocellulolytic extracts from thermotolerant strain fomes sp. eum1:

stability and digestibility of agricultural wastes, J. Agr. Sci. Tech-Iran. 15 (2013) 229-240.

[10] C.C. Lee, D.W. Wong, G.H. Robertson, Cloning and characterization of two cellulase genes from

Lentinula edodes, Fems Microbiol. Lett. 205 (2001) 355-360.

[11] V.D. Tuyen, J. Cone, J. Baars, A. Sonnenberg, W. Hendriks, Fungal strain and incubation period

affect chemical composition and nutrient availability of wheat straw for rumen fermentation, Bioresour.

Technol. 111 (2012) 336-342.

[12] D. Tuyen, H. Phuong, J. Cone, J. Baars, A. Sonnenberg, W. Hendriks, Effect of fungal treatments

of fibrous agricultural by-products on chemical composition and in vitro rumen fermentation and

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

23

methane production, Bioresour. Technol. 129 (2013) 256-263.

[13] G. Banerjee, J.S. Scottcraig, J.D. Walton, Improving enzymes for biomass conversion: a basic

research perspective, Bioenerg. Res. 3 (2010) 82-92.

[14] T.T. Teeri, Crystalline cellulose degradation: new insight into the function of cellobiohydrolases,

Trends. Biotechnol. 15 (1997) 160-167.

[15] Y.L. Li, H. Li, A.N. Li, D.C. Li, Cloning of a gene encoding thermostable cellobiohydrolase from

the thermophilic fungus Chaetomium thermophilum and its expression in Pichia pastoris, J. Appl.

Microbiol. 106 (2010) 1867-1875.

[16] F. Hao, L. Xia, Heterologous expression and production of Trichoderma reesei cellobiohydrolase

II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw,

Biomass Bioenerg. 78 (2015) 99-109.

[17] H. Billard, Optimization of a synthetic mixture composed of major Trichoderma reesei enzymes

for the hydrolysis of steam-exploded wheat straw, Biotechnol. Biofuels, 5 (2012) 9.

[18] J.W. Cone, A.H.V. Gelder, G.J.W. Visscher, L. Oudshoorn, Influence of rumen fluid and substrate

concentration on fermentation kinetics measured with a fully automated time related gas production

apparatus, Anim. Feed Sci. Tech. 61 (1996) 113-128.

[19] P.J. Van Soest, J.B. Robertson, B.A. Lewis, Methods for dietary fiber, neutral detergent fiber, and

nonstarch polysaccharides in relation to animal nutrition, J. Dairy Sci. 74 (1991) 3583-3597.

[20] M.W. Weatherburn, Phenol-hypochlorite reaction for determination of ammonia, Anal. Chem. 39

(1967) 971-974.

[21] H.P. Makkar, O.P. Sharma, R.K. Dawra, S.S. Negi, Simple determination of microbial protein in

rumen liquor, J. Dairy Sci. 65 (1982) 2170-2173.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

24

[22] L. Li, M. Qu, C. Liu, L. Xu, K. Pan, X. Song, K. Ouyang, Y. Li, X. Zhao, Expression of a

recombinant Lentinula edodes xylanase by Pichia pastoris and its effects on ruminal fermentation and

microbial community in in vitro incubation of agricultural straws, Front. Microbiol. 9 (2018) 2944.

[23] N. Segata, J. Izard, L. Waldron, D. Gevers, L. Miropolsky, W.S. Garrett, C. Huttenhower,

Metagenomic biomarker discovery and explanation, Genome Biol. 12 (2011) R60.

[24] S. Taipakova, B. Smailov, G. Stanbekova, A. Bissenbaev, Cloning and expression of Lentinula

edodes cellobiohydrolase gene in E. coli and characterization of the recombinant enzyme, Journal of

Cell and Molecular Biology 9 (2011) 53-61.

[25] A. Bissenbaev, S. Taipakova, I. Smekenov, Expression of cellobiohydrolase CEL7A gene from

Lentinula edodes in Saccharomyces cerevisiae with use of two different promoters, J. Biotechnol. 185

(2014) S69-S69.

[26] L.S. Hsieh, C.S. Yeh, H.C. Pan, C.Y. Cheng, C.C. Yang, P.D. Lee, Cloning and expression of a

phenylalanine ammonia-lyase gene in Escherichia coli and Pichia pastoris, Protein Expres. Purif. 71

(2010) 224-230.

[27] L. Marianne Wittrup, U.T. Bornscheuer, H. Karl, Expression of Candida antarctica lipase B in

Pichia pastoris and various Escherichia coli systems, Protein Expres. Purif. 62 (2008) 90-97.

[28] J.S. Eun, S.H. Hong, K.A. Beauchemin, M.W. Bauer, Exogenous enzymes added to untreated or

ammoniated rice straw: effects on in vitro fermentation characteristics and degradability, Anim. Feed

Sci. Tech. 131 (2006) 87-102.

[29] L.Y. Zhao, Y.J. Peng, J.K. Wang, J.X. Liu, Effects of exogenous fibrolytic enzyme on in vitro

ruminal fiber digestion and methane production of corn stover and corn stover based mixed diets, Life

Sci. J. 12(2015) 1-9.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

25

[30] L.A. Giraldo, M.D. Carro, M.J. Ranilla, M.L. Tejido, A.H. Mohamed, A. Priolo, L. Biondi, H.B.

Salem, P. Morandfehr, In vitro ruminal fermentation of low-quality forages as influenced by the

treatment with exogenous fibrolytic enzymes, Options Méditerranéennes Series A, 74 (2007) 263-267.

[31] Y. Wang, J.E. Ramirez-Bribiesca, L.J. Yanke, A. Tsang, T.A. Mcallister, Effect of exogenous

fibrolytic enzyme application on the microbial attachment and digestion of barley straw in vitro,

Asian-Aust. J. Anim. Sci. 25 (2012) 66-74.

[32] J. Dijkstra, Production and absorption of volatile fatty acids in the rumen, Livest. Prod. Sci. 39

(1994) 61-69.

[33] J. France, J. Dijkstra, Volatile fatty acid production, in: Dijkstra, J., Forbes, J.M., France, J. (Eds.),

Quantitative aspects of ruminant digestion and metabolism. CABI Publishing/CAB International,

Wallingford, pp: 159.

[34] Q. Zhao, P. Zheng, Y. Tian, H. Huang, Effects of NFC/NDF in diet on rumen fermentation

parameters and microbial flora diversity in calves, Животноводство и кормопроизводство 101 (2018)

7-15.

[35] X. Zhang, L. Wang, Effects of dietary neutral detergent fibre level on structure and composition of

rumen bacteria in goats, Chinese Journal of Animal Nutrition 30 (2018) 1377-1386.

[36] L. Li, M. Qu, C. Liu, L. Xu, K. Pan, K. OuYang, X. Song, Y. Li, H. Liang, Z. Chen, Effects of

recombinant swollenin on the enzymatic hydrolysis, rumen fermentation, and rumen microbiota during

in vitro incubation of agricultural straws, Int. J. Biol. Macromol. 122 (2019) 348-358.

[37] K. Singh, T. Jisha, B. Reddy, N. Parmar, A. Patel, A. Patel, C. Joshi, Microbial profiles of liquid

and solid fraction associated biomaterial in buffalo rumen fed green and dry roughage diets by tagged

16S rRNA gene pyrosequencing, Mol. Biol. Rep. 42 (2015) 95-103.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

26

[38] N.R. Krieg, J.T. Staley, D.R. Brown, B.P. Hedlund, B.J. Paster, N.L. Ward, W. Ludwig, W.B.

Whitman, Family V. Prevotellaceae fam. nov, Bergey’s Manual of Systematic Bacteriology, 4(2011) 85.

[39] D.O. Krause, S.E. Denman, R.I. Mackie, M. Morrison, A.L. Rae, G.T. Attwood, C.S. McSweeney,

Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics, FEMS

Microbiol. Rev. 27 (2003) 663-693.

[40] M.A. Cotta, Interaction of ruminal bacteria in the production and utilization of

maltooligosaccharides from starch, Appl. Environ. Microb. 58 (1992) 48-54.

[41] H. Matsui, K. Ushida, K. Miyazaki, Y. Kojima, Use of ratio of digested xylan to digested cellulose

(X/C) as an index of fiber digestion in plant cell-wall material by ruminal microorganisms, Anim. Feed

Sci. Tech. 71 (1998) 207-215.

[42] J.H. Liu, M.L. Zhang, R.Y. Zhang, W.Y. Zhu, S.Y. Mao, Comparative studies of the composition

of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of

lactating dairy cows, Microb. Biotechnol. 9 (2016) 257-268.

[43] J. Zhang, H. Shi, Y. Wang, S. Li, Z. Cao, S. Ji, Y. He, H. Zhang, Effect of dietary forage to

concentrate ratios on dynamic profile changes and interactions of ruminal microbiota and metabolites

in Holstein heifers, Front. Microbiol. 8 (2017) 2206.

[44] G. Voordouw, The genus Desulfovibrio: the centennial, Appl. Environ. Microb. 61 (1995)

2813-2819.

[45] M. Sandri, C. Manfrin, A. Pallavicini, B. Stefanon, Microbial biodiversity of the liquid fraction of

rumen content from lactating cows, Animal 8 (2014) 572-579.

[46] J. Klang, S. Theuerl, U. Szewzyk, M. Huth, R. Tölle, M. Klocke, Dynamic variation of the

microbial community structure during the long‐ time mono‐ fermentation of maize and sugar beet

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

27

silage, Microb. Biotechnol. 8.(2015) 764-775.

[47] F.A. Rainey, Family VIII. Ruminococcaceae fam. nov, Bergey’s Manual of Systematic

Bacteriology 3 (2009) 1016-1018.

[48] S. Koike, Y. Kobayashi, Fibrolytic rumen bacteria: their ecology and functions, Asian-Aust. J.

Anim. Sci 22 (2009) 131-138.

[49] H.G. Sung, Y. Kobayashi, J. Chang, A. Ha, I.H. Hwang, J. Ha, Low ruminal pH reduces dietary

fiber digestion via reduced microbial attachment, Asian-Aust. J. Anim. Sci 20 (2006) 200-207.

[50] Y. Wang, T.W. Alexander, T.A. McAllister, In vitro effects of phlorotannins from Ascophyllum

nodosum (brown seaweed) on rumen bacterial populations and fermentation, J. Sci. Food. Agr. 89(13)

(2009) 2252-2260.

[51] F.A. Rainey, Genus XXIII. Succiniclasticum van Gylswyk 1995, 298VP, Bergey’s Manual of

Systematic Bacteriology 3 (2009) 1116-1117.

[52] M. Hélène, T. Corinne, C. Josiane, J.P. Hélène, R. Frédéric, G. Bernard, C. Jean-Philippe, J.B.

Estelle, Negativicoccus succinicivorans gen. nov., sp. nov., isolated from human clinical samples,

emended description of the family Veillonellaceae and description of Negativicutes classis nov.,

Selenomonadales ord. nov. and Acidaminococcaceae fam. nov. in the bac, Int. J. Syst. Evol. Microbiol.

60 (2010) 1271-1279.

[53] Y. Song, C. Liu, S. Finegold, Genus I. Bacteroides Castellani and Chalmers 1919, 959AL emend.

Shah and Collins 1989, 85, Bergey’s Manual of Systematic Bacteriology 4 (2011) 27-41.

[54] A. Salyers, J. Vercellotti, S. West, T. Wilkins, Fermentation of mucin and plant polysaccharides by

strains of Bacteroides from the human colon, Appl. Environ. Microb. 33 (1977) 319-322.

[55] M.N.V. de Oliveira, K.A. Jewell, F.S. Freitas, L.A. Benjamin, M.R. Tótola, A.C. Borges, C.A.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

28

Moraes, G. Suen, Characterizing the microbiota across the gastrointestinal tract of a Brazilian Nelore

steer, Vet. Microbiol. 164 (2013) 307-314.

[56] M. Theodorou, J. France, Rumen microorganisms and their interactions, Quantitative aspects of

ruminant digestion and metabolism , CABI, 2005.

[57] G. Witten, F. Richardson, Competition of three aggregated microbial species for four substrates in

the rumen, Ecol. Model. 164 (2003) 121-135.

[58] X. Zhao, Z. Chen, S. Zhou, X. Song, K. Ouyang, K. Pan, L. Xu, C. Liu, M. Qu, Effects of daidzein

on performance, serum metabolites, nutrient digestibility, and fecal bacterial community in bull calves,

Anim. Feed Sci. Tech. 225 (2017) 87-96.

[59] Y. Li, Q. Liao, M. Lin, D. Zhong, L. Wei, B. Han, H. Miao, M. Yao, Z. Xie, An integrated

metabonomics and microbiology analysis of host-microbiota metabolic interactions in rats with Coptis

Chinensis-induced diarrhea, RSC Adv. 5 (2015) 79329-79341.

[60] E. Jumas-Bilak, L. Roudiere, H. Marchandin, Description of 'Synergistetes' phyl. nov. and

emended description of the phylum 'Deferribacteres' and of the family Syntrophomonadaceae, phylum

'Firmicutes', Int. J. Syst. Evol. Micr. 59 (2009) 1028-1035.

[61] L. Satter, L. Slyter, Effect of ammonia concentration on rumen microbial protein production in

vitro, Brit. J. Nutr. 32 (1974) 199-208.

[62] K. Sommart, D. Parker, P. Rowlinson, M. Wanapat, Fermentation characteristics and microbial

protein synthesis in an in vitro system using cassava, rice straw and dried ruzi grass as substrates,

Asian-Aust. J. Anim. Sci. 13 (2000) 1084-1093.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

29

Fig. 1. Analysis of recombinant LeCel7A by SDS-PAGE, Western blot (A), and

deglycosylation (B and C). (A) 1, protein marker; 2, purified LeCel7A; 3, Western blot

analysis of culture supernatant from LeCel7A transformant. (B) 1, protein marker; 2, purified

LeCel7A; 3, LeCel7A treated with N-glycosidase F; 4, N-glycosidase F; (C) 1, protein marker;

2, purified LeCel7A; 3, LeCel7A treated with O-glycosidase and neuraminidase; 4,

O-glycosidase and neuraminidase.

A B

C

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

30

Fig. 2. Determination of the optimal temperature (A) and pH (B) of the purified recombinant

LeCel7A.

A

B

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

31

Fig. 3. The hydrolysis activity of purified recombinant LeCel7A on three agricultural straws.

Asterisks indicate significant differences (P ≤ 0.05).

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

32

Table 1 Effects of LeCel7A on rumen digestion and fermentation parameters of

agricultural straws in in vitro incubation.

Item

Rice straw

SEM

Wheat straw

SEM

Corn straw

SEM

Control LeCel7A Control LeCel7A Control LeCel7A

IVNDFD (%) 62.10 67.32* 1.22 62.44 65.60 0.88 56.83 59.97* 0.86

Total VFA (mM) 54.63 66.44* 2.81 64.70 68.30* 0.88 64.57 70.77* 1.50

Acetate (mM) 40.95 50.93* 2.33 48.92 51.45* 0.62 48.34 53.44 * 1.27

Propionate (mM) 9.85 11.07 0.35 11.09 11.70* 0.16 11.21 12.41 * 0.29

Butyrate (mM) 2.57 2.43 0.12 2.93 2.89 0.04 2.90 2.63 0.09

Other VFAs

(mM)

1.25 2.00* 0.17 1.77 2.27 * 0.11 2.12 2.28 0.05

Ammonia-N (mM) 8.56 13.68* 1.15 10.95 14.63* 0.83 11.96 15.67 * 0.84

FLMCP (μg/ml) 296.94 328.22* 8.02 295.18 350.29 * 12.83 312.02 383.33 * 18.59

Note: Asterisks indicate significant differences (P ≤ 0.05); the other VFAs including

valerate, isobutyrate and isovalerate.

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

33

Fig. 4. PCoA plot of samples from in vitro ruminal incubation of rice straw (A), wheat

straw (B), and corn straw (C) based on Bray-Curtis distances.

A B

C

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

34

Fig. 5. Distribution of the predominant rumen bacteria at phylum level (A) and genus level

(B). The color-coded bar plot represent the top 10 abundant taxa in the phylum level and the

top 20 abundant taxa in the genus level.

A

B

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

35

Fig. 6. LEfSe analysis displaying the ruminal bacteria change between control group and

LeCel7A group in in vitro ruminal incubation of rice straw (A), wheat straw (B) and corn

straw (C). LDA ≥ 2.5 and P ≤ 0.05 were shown.

A

C

B

ACCEPTED MANUSCRIPT

ACC

EPTE

D M

ANU

SCR

IPT

36

Fig. 7. Effects of LeCel7A on 16S rDNA gene copy numbers of three predominant ruminal

cellulolytic bacteria from fermentation liquid of agricultural straws in in vitro cultures.

A

D C

B

ACCEPTED MANUSCRIPT