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Efficient co-production of mannanase and cellulase by transformation of a codon-optimized endo-mannanase gene from Aspergillus niger into Trichoderma reesei Xianhua Sun, Xianli Xue, Mengzhu Li, Fei Gao, Zhenzhen Hao, Huoqing Huang, Huiying luo, Lina Qin, Bin Yao, and Xiaoyun Su J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05114 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017
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Journal of Agricultural and Food Chemistry
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Efficient co-production of mannanase and cellulase by
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transformation of a codon-optimized endo-mannanase gene
3
from Aspergillus niger into Trichoderma reesei
4
Xianhua Sun1, Xianli Xue1, Mengzhu Li1, Fei Gao1, Zhenzhen Hao1, Huoqing Huang1,
5
Huiying Luo1, Lina Qin2*, Bin Yao1*, Xiaoyun Su1* 1
6
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed
7
Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
8
2
National Engineering Research Center of Industrial Microbiology and Fermentation
9
Technology; College of Life Sciences, Fujian Normal University, Fuzhou, Fujian
10
350108, China
11 12
* Address correspondence to Lina Qin: National Engineering Research Center of
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Industrial Microbiology and Fermentation Technology; College of Life Sciences,
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Fujian Normal University, Fuzhou, Fujian 350108, China. Tel: +86-591-22868612,
15
E-mail:
[email protected]; Bin Yao and Xiaoyun Su: Key Laboratory for Feed
16
Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese
17
Academy of Agricultural Sciences, No. 12 South Zhongguancun Street, Beijing
18
100081,
19
[email protected].
China.
Tel.:
+86-10-82106094.
E-mail:
20 21 22
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[email protected];
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Abstract
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Cellulase and mannase are both important enzyme additives of animal feeds.
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Expressing the two enzymes simultaneously within one microbial host could
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potentially lead to cost saving in animal feeding. For this purpose, we codon
27
optimized the Aspergillus niger Man5A gene to the codon usage bias of Trichoderma
28
reesei. By comparing the free energy and the local structure of the nucleotide
29
sequences, one optimized sequence was finally selected and transformed into the T.
30
reesei pyridine auxotrophic strain TU-6. The codon optimized gene was expressed to
31
a higher level than the original one. Further expressing the codon optimized gene in a
32
mutated T. reesei strain in fed-batch cultivation resulted in co-production of the
33
cellulase and mannanase up to 1,376 U·mL-1 and 1,204 U·mL-1, respectively.
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Key words: mannanase; cellulase; Trichoderma reesei; feed additive
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Introduction
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Glycoside hydrolases serve as routine additives to animal feeds in the current
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husbandry industry
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cellulases are of huge demand since many kinds of animal feeds are plant-derived and
40
they contain plant cell wall polysaccharides. Decomposition of these polysaccharides,
41
mainly constituted by cellulose and hemicellulose by an in vivo enzymatic manner,
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has been shown to be beneficial for growth performance of livestock and poultry 3.
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The feeds derived from certain kinds of plants, such as palm kernel cake and copra
44
meal, are rich in cellulose and mannan 4. Adding cellulase and mannanase into these
45
feed stuffs will facilitate release of the nutritional components in the gastrointestinal
46
tract.
1, 2
. Among the glycoside hydrolases used as feed additives,
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Trichoderma reesei is a saprophytic filamentous fungus with prominent ability to
48
produce producing cellulase 5. It can also be used as a host to produce heterologous
49
genes 6. Proteins such as xylanase 7, lipase 8, and β-glucosidase 9 of bacterial, fungal,
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and even mammalian origin 10 have been successfully expressed in this organism.
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Traditionally, enzymes used for animal feeds are produced individually by
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different organisms. However, production of two or even more enzymes in one
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microorganism, instead of multiple hosts, may save fermentation cost and therefore be
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economically more beneficial. Endo-mannanase is an enzyme that can randomly
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degrade the internal chain of homogenous or heterogeneous mannans
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useful in enzymatic treatment of mannan-rich animal feeds. The Aspergillus niger
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GH5 endo-mannanase AnMan5A is an excellent endo-mannanase for potential usage 3
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11, 12
. It is very
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in animal feed: it is highly active on different kinds of mannans (locust bean gum,
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konjac glucomannan, and guar gum), thermostable by retaining 38.2% residual
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activity at 75°C for 4 h, and active at 40°C (near body temperature)13. In this study,
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we carried out the expression of the A. niger Man5A in T. reesei by using both the
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original and one codon-optimized gene. The properties of the recombinant AnMan5A
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were measured. Co-expression of AnMan5A gene with cellulase in a mutated strain
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with improved cellulase production performance was carried out in fed-batch
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fermentation.
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Materials and Methods
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Microbial strains and growth conditions
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The Escherichia coli Trans1 strain (Transgen, Beijing, China) was used as a host for
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plasmid construction and maintenance in this work. The S. cerevisiae AH109
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auxotrophic strain was used as the host for assembly of the expressing cassettes by
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DNA assembler
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peptone dextrose medium supplemented with adenine (YPDA). The T. reesei strains
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used to overexpress the mannanase gene were the uridine auxotrophic TU-6 (ATCC
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MYA-256) strain and the T. reesei SUS1 strain, which is a mutant of QM9414
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developed in our lab with enhanced cellulase producing ability and lower viscosity
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after multiple rounds of mutagenesis. The T. reesei strains were maintained on potato
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dextrose agar (PDA) at 28 °C for sporulation.
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Codon optimization
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The AnMan5A gene from A. niger LW-113 was synthesized by GenScript Inc.
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(Nanjing, China). The codon of AnMan5A was optimized by using the Gene Designer
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software15
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(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=51453).
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optimized AnMan5A nucleotide sequences were uploaded into the RNA structure
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software version 5.8.1 16 for analyses of the secondary structure and the free energy.
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Plasmid construction
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We used DNA assembler, a method allowing quick assembly of several DNA
89
fragments
14
. Then the yeast AH109 strain was cultivated at 30 °C in yeast
according
to
the
codon
usage
14
bias
of
T.
reesei Ten
, to construct the plasmid for expressing AnMan5A in TU-6. The 5
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EcoRI-restriction digested pRS424 plasmid (New England Biolabs, Beverly, MA) was
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mixed with the cbh1 promoter (amplified using Trcbh1pF/Trcbh1pR primer pairs
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from the genomic DNA of TU-6, Table S1), cbh1 terminator (amplified using
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Trcbh1tF/Trcbh1tR primer pairs from the genomic DNA of TU-6, Table S1), and the
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AnMan5A gene (amplified using Trman5AF/Trman5AR primer pairs from
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synthesized gene, Table S1) and these DNA fragments were co-transformed into
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AH109. The transformants were selected on yeast synthetic dropout medium without
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tryptophan. Assembly of the fragments in the pRS424 were verified by PCR from the
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transformant colonies with the gene specific primers of Yz-pcbh1pF/Yz-man5AR
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(Table S1) spanning the joint region of cbh1 promoter and AnMan5A. For the
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codon-optimized AnMan5A, our aim is to first evaluate its expression in the model
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strain TU-6 and then test its co-production with cellulase in the mutant strain SUS1.
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Unlike TU-6, the latter strain is resistant to co-transformation of two plasmids
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containing the gene of interest and the selection marker, respectively, which is
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commonly used in gene transformation of T. reesei
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manipulation, its expressing cassette was amplified using the primers of
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Trman5A(opt)F/Trman5A(opt)R and inserted into the BamHI and EcoRI sites of a
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plasmid pAPA, which was constructed in our lab and contained a pyr4 marker gene
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flanked by two tandem repeated DNA fragments (ampicillin-resistance genes). The
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repeated DNA fragments were designed for potential use in looping out the pyr4
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selection marker gene in the T. reesei transformants.
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Transformation of T. reesei 6
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. Therefore, for ease of
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The transformation of T. reesei was basically the same as that described in 17. Briefly,
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young mycelia cultured in MM-glucose (2 %) for 20 h were harvested and 10 mg/mL
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of Lysing Enzymes from Trichoderma harzianum (L1412, Sigma-Aldrich, St. Louis,
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MO) were added to release protoplasts from the mycelia. A pSKpyr4 plasmid
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harboring the pyr4-expressing cassette
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AnMan5A-bearing expression plasmid into TU-6 and selected on MM-glucose plates
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without addition of uridine. For the codon-optimized AnMan5A, the expressing
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plasmid was transformed into TU-6 and SUS1, respectively. The transformants were
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also selected on MM-glucose plates without uridine. After 5-7 days of cultivation at
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28ºC, transformants could be clearly visualized and they were screened for integration
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of the AnMan5A-expressing cassette in the chromosome by PCR using the primers
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Yz-pcbh1pF/Yz-man5AR (Table S1).
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Induction of AnMan5A expression in T. reesei
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For induction of recombinant AnMan5A (both the wild-type and codon-optimized)
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expression in T. reesei in shake flask fermentation, 107 fresh spores were inoculated
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into 50 mL of liquid MM supplemented with 2 % glucose and shaken at 28 ºC for 48
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h. The mycelia were filtered through a 200-mesh sifter and washed with sterile water
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to remove residual glucose. Then equal amounts of mycelia based on wet weight from
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the
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codon-optimized one) were transferred to 100 mL MM-Avicel (2%) and the culture
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was continued at 28ºC for induction of mannase expression. From day 2 to 7 post
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induction, 2 mL of the culture supernatants were periodically collected for assay of
parent
strain
TU-6
and
its
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was co-transformed with the original
AnMan5A transformants
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(wild-type
and
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enzymatic activities and protein concentrations. The fed-batch cultivation of T. reesei
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was essentially the same as that carried out in
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Avicel cellulose The operating volume was 10 L. Ammonium hydroxide was used to
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provide nitrogen and maintain the pH at 5.5. The fermentation temperature was 28°C.
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Assay of mannanase and cellulase activities and protein concentration
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For assay of the endo-mannase activity, 100 µL of appropriately diluted crude enzyme
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(fermentation broth) were mixed with 5 mg/mL of locust bean gum in 0.2 M
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Na2HPO4-0.1 M citric acid buffer (pH 5.0) with a total volume of 1 mL. The reaction
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was incubated at 50 ºC for 15 min. The reducing sugars released were determined
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using the 3,5-dinitrosalicylic acid (DNS) method 19. One unit of mannanase activity
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was defined as the amount of enzyme liberating 1 µmol of reducing sugar in one
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minute. The cellulase activity of T. reesei can be presented as the overall cellulase
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activity or divided into endo-glucanase, exo-glucanase, and β-glucosidase activities 20.
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For assay of the overall cellulase activity, the reaction included one strip of Whatman
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No.1 filter paper (6 × 1 cm) and 100 µL of culture supernatant in a 50 mM acetate
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buffer (pH 4.8) and was incubated at 50 ºC for 1 h.21 One unit of the overall cellulase
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activity was defined as the amount of enzyme that released 1 µmol of reducing sugar
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per hour under the assay conditions. For endo-glucanase, sodium carboxymethyl
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cellulose (CMC-Na, 10 mg/mL, purchased from Sigma-Aldrich, St. Louis, MO) was
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used as the substrate and the reaction was carried out in the McIlvaine buffer (pH 5.0)
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and incubated at 50 ºC for 30 min. One unit of the endo-glucanase activity was
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defined as the amount of enzyme that released 1 µmol of reducing sugar per min
18
with the change of Solka Floc to
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under the assay conditions. The assay of cellobiohydrolase I (CBHI) activity was
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essentially
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4-methylumbelifery-β-D-lactopyranoside (MUL) as the substrate and incubated at 50
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ºC for 20 min 22. One unit of CBHI activity was defined as the amount of enzyme that
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released 1 µmol of 4-Methylumbelliferyl (4-MU) per min under the assay condition.
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The
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(p-Nitrophenol-β-D-glucopyranoside) as the substrate and incubated at 50 ºC for 10
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min. One unit of β-glucosidase activity was defined as the amount of enzyme that
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released 1 µmol of pNP in one hour. The protein concentration was determined with a
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BCA-200 Protein Assay Kit (Pierce, Rockford, IL) by using bovine serum albumin as
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the standard.
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Assay of specific enzyme production rates
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The specific mannanase production rate from day 1 to 6 was calculated. Because the
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inducing culture contains insouble cellulose Avicel prohibiting us from directly
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measuring the mycelia weight, we used the mycelia protein as a representative of the
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fungal biomass. The mycelia of the strains expressing AnMan5A and codon optimized
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AnMan5A were collected in different time periods from day 1 to 6 and the method
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described by Jayaraman 23 was adopted to assay the mycelia proteins.
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Effect of pH and temperature on the biochemical properties of AnMan5A
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To determine the optimal pH of recombinant AnMan5A, the fermentation broth of the
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codon-optimized AnMan5A transformant on 5 days post induction was incubated with
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5 mg/mL of locust bean gum in 0.2 M Na2HPO4-0.1M citric acid buffer with the pH
the
same
β-glucosidase
as
that
activity
described
was
by
Beiley
determined
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et
al.
using
using
pNPG
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ranging from 2.5 to 7.5. The optimal temperature of recombinant AnMan5A was
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determined at the optimal pH with the temperatures changing from 30 to 90 °C. To
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determine the pH-stability, the crude enzyme was first pretreated in a buffer with
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varying pHs (pH 1.0–2.0: 100 mM Glycine-HCl; pH 3.0–8.0: 100 mM McIlvaine
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buffer; pH 9.0–12.0: 100 mM Glycine-NaOH) at 37°C for 1 h. To determine the
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thermostability, the crude enzyme was pre-incubated at 75°C and 80°C for different
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periods of time (10 min, 20 min, 30 min, 40 min, 50 min, and 1 h). For both
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pH-stability and thermostability assays, the residual activity after pretreatment was
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determined under its optimal conditions (pH 4.5 and 80 °C). The released reducing
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sugars were measured using the DNS method.
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Mass spectrometry analysis
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The fermentation broth for TU-6 expressing the wild-type AnMan5A was first
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resolved on the SDS–PAGE gel and the protein bands at ~43 kDa were excised and
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used for in-gel trypsin digestion. The digestion solution was first incubated for 60 min
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at 4 °C and then the sample was incubated for 14 h at 37 °C. To extract the peptide
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fragments from the tryptic digests, 20 µl of 5% (v/v) TFA were added and incubated
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for 60 min at 37 °C. Thereafter, 20 µl of 50% (v/v) acetonitrile (containing 2.5% (v/v)
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TFA acid) was added to gel pieces and incubated for 60 min at 30 °C. The extracted
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peptides were dissolved in 0.1% formic acid in distilled water. LC−MS/MS analysis
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was run on Easy-nLC 1000 (Thermo Fisher Scientific, Bremen, Germany) coupled
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Q-Exactive HF (Thermo Fisher Scientific) mass spectrometer. Samples were loaded
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onto a reversed-phase trap column at a flow rate of 5 µL/min. Buffer A (0.1% formic 10
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acid in water) and buffer B (0.1% formic acid in acetonitrile) were used as mobile
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phase buffer. Peptides were separated on an analytical column at a flow rate of 350
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µL/min using the following gradients: from 3 to 8% buffer B in 1 min, from 8 to 28%
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buffer B in 10 min, from 28 to 90% buffer B in 2 min, and remaining at 90% buffer B
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for 7 min. The peptides were eluted from the analytical column and directly injected
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into the mass spectrometer via nano-ESI source. Ion signals were collected in a
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data-dependent mode. The RAW MS/MS data were searched against a composite
207
database for protein identification using in-house PEAKS software (version 8.0).
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Quantitative reverse transcription PCR analysis
209
For quantitative reverse transcription PCR (qRT-PCR), the mycelia of TU-6 and of the
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strains expressing wild-type AnMan5A and codon-optimized AnMan5A, respectively,
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in shake flask fermentation were collected at 24 h post Avicel induction. The mycelia
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were pulverized in liquid nitrogen using a pestle and mortar. Total RNA was extracted
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from frozen lyophilized mycelia using the TRIzol reagent (Thermo Fisher Scientific,
214
Waltham, MA). One µg of RNA was treated with DNAse I and reverse-transcribed to
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cDNA using the First Strand cDNA Maxima Synthesis kit (TOYOBO, Shanghai,
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China). qRT-PCR was performed in a QuantStudio 6 Flex Real-Time PCR System
217
(Applied Biosystems, San Diego, CA) using a TransScript Green One-Step qRT-PCR
218
SuperMix (TransGen, Beijing, China). The actin(act) gene was used as an
219
endogenous reference gene. The primers used for qRT-PCR were actF/R, Man5AF/R1,
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Pcbh1F/R, Pcbh2F/R, Pegl1F/R, and Pbgl1/F/R and their sequences were given in
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Table S1. The following amplification conditions were used:95 ℃for 10 min, then 40 11
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cycles including 94℃ for 10s, 60 ℃ for 20 s, and 72 ℃ for 30 s.
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Deglycosylation
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Endo-β-N-acetylglucosaminidase H (Endo H) from New England Biolabs (Ipswich,
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MA) was used to remove N-glycosylation. The reaction system containing 9 µL
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protein (fermentation broth of TU-6 expressing original and codon-optimized
227
AnMan5A and of Pichia pastoris expressing AnMan5A) and 1 µL Denaturing Buffer
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(0.5% SDS, 40 mM DTT) was boiled at 100 ºC for 10 min. Then, 2 µL GlycoBuffer 3
229
(50 mM sodium acetate, pH 6) and 1 µL Endo H were added and the mixture was
230
incubated at 37 ºC for 2 h. The Endo H-treated enzymes were analyzed by SDS-PAGE.
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For expression of AnMan5A in P. pastoris, the synthesized gene was cloned into the
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EcoRI and NotI restriction sites of pPIC9 (Invitrogen, Carlsbad, CA) and the
233
recombinant plasmid was transformed into GS115.
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Results
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Codon optimization of AnMan5A
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To improve expression of a heterologous gene, codon optimization is commonly
237
employed to avoid rare codons that may appear in the gene of interest. For this
238
purpose, the AnMan5A gene was codon optimized using the Gene Designer software
239
15
240
more parameters were considered in addition to the algorithms of the software. These
241
included i) the free energy of the mRNAs, which is indicative of their stability and ii)
242
the local secondary structure near the start codon (ATG) of the transcripts. For this
243
consideration, we generated 10 codon-optimized AnMan5A gene sequences by
244
repeated running Gene Designer. Even with the same seed AnMan5A amino acid
245
sequence, codon usage table, and algorithm, the software calculated quite different
246
nucleotide sequences in each run. Since the cbh1 promoter and terminator were used
247
to drive expression of AnMan5A gene, these ten nucleotide sequences were
248
computationally placed between the 5’ untranslated region (UTR)-the cbh1 signal
249
sequence and the 3’ UTR. These combinatorial mRNA sequences were calculated for
250
their secondary structure and free energy using the RNA structure software. From the
251
calculation, the optimized gene AnMan5A-7 was selected for further expression
252
analysis, which had a relatively low free energy and loose local structure at the start
253
codon (Fig. 1 and Fig. S1).
254
Construction of plasmids for AnMan5A expression in T. reesei
255
Both the original and the codon-optimized AnMan5A gene were synthesized by the
according to the codon bias of T. reesei. In the codon optimization process, two
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GenScript company (Nanjing, China). The original AnMan5A gene was mixed with
257
the T. reesei cbh1 promoter and terminator, and transformed together with EcoRI
258
linearized pRS424 into the S. cerevisiae AH109 strain. With this DNA assembly
259
method via yeast in vivo homologous recombination, the cbh1 promoter, AnMan5A
260
gene, and cbh1 terminator were one-step assembled in the pRS424 plasmid (Fig. 2A).
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The codon-optimized AnMan5A gene was also assembled into pRS424, and then the
262
expressing cassette for AnMan5A was further amplified and inserted into the BamHI
263
and EcoRI sites of a plasmid pAPA for transformation of a mutant T. reesei strain
264
using the Gibson assembly method (Fig. 2B). The plasmid pAPA harbors a
265
pyr4-expressing cassette conferring uridine autotroph, which is flanked by two
266
ampicillin-resistance encoding genes. The repeating ampicillin-resistance DNA
267
fragments can be used for future potential looping out the pyr4 gene in the T. reesei
268
transformants.
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Expression of AnMan5A in T. reesei
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The whole expression plasmids bearing the original and codon-optimized AnMan5A
271
genes were individually transformed into TU-6, a uridine auxotroph mutant of T.
272
reesei QM9414
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AnMan5A-expressing cassettes and then cultivated in shake-flasks for initial screen of
274
the mannanase-producing strains. For both the original and codon-optimized
275
AnMan5A genes, one representative transformant was selected, which had the highest
276
mannanase activity in the initial screen. These two transformants, with the TU-6 host
277
strain, were cultivated for detailed analysis of the mannanase and cellulase activities
24
. The transformants were first detected for integration of the
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and protein production.
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The original AnMan5A gene was successfully expressed in T. reesei. On
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SDS-PAGE gel, it appeared as a smear centering at around 43 kDa (Fig. 2C). Mass
281
spectrometry analysis unequivocally supported this conclusion (Fig. S2). Interestingly,
282
two clear bands, but not a smear, were observed on the SDS-PAGE gel for the
283
codon-optimized mannanase (Fig. 2C). From the SDS-PAGE analysis, codon
284
optimization apparently enhanced expression of this gene. In accordance with this
285
analysis, while only low mannanase activity was observed for TU-6, the transformant
286
bearing the original AnMan5A had much higher mannanase activity, peaking at day 4
287
with activity of 2.9 U·mL-1. Codon optimization largely improved the mannanase
288
activity, with highest activity appearing at day 6 with 9.3 U·mL-1 (Fig. 3A). The
289
specific mannanase production rate of codon-optimized AnMan5A from day 1 to day
290
6 was 133.3 U·g mycelia protein·h-1, higher than 32.7 U·g mycelia protein·h-1 for the
291
original AnMan5A. The extracellular protein concentrations of the three strains had
292
the same pattern with that of the mannanase activity (Fig. 3B). The difference in the
293
CBH1 (Fig. 3C) and endo-glucanase (Fig. 3D) activities of TU-6 and the two strains
294
were not large. However, it appeared that the more AnMan5A was expressed, the less
295
β-glucosidase activity was observed (Fig. 3E). As the overall cellulase activity is the
296
synergy among the exo-glucanase, endo-glucanase, and β-glucosidase, the overall
297
cellulase activity of TU-6 expressing codon-optimized AnMan5A was the lowest
298
among the three strains (Fig. 3F).
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Transcript levels of AnMan5A and main cellulase genes 15
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Total RNA was extracted from the mycelia collected at 24 h post Avicel cellulose
301
induction and the transcript levels of AnMan5A and main cellulase genes were
302
quantified by qRT-PCR. As shown in Fig. 4, the transcript level of AnMan5A(opt)
303
was nearly the same as that of wild-type AnMan5A. Therefore, the transcription could
304
not account for the large elevation in the mannase activity. The observed higher
305
mannanase activity for AnMan5A(opt)-expressing strain was most likely from
306
enhanced translation efficiency caused by codon optimization. Further analysis
307
indicated that two cellobiohydrolases (cbh1 and cbh2) and one main endo-glucanase
308
(egl1) did not change much in TU-6 and the two strains expressing AnMan5A, in
309
accordance to the comparable cellobiohydrolase and endo-glucanase activity in the
310
three strains (Fig. 3C, D). Interestingly, we noted that, while the extracellular
311
β-glucosidase activity decreased significantly in AnMan5A-expressing strains, the
312
mRNA level of the major extracellular β-glucosidas gene bgl1 was very similar in
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these three strains.
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Biochemical properties of recombinant AnMan5A
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Since the parent strain TU-6 displayed very low endo-mannanase activity (Fig. 3A),
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the biochemical properties of the recombinant AnMan5A could be estimated using the
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crude enzyme, i.e. the culture supernatant. The codon-optimized AnMan5A had a
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maximal activity at pH4.5 and exhibited over 50% activity between pH 3.5 to 6.0 (Fig.
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5A). It had an optimal temperature of 80 °C and retained 27% activity at 40°C, a
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temperature close to animal body temperature (Fig. 5B). The recombinant enzyme
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was quite stable: over 60% residual activity was retained after pretreatment of 16
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AnMan5A between pH 2.0 to 12.0 for 1 h (Fig. 5C). Moreover, the residual activity of
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AnMan5A only slightly reduced to 78% after treatment at 75 °C for 1 h (Fig. 5D).
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Co-production of cellulase and mannanase by recombinant T. reesei in fed-batch
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cultivation
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The codon-optimized AnMan5A gene was further transformed and expressed in SUS1,
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a mutant strain of QM9414 with improved cellulase activity. In the fed-batch
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cultivation, the maximal endo-glucanase activity reached 1,376 U·mL-1 (Fig. 6A) and
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the endo-mannanase activity maximized to 1,204 U·mL-1 (Fig. 6B), respectively.
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Discussion
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We proposed that co-production of two or more feed enzymes could potentially save
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costs of animal husbandry. Among the commonly used microbes, T. reesei is a good
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candidate for co-expression of cellulase with another feed enzyme for the following
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two reasons: i) it is by itself a prominent cellulase producer
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could be over 100 g·L-1 5. Indeed, by simply transforming an endo-mannanase gene
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into T. reesei, we were able to express cellulase and mannanase simultaneously within
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one microbe with enzyme activities up to 1,376 U·mL-1 and 1,204 U·mL-1,
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respectively. Both enzymes have great potential as feed enzymes and have been at
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least tested or commercialized for their usage in the feed industry. Therefore, to the
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best of the authors’ knowledge, this is the first report of co-expression of two feed
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enzymes, specifically cellulase and mannanase, in one microbe. However, we noticed
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that expression of β-glucosidase was negatively affected by the over-expression of
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AnMan5A. Higher expression of AnMan5A led to lower β-glucosidase activity
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(compare Fig. 3A and Fig. 3E). Given that the transcription of bgl1 was similar in all
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three strains studied (Fig. 4), one reason could be that the recombinant AnMan5A
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competed for certain secretory pathway components which were also used by the
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β-glucosidase for secretion. However, more evidence is needed to support this
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hypothesis in the future.
20
and ii) its productivity
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The recombinant AnMan5A produced in T. reesei had an optimal pH of 4.5 and
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temperature of 70°C, while these parameters for AnMan5A expressed in P. pastoris
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were pH3.5 and 70°C, respectively13. The AnMan5A expressed in P. pastoris has 18
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38.2% residual activity after 1 h of incubation at 75°C. The AnMan5A expressed in T.
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reesei is also thermostable by keeping 78% residual activity after pretreating at 75 °C
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for 1 h, a feature favorable for application in feed industry since the feed pelleting
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involves processing at high temperatures
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result of different glycosylation patterns, which may have profound effects on
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enzyme’s thermostability
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broth of recombinant TU-6 expressing wild-type and codon-optimized AnMan5A and
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of the AnMan5A expressed in Pichia pastoris indicated that the mannanase was
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differently glycosylated (Fig. S3). It is not known why the electrophoresis patterns of
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AnMan5A expressed from the original and codon-optimized genes were slightly
363
different.
25
. The different thermostability may be a
26
. Indeed, deglycosylation analysis of the fermentation
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Codon optimization successfully improved expression of the Man5A gene from A.
365
niger in T. reesei. Many factors could affect heterologous gene expression in a
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microbial host 6. For example, scrutinizing the local structure at the start codon and
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mutating the corresponding coding nucleotides have been successfully used for
368
improving expression of a 1,3-1,4-β-D-glucanase from Fibrobacter succinogens in P.
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pastoris 27. Since there is no such study reported for manipulating heterologous gene
370
expression in T. reesei, we employed this strategy and combined it with considering
371
the free energy of the transcript. We found that repeated running of Gene Designer
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produced optimized genes with much differing nucleotide sequences. These
373
sequences were calculated to have varying free energies and secondary structures near
374
the start codon. From these calculations, we selected one optimized gene with 19
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apparently more favorable parameters and this simple method proved to be useful for
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codon optimization of AnMan5A. This strategy could be also used for testing
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expression of other heterologous genes in T. reesei in the future.
378
In conclusion, an endo-mannanase gene from A. niger was transformed in T.
379
reesei TU-6. Its expression was successfully improved by codon optimization which
380
was carried out by comparing the free energies and local secondary structures near the
381
start codon of the computationally designed transcripts. Introduction of the
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codon-optimized AnMan5A expression cassette in a T. reesei mutant resulted in
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co-production of cellulase and mannanase at high titers of 1,376 U·mL-1 and 1,204
384
U·mL-1, respectively.
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Supporting Information Available
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Primers used in this study (Table S1), Predicted secondary structures of the transcripts
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of codon-optimized AnMan5A gene (Figure S1), Mass spectrometry analysis of the
389
expressed recombinant AnMan5A (Figure S2), and Deglycosylation analysis of
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AnMan5A (Fig. S3).
391 392
Funding sources
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This research was supported by a grant from the National Key R&D Program of
394
China (2016YFD0501409-02), the National Natural Science Foundation of China
395
(31400067), the China Modern Agriculture Research System (CARS-42), and the
396
Elite Youth Program of Chinese Academy of Agricultural Sciences.
397 398
CONFLICTS OF INTEREST
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The authors declare that they have no conflicts of interest with the contents of this
400
article.
401 402
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Figure legends
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Figure 1. Codon optimization of AnMan5A gene for expression in T. reesei. The free
405
energy and local secondary structure as analyzed by the RNAstructure software were
406
shown in (A) and (B), respectively. The arrow indicated the start codon, where the
407
translation initiates.
408
Figure 2. Expression of AnMan5A in T. reesei. A and B: Schematic diagrams for the
409
expression cassette of the wild-type AnMan5A (A) and the codon-optimized
410
AnMan5A gene (B). C: SDS-PAGE analysis of the fermentation broth of T.
411
reesei-expressing AnMan5A. M: protein molecular mass marker; lane 1: TU-6; lane 2:
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T. reesei expressing the wild-type AnMan5A; lane 3: T. reesei expressing the
413
codon-optimized AnMan5A. The arrows indicate the recombinant AnMan5A.
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Figure 3. Endo-mannanase and cellulase activities of the fermentation broth in the
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shake flask culture of the recombinant T. reesei strains harboring the wild-type or
416
codon-optimized AnMan5A. A: The endo-mannanase activity using locust bean gum
417
as the substrate. B: The overall extracellular protein concentration. C: The CBH1
418
activity. D: The endo-glucanase activity. E: The β-glucosidase activity. F: The overall
419
cellulase activity measured using filter paper as the substrate. All enzymatic activities
420
were measured at 50°C and pH 5.0. TU-6, AnMan5A, and AnMan5Aopt: the crude
421
enzymes from the parent strain TU-6, the TU-6 expressing the wild-type AnMan5A,
422
and that expressing the codon-optimized AnMan5A, respectively.
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Figure 4. Relative transcript level of AnMan5A and main cellulase genes in TU-6 and
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the two T. reesei strains expressing the wild-type AnMan5A and codon-optimized 22
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AnMan5A, respectively, as analyzed by qRT-PCR. The mycelia collected at 24 h post
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Avicel induction were used for RNA extraction and reverse transcription quantitative
427
PCR. For AnMan5A, its relative expression level in the T. reesei strain expressing the
428
wild-type AnMan5A was set as 1.0. For cbh1, cbh2, egl1, and bgl1, the relative
429
expression level of the genes in TU-6 were set as 1.0.
430
Figure 5. Biochemical properties of the AnMan5A crude enzyme in the fermentation
431
broth of TU-6 expressing the codon-optimized gene. A: The optimal pH of the
432
recombinant AnMan5A. B: The optimal temperature. C: The stability of AnMan5A at
433
different pHs. D: Thermostability. For determination of optimal pH and temperature,
434
the maximal activities were set as 100%. For pH and thermostability assays, the
435
activity of the enzyme prior to pretreatment was set as 100%.
436
Figure 6. Co-production of cellulase and endo-mannanase by T. reesei SUS-1
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harboring the codon-optimized AnMan5A gene in fed-batch cultivation. A: The
438
endo-glucanase activity. B: The endo-mannanase activity.
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