Abstract
Feather waste is the highest protein-containing resource in nature and
is poorly reused. Bioconversion is widely accepted as a low-cost and
environmentally benign process, but limited by the availability of safe
and highly efficient feather degrading bacteria (FDB) for its
industrial-scale fermentation. Excessive focuses on keratinase and
limited knowledge of other factors have hindered complete understanding
of the mechanisms employed by FDB to utilize feathers and feather
cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently
degrade feather to products with high amino acid content, useful as a
nutrition source for animals, plants and microorganisms. Using multiple
omics and other techniques, we reveal how SCUT-3 turns on its feather
utilization machinery, including its colonization, reducing agent and
protease secretion, peptide/amino acid importation and metabolism,
oxygen consumption and iron uptake, spore formation and resuscitation,
and so on. This study would shed light on the feather utilization
mechanisms of FDBs.
Subject terms: Applied microbiology, Biocatalysis
__________________________________________________________________
Li et a. report a new Streptromyces isolate, SCUT-3 which can
efficiently degrade feather into products with high amino acid content,
useful as feed for plants, animals and microbes. Using multiple omics
and other techniques, they report how SCUT-3 turns on its feather
utilization machinery and suggest a number of expressed genes most
likely implicated in feather degradation.
Introduction
Feathers predominantly comprise beta-keratin (approximately 85%), and
exists in the currently living birds and their evolutionary ancestors,
the feathered dinosaurs. The poultry industry generates increasing
amounts of feather waste; according to the Alltech Global Feed Survey,
broiler feed production in 2018 was 0.3 billion tons, which estimated
to produce about 0.2 billion tons of chicken and more than 10 million
tons of chicken feathers. Numerous disulfide bonds make the feather
matrix highly durable and resistant to chemical and physical
factors^[38]1.
Traditional hydrothermal and chemical treatments currently used in the
feather meal industry are energy exhausted and the produced feather
meal has low solubility and is difficult to be digested by animals.
These high temperature and pressure treatments release large amounts of
sulfur and ammonia waste gases, making them unsustainable and
polluting. The most promising alternative approaches involve
biodegradation using keratinases or bacteria. Numerous feather
degrading bacteria (FDB) have been isolated from various genera,
including Bacillus, Staphylococcus, Enterococcus, Streptomyces, and
Pseudomonas sp., among others^[39]2,[40]3. Most FDB need ≥ 5–6 days to
completely degrade 1% feather-containing medium and this low efficiency
limits their industrial application. A Pseudomonas otitis isolate, H11,
was recently reported to degrade 1% feather medium with 88.8%
efficiency after 48 h, which is the highest efficiency reported to
date^[41]4. Most research on FDB remains at an experimental stage,
requiring adjustment before industry-scale applications can be
developed.
To understand the feather-degrading mechanisms of FDB, the keratinases
they secrete have been studied intensively^[42]2 and bacteria
expressing recombinant keratinases constructed. A recombinant KerK
strain, Bacillus amyloliquefaciens K11, can completely degrade feather
plumes in 12 h, but not the feather shaft^[43]5. While keratinases do
have a vital role in feather degradation, the abundant disulfide bonds
in feather keratin lead to a tightly packed structure, inaccessible to
these enzymes and disulfide bond reduction mechanisms used by FDB are
poorly understood. Only metabolically active Streptomyces pactum cells
have been reported to produce soluble reducing agents; however, the
specific reducing agents involved were not identified^[44]6. Sulfide
production is indispensable for dermatophyte nail infection^[45]7 and a
free cysteinyl group is essential for feather degradation by
Escherichia coli expressing recombinant keratinase^[46]8, indicating
that similar reducing agents may be produced by FDB.
FDB are opportunistic species and do not need feathers to survive in
soil. Their adjustment to feather utilization mode when they encounter
feather in the soil is a systematic process involving colonization,
secretion of reducing agents and keratinases, matter transportation,
and alterations in metabolism/replication/transcription/translation
processes. Merely describing keratinases, as in previous reports of
FDB, is far from understanding the feather utilization processes of
these organisms. Here, a new FDB, Streptomyces sp. SCUT-3, was isolated
and found to exhibit high-efficiency industrial feather degradation.
The product of its feather fermentation can be used as safe fish feed
protein source, plant fertilizer, and microorganism culture medium.
Ultra-microstructural analysis revealed the colonization and spore
dispersion of SCUT-3 on feathers. Further, we sequenced the genome and
compared the transcriptomes of SCUT-3 bacteria cultured on LB and
feather to identify factors involved in feather degradation and reveal
the mechanisms underlying its feather utilization.
Results
SCUT-3 efficiently degrades feather to peptides and amino acids
Using feather powder plates, we identified 27 isolates from soil under
a feather pile in Shaoguan (Guangdong, China) able to degrade feather.
Among these isolates, Streptomyces sp. SCUT-3 was the most efficient;
it could completely degrade both the plumage and shafts of white
chicken feathers in 36 h in chicken feather medium (CFM) comprising a
1% feather:liquid ratio (Fig. [47]1b, c), resulting in 93.6 ± 1.5%
solid feather matter weight loss (Fig. [48]1a). White feather matter
protein content was determined as 85% using the Kjeldahl method^[49]9,
and the total protein content of feather products did not change before
and after fermentation in any following experiments. In 1% CFM, of the
85% total dry feather protein, 31.6% was converted to soluble amino
acids, 12.7% to soluble peptides, and the remaining 40.7% remained in
the undegraded feather residue and bacterial cells (Fig. [50]1a).
Different from other reported FDB, SCUT-3 produced higher levels of
soluble amino acids than peptides, indicating that it could secrete
efficient terminal peptidases, as well as endopeptidase. These
hydrolyzed amino acids and peptides could be used by SCUT-3 to sustain
its growth and proliferation.
Fig. 1. High-efficiency degradation of feather by SCUT-3 and application of
fermented feather products in animal, plant, and microorganism cultures.
[51]Fig. 1
[52]Open in a new tab
a Feather degradation rate of 1%, 10%, and 40% CFM by SCUT-3, presented
as weight loss (red columns), and composition of amino acids, peptides,
and insoluble proteins remaining undegraded in feather and bacteria of
products (blue columns); n = 3/group. b, c 0 and 36 h SCUT-3 culture in
1% CFM, showing complete feather degradation. d, e 0 and 6 d SCUT-3
culture in 40% CFM. No feather plumage or shafts were visible in the
sticky gruel after 6 days of feather fermentation. f Rice growth in
control (left) and 50 mg amino acids added (from 10% SCUT-3 cultured
CFM broth) (right) soil (50 g), showing the robust leaves and shorter
roots with more root hairs. g Succulent growth in the control (left)
and amino acids added (right) soil; n = 3/group, *P < 0.05. h Feed
coefficients of full fish meal feed (45% fish meal, FM) and half
fermented feather meal substitute feed (22.5% fish meal + 22.5%
fermented feather meal, FM + FFM) after use for eight weeks in tilapia
culture; n = 20/group, *P < 0.05. i Fresh weight of rice grown in
control and supplemented soil; n = 6/group, *P < 0.05. j The effect of
feather degradation products on microbial recombinant protein
production; n = 3/group, *P < 0.05. The experimental group added 3 mg
amino acids (from 10% SCUT-3 cultured CFM broth) to the control LB
medium. Asterisks indicate significant differences in feed coefficient,
fresh weight and recombinant protein production. P-values between
groups were obtained by unpaired two-tailed Student’s t test. All data
were presented as mean ± SD.
The feather degradation efficiency of most FDB isolates is tested using
1% CFM, with few reports of their performance in higher feather: liquid
ratio media. For industrial application of FDB, low feather: liquid
ratios are inefficient, with resulting products requiring further
concentration or drying. We found that SCUT-3 could completely degrade
feather in 10% CFM in 8 days, with a 50.3 ± 1.4% degradation rate on
day 4. Further, in 40% CFM, a 57.3 ± 2.3% degradation rate was observed
on day 6 (Fig. [53]1a) and no feather plumage or shafts were visible in
the fermentation broth, which had the appearance of sticky gruel
(Fig. [54]1d, e). The excellent performance of SCUT-3 in high feather:
liquid ratio fermentation makes it a competitive reagent for
application in the feather biodegradation industry. Further, the high
soluble free amino acid and peptide content of its feather fermentation
products make them good candidates for use in animal feed, plant amino
acid-containing fertilizer, and as a microbial nitrogen source. The
amino acid composition, including free amino acids, free amino
acids/peptides, and whole proteins in the 40% fermented feather meal
produced by SCUT-3, was compared with that of untreated feather
(Supplementary Table [55]1); cystine content reduced from 8.20% to
5.88%, while methionine increased from 0.46% to 1.38%, indicating
disruption of disulfide bonds during feather fermentation.
To determine the safety and nutritional value of the feather meal
produced by 40% fermentation for use in animal feed, it was added to
tilapia feed. After 4 weeks, all fish survived in the group receiving
fermented feather meal as a sole protein source (45% addition) and all
tissues and organs were normal on anatomical examination, indicating
the safety of the fermented feather meal. The feed coefficient of the
fermented feather meal group was high (1.90), which is not unexpected,
due to its unbalanced amino composition, solely derived from feather
protein. The feed coefficient of the fermented feather meal: fish meal
(FFM: FM, 22.5%:22.5%) group was 0.85, close to the full fish meal
group (0.71) (Fig. [56]1h), demonstrating that this fermented feather
meal is a relatively good protein source for use in animal feed.
Additional nutritional tests are needed in future studies to support
its rational addition to feed. To determine whether 10% fermented
feather broth is good plant fertilizer, it was added to soil-grown
(50 mg amino acids of 10% fermented broth in 50 g soil) and hydroponic
(50 mg L^−1 amino acids) rice. Using both methods, the rice grew much
more strongly in the group with feather broth added, with more robust
leaves (Fig. [57]1f). The fresh weight of rice cultured in soil with
feather broth addition on week 3 was 2.09 times that of controls
(Fig. [58]1i). Interestingly the roots of rice in the fermented feather
broth added group were shorter, with many more root hairs, compared
with controls (Fig. [59]1f). Succulent plants also exhibited earlier
budding and better growth in broth supplemented with fermented feather
(Fig. [60]1g). These experiments show that the SCUT-3 fermented feather
broth is a good plant amino acid liquid fertilizer. To determine
whether the 10% fermented feather broth is benefit for microbial growth
and recombinant protein production, it was added to the culture of KerK
recombinant B. subtilis (3 mg amino acids of 10% fermented broth in
20 mL LB medium). No significant difference (p = 0.08) of microbial
growth was observed, while the production of recombinant KerK was
improved by 45.5% (Fig. [61]1j); a similar phenomenon was observed in
another recombinant esterase B. subtilis (Supplementary Fig. [62]1).
These experiments show 10% fermented feather broth can improve
microorganism’s recombinant protein production, probably by supplying
amino acids for protein synthesis.
SCUT-3 is a new Actinomyces species with penetration ability
The 16 s rRNA of SCUT-3 was sequenced (Gene Bank accession number
[63]MK743936.1) and a phylogenetic tree generated, demonstrating that
SCUT-3 is a species of the Streptomyces genus, genetically closest to
Streptomyces thermolineatus (Supplementary Fig. [64]2). We then
assembled the genome of SCUT-3 from Single Molecule Real Time
Sequencing data, revealing that its size is approximately 6.08 Mb;
further, 5811 genes were annotated (Gene Bank accession number:
[65]CP046907). Calculation of average nucleotide identity (ANI) showed
that the highest ANI value for SCUT-3 (78.51%) was with S. cattleya DSM
46488, among 1222 Streptomyces genomes available (Supplementary
Table [66]2), indicating that SCUT-3 is a new species, different from
all those tested. The physiological and biochemical characteristics of
SCUT-3 were tested and compared with those of S. thermolineatus A1484,
according to Bergey’s Manual of Systematic Bacteriology (Supplementary
Table [67]3). Unlike S. thermolineatus, SCUT-3 was positive for
hydrogen sulfide production, while mannitol use was negative. SCUT-3
was a Gram-positive bacterium with white colonies on Gauze No. 1 medium
plates at day 2, becoming gray-green by day 5. The surface of the
colony was dry and had an earthy smell. We compared the
feather-degrading capacity of SCUT-3 with those of three other
Streptomyces genus FDB species: S. thermolineatus, S. fradiae, and S.
cattleya; SCUT-3 had the highest feather-degrading ability
(Fig. [68]2g), the highest keratinase activity (Fig. [69]2h), and
second most potent disulfide bond reduction activity (after S.
thermolineatus) (Fig. [70]2i). These data suggest that SCUT-3 is a new
Streptomyces species, which we named Streptomyces sp. SCUT-3. The
strain has been deposited in the Guangdong Provincial Center for
Microbial Strains and the strain number is GDMCC No: 60612.
Fig. 2. Scanning electron microscopy observation of SCUT-3 cultured on
feather and comparison of feather degradation by SCUT-3 and three other
Streptomyces sp. FDB.
[71]Fig. 2
[72]Open in a new tab
a Untreated chicken feather control. b Feather cultured with SCUT-3 for
36 h, showing the mycelium tangled around barbules. c Feather cultured
with SCUT-3 for 60 h, showing the compact attachment of the mycelium
and the digestion and slit of the barbule surface by SCUT-3. d, e
Feather cultured with SCUT-3 for 84 h, showing the formation, diffusion
of SCUT-3 spores. f Spores diffusion is accompanied by spore
resuscitation, beginning a new growth cycle. g–i Comparison of the
degradation rate, keratinase activity, reducing power; n = 3/group,
*P < 0.05. j Penetration of SCUT-3 and three other Streptomyces sp. FDB
in 1% and 4% LB agar. P-values between groups were obtained by unpaired
two-tailed Student’s t test. All data were presented as mean ± SD.
SEM examination showed that SCUT-3 produced tightly tangled filamentous
mycelium around feather barbules on days 1–2 (Fig. [73]2b). Barbule
surfaces were compactly attached by the hyphae, then disintegrated and
digested by SCUT-3 on days 2–3 (Fig. [74]2c). Numerous smooth spores
were generated along the filaments at regular intervals on day 3, which
then dispersed onto the surface of a different barbule and quickly
germinated to form new filamentous mycelia (Fig. [75]2d–f). Penetration
testing showed that SCUT-3 had the strongest mechanical penetration
force, followed by S. thermolineatus, S. cattleya, and S. fradiae
(Fig. [76]2j), as it could penetrate most deeply into 4% agar. The
efficient elongation and tangling of its mycelium, as well as effective
spore generation and diffusion, rapid spore germination, and strong
mechanical penetration, may help SCUT-3 to quickly and competitively
occupy the feather niche and become the dominant flora after
encountering feather in the soil.
Transcriptomes showed SCUT-3’s adaption to feather medium
Most FDB are opportunistic species that can survive without feather.
Hence, it is reasonable to suppose that they live in different modes in
the presence or absence of feather; however, no previous report has
compared transcriptome differences in detail, according to growth on
feather-containing or other media. Here, we found that SCUT-3 only
degraded the feather in the feather medium, but not feather added to LB
medium, indicating that SCUT-3 does not use feather when other
nutrition is available, and that feather utilization-associated
machines are shut off in the LB-cultured SCUT-3. Transcriptome of
SCUT-3 cultured for 24 h in LB or 1% feather medium were sequenced and
compared. The RNA-seq data used in this study are deposited in the
National Center for Biotechnology Information SRA database (SRA
accession no. PRJNA611875). Then, contigs were assembled and annotated,
with reference to the SCUT-3 genome data. We detected 775 up-regulated
and 623 down-regulated differentially expressed genes (DEGs) (volcano
plot, Supplementary Fig. [77]3). Significant DEGs (p < 0.05) were
enriched for specific GO terms (Supplementary Figs. [78]4 and [79]5)
and KEGG pathways (Supplementary Fig. [80]6). Next, we mapped the
significant DEGs to the SCUT-3 genome, revealing that many were
regulated in operon mode (see subsequent results). Sixteen of the top
20 differential pathways were metabolism-related, indicating that
SCUT-3 invokes an alternative metabolism strategy when using feather as
its sole carbon and nitrogen source, relative to LB medium. The
remaining four of the top 20 pathways comprised a two-component system,
quorum sensing, oxidative phosphorylation, and ABC transport,
reflecting the activities of SCUT-3 in sensing its circumstances,
active oxidative respiration, and matter transportation, during feather
utilization. Additional DEGs and their organization in the genome are
described in detail below, along with further experimental data,
revealing the feather utilization mechanisms likely used by SCUT-3
bacteria.
Disulfide bond reduction is the first key step
Although few details are known about the mechanisms involved in
disulfide bond reduction by FDB, the importance of disulfide bond
sulfitolysis for feather degradation is established. Based on release
of sulfhydryl content from oxidized glutathione, we found that only
living SCUT-3 cells could reduce disulfide bonds, while its
extracellular secretory supernatant or intracellular cell lysate could
not (Fig. [81]3b), similar to previously reported FDB^[82]6.
Fig. 3. Feather disulfide bond reduction and up-regulation of reducing agent
production-associated genes in feather medium-cultured SCUT-3.
[83]Fig. 3
[84]Open in a new tab
a Sulfite detection in SCUT-3-cultured CFM supernatant by BaCl[2]
precipitation, HCl bubble production, and KMnO[4] bleaching tests. b
Evaluation of SCUT-3 reducing power by detection of sulfhydryl content
release from oxidized GSH, showing that only the living cells, but not
its intracellular or extracellular components, exhibited reducing
power; n = 3/group. c Sulfite improvement of KerK (pink) and DTNB
inhibition of SCUT-3 (red) feather degradation, showing the function of
sulfite and sulfhydryl groups in feather disulfide bond breakdown;
n = 3/group, *P < 0.05, P-values between groups were obtained by
unpaired two-tailed Student’s t-test. d qRT-PCR verification of the
up-regulation of sulfite production (cdo1/cdo2/mdeA), sulfite
exportation (tauE), and mycothiol synthesis (mshA/mshD) genes
(n = 3/group). e The operon structure of glutathione transporter genes
(red numbers above gene symbols are log[2] FC values for each gene).
All data were presented as mean ± SD.
Production of sulfite to reduce disulfide bonds is important for
dermatophyte infection of nails^[85]7. Here, we found that 80 mM
Na[2]SO[3] addition could efficiently improve feather degradation of
the recombinant keratinase KerK in vitro (from 11.5 ± 0.7% to
15.5 ± 1.1%, p = 0.006) (Fig. [86]3c), the degradation rate of 8 M
Na[2]SO[3] addition could reached much higher of 43.5 ± 0.7%, while
there was no effect using <80 mM Na[2]SO[3] (Supplementary Fig. [87]7).
Sulfite production in feather cultured SCUT-3 supernatant was confirmed
by BaCl[2] precipitation, HCl bubble production, and KMnO[4] bleaching
(Fig. [88]3a), suggesting that this process likely accompanies
disulfide bond destruction by SCUT-3. Although the concentration in the
culture supernatant may not reach as high as 80 mM, we believe
comparatively high sulfite concentrations could be produced locally in
the compact contact surfaces between SCUT-3 mycelia and feather
barbules detected by SEM (Fig. [89]2b–c). Dermatophyte mutants of cdo
and ssu (genes encoding proteins involved in sulfite production and
exportation) lose their nail infection ability^[90]7. Two cysteine
dioxygenase genes, cdo1 and cdo2, were up-regulated in feather
medium-cultured SCUT-3 according to transcriptome data and confirmed by
real-time RT-PCR, with fold-change values, 50.2 and 8.3, respectively.
An L-methionine γ-lyase, mdeA, was also up-regulated (28.6-fold)
(Fig. [91]3d). Cdo and MdeA can oxidize the sulfhydryl group of
cysteine and the methyl thioether of methionine, respectively, to
generate sulfite^[92]10,[93]11. No ssu gene was detected in the SCUT-3
genome; however, another sulfite exporter, tauE, was found and
up-regulated approximately 2.0-fold (Fig. [94]3d). These data indicate
that sulfite production may be involved in SCUT-3 breakdown of feather
disulfide bonds.
Besides sulfite, free cysteinyl groups are important reducing agents in
feather sulfitolysis. Cysteinyl-glycine produced from GSH is required
for feather degradation by E. coli expressing recombinant
keratinase^[95]8. DTNB assays revealed the participation of free
cysteinyl in SCUT-3-mediated sulfitolysis, with addition of DTNB (5 mM)
reducing the SCUT-3 feather degradation rate (Fig. [96]3c). Many
actinomycetes lack GSH, with mycothiol (AcCys-GlcN-Ins, MSH)
functioning as a surrogate^[97]12. Two genes (mshA and mshD) involved
in mycothiol synthesis were up-regulated 4.3- and 9.8-fold,
respectively, in feather medium-cultured SCUT-3 (Fig. [98]3d). No MSH
transporter is yet defined^[99]13. A GSH transporter operon, gsiDBCA,
was annotated in the SCUT-3 genome and its expression up-regulated in
feather medium-cultured SCUT-3 (Fig. [100]3e). Whether MSH is the
cysteinyl group involved in feather sulfitolysis by SCUT-3 and whether
gsiDBCA is responsible for free cysteinyl group transportation in
SCUT-3 requires further study.
Protease hydrolyzation and peptide/amino acid transportation
Following disulfide bond reduction, feather is further hydrolyzed by
secretory proteases, to generate peptides and free amino acids. SCUT-3
secreted abundant proteases and the keratinase activity of 24 h 1%
feather medium-cultured SCUT-3 supernatant was 65.8 U mL^−1, about 4.6
times that of LB-cultured SCUT-3 (14.3 U mL^−1) (Table [101]1). In
transcriptome data, 19 of 22 significantly (p < 0.05) differentially
expressed extracellular protease genes were up-regulated in feather
medium-cultured SCUT-3, with fold-change values much higher than the
remaining three down-regulated proteases (Fig. [102]4a). The 19
up-regulated proteases were serine-type, cysteine-type, and
metalloproteases; most were endopeptidases, and one
dipeptidyl-peptidase and one carboxypeptidase were identified. These
proteases’ classifications according to MEROPS database and Pfam
peptidase domains were shown in Supplementary Table [103]4. Unlike
changes to the expression pattern of extracellular proteases, 10
up-regulated and 11 down-regulated intracellular proteases were
detected. Expression levels of the six most up-regulated proteases were
verified by qRT-PCR, with the highest fold-change in expression for
Sep39 protease, of 451.9-fold (Fig. [104]4b). These extracellular
proteases are likely involved in keratin hydrolysis to produce peptides
and amino acids by SCUT-3.
Table 1.
Overexpression of proteinase Sep39 in Streptomyces sp. SCUT-3.
Medium Strain Relative mRNA level Keratinase activity (U mL^−1)
Degradation rate (%) Peptide content (mg mL^−1) Amino acid content
(mg mL^−1)
LB(24 h) SCUT-3 1 14.3 ± 1.9 – – –
SCUT-3-sep39 5.6 ± 0.1* 64.4 ± 2.1* – – –
1%CFM (24 h) SCUT-3 261.1 ± 41.3 65.8 ± 5.0 64.5 ± 1.1 1.3 ± 0.1
1.4 ± 0.1
SCUT-3-sep39 618.9 ± 91.5* 102.5 ± 3.8* 71.0 ± 1.0* 1.5 ± 0.1*
1.7 ± 0.1*
5%CFM (48 h) SCUT-3 – 69.1 ± 3.7 45.5 ± 1.1 4.5 ± 0.2 6.5 ± 0.2
SCUT-3-sep39 – 117.1 ± 3.1* 53.3 ± 0.8* 5.8 ± 0.6* 8.7 ± 0.6*
[105]Open in a new tab
Note: Asterisks indicate significant differences compared to wild-type
SCUT-3; n = 3/group, *P < 0.05. P-values between groups were obtained
by unpaired two-tailed Student’s t test. All data were presented as
mean ± SD.
Fig. 4. Up-regulation of protease secretion and amino acid/peptide
transporter genes in feather medium-cultured SCUT-3.
[106]Fig. 4
[107]Open in a new tab
a Significantly regulated extracellular and intracellular protease
genes identified by transcriptome analysis, showing the significant
up-regulation of most secretory proteases. b qRT-PCR verification of
the top 6 significantly up-regulated protease genes; n = 3/group. c
qPCR results of nine protease genes and one disulfide reduction related
gene at 0, 3, 6, 12, and 24 h; n = 3/group. d The up-regulation of two
PTR di- and tri-peptide transporter genes (cstA and dtpT), two peptide
ABC transporter operons (oppABCDF I and oppABCDF II), and two amino
acid ABC transporter operons (metNIQ and livJHMGF). Numbers above genes
are log[2] FC values for each gene; red > 1.0, pink < 1.0. P-values
between groups were obtained by unpaired two-tailed Student’s t test.
All data were presented as mean ± SD.
To elucidate the expression pattern of these proteinases during the
feather degradation, qPCR of the nine most up-regulated proteases were
detected at 0, 3, 6, 12, and 24 h in 1% CFM medium culture. As we
showed in the heatmap of Fig. [108]4c, all nine tested proteases were
quickly and up-regulated at 3 h. Among them, the up-regulation fold of
sep39 kept rising until 24 h, that of cp17 continued to rise until 12 h
and went down at 24 h. The up-regulation folds of the other seven
proteases sustained at their 3 h levels and went down at the later
different time points. The different up-regulation mode of these
proteases reflected their cooperation mode during the feather
degradation. Besides proteases, we also detected the cdo1’s expression
(Fig. [109]4c) and found up-regulation folds of Cdo1 kept rising during
24 h as Sep39, which indicates the disulfide reduction accompanies the
peptide bond hydrolysis in the feather degradation process.
To further confirm the function of the up-regulated proteases in the
feather degradation, we overexpressed them in SCUT-3. In this article,
we showed the overexpression results of the highest-up-regulated
protease Sep39. As we showed in Table [110]1, the 24 h sep39 mRNA level
of the overexpression strain SCUT-3-sep39 cultured in LB medium was 5.6
times of wild-type SCUT-3 and its corresponding keratinase activity was
about 4 times as wild-type bacteria, which indicates sep39 had been
successfully overexpressed in SCUT-3-sep39. Applied this reconstructed
SCUT-3-sep39 in 1% and 5% CFM’s feather degradation, we found that not
only the sep39 mRNA level and keratinase activity were up-regulated
compared to wild-type strain in both trials, but also the degradation
rate and peptide/amino acid content in the degraded feather medium
supernatant were improved in SCUT-3-sep39 group (Table [111]1). These
data confirmed Sep39 is an important keratinase for SCUT-3’s feather
degradation and its overexpression can improve SCUT-3’s feather
degradation efficiency. Other proteases’ overexpression strains are now
under construction in our lab and the pilot experiments showed their
overexpression could make different degree’s improvement of the
SCUT-3’s feather degradation efficiency. Another exciting phenomenon is
that co-overexpression of Sep39 and Cdo1 in SCUT-3 achieved a higher
degradation efficiency than single overexpression of Sep39 itself,
which also indicated the cooperation of disulfide bond reduction and
peptide bond hydrolysis during feather degradation. These data will be
published in our next article.
Hydrolyzed peptides and amino acids can be imported by bacteria as
nutrients via two categories of transporters: the proton motive
force-driven transporters (PTR transporters) and ATP binding
cassette-containing transporters (ABC transporters)^[112]14. In this
study, two PTR di- and tri-peptide transporters, cstA and dtpT, were
identified as up-regulated in feather medium-cultured SCUT-3
(Fig. [113]4d). Further, two peptide ABC transporter operons, oppABCDF
I and oppABCDF II, were up-regulated (Fig. [114]4d). Two amino acid ABC
transporter operons, metNIQ and livJHMGF, responsible for Met and
branched chain amino acid (Ile, Leu and Val) importation, were also
up-regulated (Fig. [115]4d). Up-regulation of these transporter genes
and operons indicates efficient peptide and amino acid importation by
SCUT-3 to maintain its growth on feather.
Iron uptake are regulated by the Fur regulon
Iron is essential element for organisms, participating oxygen
transport, ATP generation, cell growth and proliferation,
ribonucleotide reduction, and so on. Ferric uptake regulator (Fur) is
an important regulator for bacterial iron uptake. Holo-Fur with Fe^2+
binds its target DNA operators and functions as a repressor. Under iron
limited condition, apo-Fur dissociates from DNA and the repression is
relieved. In this study numerous genes encoding iron-utilizing proteins
in the Fur regulon were up-regulated in white CFM-cultured SCUT-3,
including the siderophore staphyloferrin B synthetase SbnA, siderophore
bacillibactin synthesis operon DhbACEBF, siderophore enterobactin
secretion exporter entS, catecholate siderophore detoxification gene
catE, elemental iron transporter operon EfeUOB, ABC transporter
yfiYZ/yusV ATPase^[116]15–[117]20 (Fig. [118]5a, c), indicating that
there was insufficient iron for SCUT-3 growth in white feather medium.
To determine whether iron uptake could improve SCUT-3 feather
degradation, we added 1 μM FeSO[4] to white chicken or duck feather,
and brown chicken feather-containing media. Interestingly, iron
addition improved the rate of white chicken (77.0%, p = 0.00003) and
white duck (50.8%, p = 0.00005), but not brown chicken (p = 0.5),
feather degradation by SCUT-3 (Fig. [119]5d), as the brown color of the
feather is caused by iron. This finding is important because white
feather broilers are the most cultivated globally.
Fig. 5. Iron uptake and oxygen consumption of feather medium-cultured SCUT-3.
[120]Fig. 5
[121]Open in a new tab
a, b Up-regulation of the siderophore synthesis (DhbACEBF), iron
transporter (EfeUOB), NADH dehydrogenase 1 (NuoA-N), and ATP synthase
(atpIBEFHAGDC) operons; numbers above genes are the log[2] FC values
for each gene (red > 1.0, pink < 1.0). c qRT-PCR verification of
siderophore synthesis and exporter (SbnA, SbnA, catE) and iron
transporter (yfiY, yfiZ, yusV) gene expression; n = 3/group. d FeSO[4]
addition (1 μM) improved degradation of white chicken and duck feathers
by SCUT-3, but not brown chicken feathers (NS means no significant,
P = 0.52); n = 3/group, *P < 0.05. e qRT-PCR verification of oxygen
sensor (AcrB/dosT), NADH dehydrogenase 2 (ndh2), and cytochrome c
oxidase (ctaD) gene expression; n = 3/group. f Aeration improved SCUT-3
feather degradation in 1% and 10% CFM; n = 3/group, *P < 0.05. P-values
between groups were obtained by unpaired two-tailed Student’s t test.
All data were presented as mean ± SD.
Aeration improves SCUT-3 feather degradation efficiency
Most Streptomyces are aerobic bacteria. A series of genes and operons
involved in oxygen sensing and the respiratory chain were up-regulated
in feather medium-cultured SCUT-3 (Fig. [122]5b, e). The oxygen sensor,
AcrB^[123]21, and the hypoxia/oxygen sensor, dosT^[124]22, were
up-regulated. In addition, the following electron transport
chain-associated genes were up-regulated: the 14-gene NADH
dehydrogenase 1 operon, NuoA-N (complex I); the NADH dehydrogenase 2
gene, ndh2; cytochrome c oxidase, ctaD; and the ATP synthase,
atpIBEFHAGDC (complex V)^[125]23,[126]24. Besides these genes, enhanced
iron uptake (as described above) may also provide ferric ion for the
Fe-S clusters of those proteins involved in the electron transport
chain. An aerating experiment showed that aeration could improve SCUT-3
feather degradation efficiency by 27.5% in 1% feather medium and 99.3%
in 10% feather medium (Fig. [127]5f), consistent with up-regulation of
electron transport chain genes, demonstrating that SCUT-3 feather
degradation is an oxygen-consuming process.
Active metabolism, spore formation/resuscitation, and division
In addition to disulfide bond reduction, protease hydrolyzation,
peptide and amino acid uptake, iron uptake, and oxygen consumption by
SCUT-3 during feather degradation, more interesting details regarding
how SCUT-3 uses feather material for its growth and cell division were
deduced from comparative analysis of transcriptome data. We determined
that the following metabolic processes were active in SCUT-3 grown in
feather medium. Uptake and synthesis genes for vitamins involved in
catabolism were up-regulated, including the thiamine (vitamin B1, for
dehydrogenase) transporter, ykoEDC; the thiamine synthesis genes,
thiC/thiG/thiS; the riboflavin (vitamin B2, for redox reaction)
synthesis operon, ribDEBAH; the pyridoxal phosphate (vitamin B6, for
transamination) synthesis genes, pdxH1/pdxH2; the nicotinamide riboside
(vitamin B3, for redox reaction) transporter, PnuC; the NAD
biosynthesis gene, NadR^[128]25; the pantothenate (vitamin B5,
precursor for CoA) synthesis genes, panB/panC/panD; and the CoA
synthesis gene, coaBC (Supplementary Fig. [129]8a). Genes involved in
amino acid catabolism pathways were also up-regulated, including the
arginine: pyruvate transaminase, aruH; the aromatic amino acid
metabolism genes, paaK/paaABCDE/paaI/paaJ; and genes involved in
branched chain amino acid metabolism, bkdABC (Supplementary
Fig. [130]8b).
In addition to its 85% keratin content, feather also comprises
approximately 5% lipids. Genes involved in lipid digestion and
catabolism were also up-regulated on feather medium, including
extracellular esterase (estB), cell-wall associated carboxylesterase
(caeB), the glycerol uptake operon (glpFKD), the long chain fatty
acid-CoA ligases (lcfB4/lcfB8/fadD15), fatty acid β-oxidation
(fadA/fadE10), Acetyl CoA synthetase (acsA3/acsA4/acsA5), and steroid
and cholesterol catabolism
(ksdD/hsaA1/hsaA2-pchF-ntaB/hsaA3-hsaC/hsaB)^[131]26 (Supplementary
Fig. [132]8c). Based on these data, alongside the active electron
transport and oxidative phosphorylation, we conclude that SCUT-3 can
efficiently catabolize both amino acids and lipids to provide energy
and metabolites for its growth in feather medium.
Numerous genes involved in DNA replication and cell division were also
up-regulated on feather medium, including the purine uptake permease,
pbuG; genes required for de novo purine synthesis,
purS-purQ-purL/guaB1/guaB4/guaD; the pyrimidine de novo synthesis
operon, pyrB -pyrC-carA-carB-pyrD-pyrF; DNA helicases, helD/dnaB; DNA
polymerases, dnaX/dnaQ; dimer chromosome segregation genes, xerC/xerD;
and the cell division regulator, yofA (Supplementary Fig. [133]8d).
Aerial mycelium, septum, and spore formation associated genes
(afsR1/afsR3/asfR8/afsK/ramA/whiB1/whiB7)^[134]27–[135]29 were also
up-regulated. Three spore resuscitation-promoting factor
(rpf1/rpf2/rpfA) implicated in the cleavage of dormant cell walls and
subsequent promotion of growth and metabolic reactivation^[136]30 were
up-regulated. Combined with observation of SCUT-3 morphology by SEM, it
is clear that this bacterium can grow efficiently on feather by prompt
mycelium formation, efficient DNA replication and spore formation, and
rapid spore diffusion and resuscitation. Rapid DNA replication can also
lead to intensive DNA errors. LexA is a transcriptional repressor that
inhibits SOS response genes. We found that lexA was down-regulated and
that the DNA damage repair associated gene, dinF, the recombination
repair genes, recA/recX/recD, the uvrABC system genes,
uvrA1/uvrA2/uvrA4, the mismatch repair gene, mutL, and the
non-homologous end-joining repair gene, ligA, were up-regulated to
ensure genetic fidelity during rapid cell division (Supplementary
Fig. [137]8d).
In addition to DNA replication genes, factors associated with
transcription and translation were also up-regulated on feather medium,
including the transcription termination gene, rho; various amino acid
tRNA genes, tRNA-Ala/Arg/Met/Glu/Gln/Leu/Thr/Pro; tRNA pseudouridine
synthase, truB; tRNA processing, rbn^[138]31; tRNA repair,
rtcB^[139]32; amino acid tRNA ligase, leuS/hisS; misacylated tRNA
proofreading, ybaK^[140]33; ribosomal proteins, rpsO (S15), rpmF (L32),
rpmB (L28), rpmH (L34), and rplM (L13); ribosome maturation,
rbfA/rsgA/rimM (30 S), rlmCD (23 S rRNA); ribosomal protein
acetylation, ydaF2/ydaF3/ydaF7/ydaF8; ribosomal assembly and
disassembly, hflX/rhlE3/rhlE4^[141]34,[142]35; translation initiation,
infB; stalled ribosome rescue, arfB^[143]36; and translation
termination and protein release, prmC^[144]37 (Supplementary
Fig. [145]8e). Up-regulation of these genes indicates the intensive
protein synthesis required for SCUT-3 growth on feather.
Discussion
The traditional physical/chemical treatment methods for feather waste
are gradually being abandoned because of the resulting pollution and
amino acid destruction. Keratinases are considered an alternative green
approach and have attracted intensive research^[146]38. Our group also
spent an inordinate amount of time developing a high-efficiency
recombinant keratinase, which was ultimately not a fruitful endeavor.
We produced high levels of the keratinase, KerK (approximately 1000 U
mL^−1) as did another group^5; however, this approach was ultimately
disappointing, since without a sulfitolysis agent, keratinase feather
hydrolyzation has very low efficiency and the cost of recombinant
keratinase preparation is considerable. Microbial fermentation is the
most economical method and there have been many attempts to isolate
natural FDB; however, most FDB is insufficiently efficient for
application in industry-scale feather
hydrolysis^[147]1,[148]39,[149]40. Keratinase overexpressing bacteria
have been generated that can hydrolyze feather using their own reducing
power, with limited success. The KerK overexpressing B. subtilis
constructed by our group hydrolyzes feather much more efficiently than
wild-type B. subtilis; however, its efficiency remains unsatisfactory.
Microbial feather utilization is a systematic process, involving
disulfide bond reduction and keratinase hydrolyzation, bacterial
colonization, import of hydrolyzed peptides and amino acids, and
metabolism and growth on feather material. Many of these processes have
been overlooked with the excessive focus on keratinases.
Based on our findings, we present a schematic illustrating the possible
feather utilization mechanisms of the new isolate, Streptomyces sp.
SCUT-3 (Fig. [150]6), following the logic outlined below. SCUT-3 does
not degrade feather in nutritionally rich LB medium; therefore, we
speculate that SCUT-3 initiates activation of its feather degradation
machinery in the comparatively limited nutrition in soil when it
encounters a molted bird feather. Sulfite and free cysteinyl groups are
secreted to reduce the disulfide bonds in the feather keratin and
proteases released to access the peptide bonds of keratin, hydrolyzing
them to generate peptides and amino acids, which are imported into the
cell via the up-regulated peptide and amino acid transporters, where
peptides can be further hydrolyzed by intracellular proteases. Feather
lipids are also hydrolyzed by secreted or cell-wall bound esterase,
while glycerol, fatty acids, and other lipids (such as cholesterol) are
imported by their associated transporters. Some amino acids and lipids
are catabolized to generate different metabolites for anabolism and
NADH for electron transportation and oxidative phosphorylation, to
yield energy for further cell division and growth. Purine and
pyrimidine are synthesized for DNA replication and RNA (rRNA, tRNA, and
mRNA) synthesis. Spores are formed and dispersed to new feather
barbules and resuscitated to undergo further cycles of feather
degradation. Many details in our schematic require verification. The
inclusion of genes that may be involved in these processes was prompted
by their up-regulation; hence they are candidate targets for genetic
manipulation of this isolate, to further enhance its feather
degradation efficiency. The model we present provides a reference for
mechanisms of feather degradation by other soil-borne FDB, and will
assist understanding of the cycle of the feather in the biosphere.
Fig. 6. Schematic diagram of SCUT-3 feather utilization mechanisms.
[151]Fig. 6
[152]Open in a new tab
Reactions framed in red are processes potentially involved in feather
disulfide bond breakdown, peptide bond hydrolysis, and amino acid
importation. The hydrolyzed feather products could be used as a
nutrition source for plants, animals, and microorganisms (blue arrows).
Reactions framed in blue include catabolism of amino acids and lipids
from feather to produced metabolites and NADH. Reactions framed in
green include oxidation of NADH and ATP synthesis, while those in the
purple frame are involved in gene transcription regulation.
Most importantly, this study presents a promising bacterium for green
industrial processing of both chicken and duck feather waste. Using 10%
submerged and 40% solid-stage fermentation, we achieved high
degradation rates of 50.3 ± 1.4% and 57.3 ± 2.3% in 4 days and 6 days,
respectively (Fig. [153]1a), which is unprecedented in previous
reports. Compared with the high price of fish meal (1,200 USD ton^−1)
and soybean meal (500 USD ton^−1) in China, the price of feathers is
<100 USD/ton. The estimated cost of feather meal produced by 40% solid
fermentation is <200 USD ton^−1. Further, the meal generated contains a
high soluble amino acid (22.4%) and peptide (10.3%) content, and could
efficiently replace fish meal in fish feed, as we demonstrated using
cultured tilapia. No bio-degraded feather meal has previously been
reported to be used in animal feed. Relative to the high price (about
700 USD ton^−1) and low feed addition effects of feather meal produced
using traditional physical-chemical treatment methods, the feather meal
produced by our approach is much more profitable. The use of
microbe-inoculated feather compost as plant fertilizer was recently
reported for growing cherry tomato, resulting in higher fruit yield and
better taste, while the duration of composting was very long (90
days)^[154]41. Here we produced 10% submerged feather fermentation
broth in 4 days, which could double the fresh weight of rice plants,
indicating great potential as plant amino acid liquid fertilizer.
Moreover, 10% submerged feather fermentation broth was also
demonstrated to improve the recombinant protein production of
microorganisms.
Two key factors, iron addition and aeration, improved the efficiency of
SCUT-3 feather degradation. Additional optimization of fermentation,
such as agitation and improved methods for supplying air to the
fermenter, can likely increase degradation efficiency further. Ongoing
experiments in our laboratory include modification of potential SCUT-3
target genes identified in this study, including those involved in
sulfite exportation and proteases, among others. These genetic
manipulations may also improve the feather degradation efficiency of
SCUT-3.
Methods
Strains, media, materials, and reagents
Streptomyces sp. SCUT-3, isolated from soil containing disposed
feathers by our laboratory was used in the present research.
Streptomyces fradiae ATCC 10745, Streptomyces thermolineatus ATCC
51534, and Streptomyces cattleya ATCC 35852 were obtained from China
General Microbiological Culture Collection Center (CGMCC) and Guangdong
Microbial Culture Collection Center (GDMCC), and maintained under their
respective designated culture conditions. SCUT-3 was cultured in Gauze
No. 1 Medium (containing, per liter distilled water: 20.0 g soluble
starch, 0.5 g sodium chloride, 0.01 g ferrous sulfate, 1.0 g potassium
nitrate, 0.5 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate,
15.0 g agar; pH 7.2). Bacillus subtilis WB600-kerK, a strain expressing
recombinant keratinase constructed and stored in our laboratory.
Bacillus subtilis L25, a strain expressing recombinant esterase
constructed and stored in our laboratory. Overexpression plasmid
pSET152 and conjugation strain E. coli ET12567/PUZ8002 were deposited
in our laboratory.
Chicken feathers (white and brown) and duck feathers (white) were
obtained from the local market, rinsed in double distilled water until
completely clean, dried and stored for further study^[155]42. FDB
enrichment media contained (g L^−1) NH[4]Cl 0.5 g, NaCl 0.5 g,
K[2]HPO[4] 0.3 g, KH[2]PO[4] 0.4 g, MgCl[2] 0.1 g, yeast extract 1.0 g,
chicken feather 10.0 g; pH 7.5. Feather powder plates contained (g
L^−1) K[2]HPO[4] 1.5 g, MgSO[4]•7H[2]O 0.025 g, CaCl[2] 0.025 g,
FeSO[4]0.015 g, chicken feather powder 10.0 g, agar powder 20.0 g; pH
7.5. Basal medium (BM) contained (g L^−1) NaCl 0.5 g, KH[2]PO[4] 0.4 g,
K[2]HPO[4] 0.3 g; pH 7.2–7.5. CFM was obtained by adding different
amounts of chicken feathers to BM (g/100 mL).
Rice (Oryza sativa) seeds, succulent (Sedum Alice Evans) and cultivated
soil were purchased from the local market and farm. Nile Tilapia
(Oreochromis niloticus) was purchased from Guangdong Tilapia Fine
Germchit Field (Guangdong, China). Tilapia nutrition experiment was
performed in the Aquaculture Laboratory of College of Marine Science,
South China Agricultural University, and was approved by the
Experimental Animal Ethics Committee of South China Agricultural
University.
Keratin was obtained from J&K Chemical Co., Ltd. (Shanghai, China).
Folin phenol reagent, dithiothreitol (DTT), oxidized glutathione
(GSSG), 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB), ninhydrin, and
trichloroacetic acid (TCA) were from Sigma (Shanghai, China). Unless
specified, all other substrates, chemicals, and primers were purchased
from Sangon Biotech (Shanghai, China).
Screening of efficient feather-degrading strains
Feather-degrading bacteria were isolated from a feather waste dumping
site in Shaoguan (Guangdong, China). Soil samples were serially
diluted, inoculated in enriched medium, and incubated in a rotary
shaker at 37 °C for 3 days. Bacterial suspensions were further plated
onto feather powder plates and cultured at 37 °C for 5 days. Strains
with strong growth were streaked onto feather meal plates, cultured at
37 °C, and single colonies picked. Isolates were then transferred to 1%
CFM to test their feather degradation ability.
Analysis of feather degradation rate
Feather degradation rates were evaluated using the weight loss
method^[156]43. After feathers were degraded by FDB, the fermented
medium was filtered through Whatman No. 1 filter paper. Feather residue
was thoroughly washed with double distilled water, dried at 65 °C, and
then weighed to calculate weight loss. Results are expressed as
percentage weight loss relative to the initial dry feather weight. All
experiments were performed in triplicate.
Determination of keratinase activity
Keratinase activity was tested using 1% soluble keratin as substrate,
according to a previously reported method, with some
modification^[157]44. Soluble keratin (100 μL of 1% (w v^−1)) was added
to 100 μL of diluted crude keratinase solution and incubated at 50 °C
for 20 min. Hydrolyzation was stopped by the addition of 200 μL TCA and
tubes centrifuged at 12,000 × g for 5 min. Aliquots of supernatant
(100 μL) were pipetted into separate tubes containing 500 μL of 0.4 M
Na[2]CO[3] and 100 μL of Folin phenol reagent. The mixture was then
incubated at 40 °C for 20 min and OD values measured at 660 nm. A
tyrosine standard curve was constructed for quantification. One unit of
keratinase activity was defined as the amount of enzyme needed to
release 1 μg tyrosine from keratin per min.
Determination of amino acid, protein, and sulfhydryl content
Amino acid concentration was tested using ninhydrin reagent^[158]45.
FDB culture broth was precipitated using 20% TCA and 200 μL of
supernatant mixed with 50 μL phosphate buffer (pH 8.04) and 50 μL 2% (w
v^−1) ninhydrin reagent. The mixture was heated in water bath at 90 °C
for 30 min, followed by addition of 950 μL distilled water. Absorbance
was read at 570 nm to quantify the amino acids present in the
hydrolysate according to a prepared isoleucine standard curve. Amino
acid composition and content were further evaluated and quantified
using an Amino acid analyzer A-300 advanced (MembraPure, Germany). A
bicinchoninic acid (BCA) assay kit from TaKaRa (Shanghai, China) was
used to determine the soluble protein concentrations in fermentation
solutions, using bovine serum albumin as the standard. Each sample was
assessed in triplicate.
The release of sulfhydryl groups into the FDB culture medium was
determined spectrophotometrically, according to the method of
Ellman^[159]46. DTNB (10 μL) and 0.1 M phosphate buffer (500 μL; 1 mM
EDTA, pH 8.0) was added to 50 μL of extracellular broth mixture.
Absorbance was measured at 420 nm and the concentration of sulfhydryl
groups calculated.
Detection of sulfite in feather culture medium
Sulfite was detected by observation of white precipitate formation on
BaCl[2] addition and bubbles on HCl addition. The presence of sulfite
was further confirmed by KMnO[4] decolorization^[160]47.
Applications in plant, animal, and microorganism culture
Tilapia was used for animal culture experiments as follows. Healthy
tilapia (weight: 6.9 ± 0.2 g) was randomly divided into three groups
(three parallels in each group and 20 fish per parallel), fed with 45%
feather meal (40% SCUT-3 cultured feather) feed, 45% full fish meal
feed, and 22.5% fish meal plus 22.5% feather meal feed. Besides the 45%
protein source, 53% flour, 1% CaHPO[4] and 1% vitamin and mineral
premix were added into the feed mixtures. After eight weeks of culture,
fish were weighed and the feed coefficients calculated for each group
using the following formula:
[MATH: Feedcoefficient=Feedconsumption/weightgain×100%. :MATH]
Rice and a succulent plant were used in the plant growth experiments
with addition of 10% SCUT-3 cultured feather broth. Rice plants (nine
per pot) and succulents (one per pot) were planted with the addition of
50 mg amino acids in 10% cultured feather broth in 50 g soil. Plants
were watered every 3 days to keep the soil moist. The fresh weight of
rice (g) was measured after 3 weeks.
Bacillus subtilis WB600-kerK was used in the microbial growth
experiment with addition of 10% SCUT-3 cultured feather broth. Bacillus
subtilis WB600-kerK was inoculated in 20 mL LB medium with addition of
3 mg amino acids in 10% cultured feather broth. After 24 h culture, the
OD[600] value and the KerK enzyme activity were tested and record.
SCUT-3 species identification
The physical and chemical characteristics of SCUT-3 were evaluated
according to the instructions in Bergey’s Manual of Systemic
Bacteriology. The ultra-scope images of 40% SCUT-3 cultured feather on
different days were acquired using an ultra-high-resolution field
emission scanning electron microscope (SEM) (Zeiss, German).
The SCUT-3 genome was sequenced by GENE DENOVO (Guangzhou, China).
SCUT-3 16 S rRNA sequences were retrieved from the genome annotation
results for constructions of the phylogenetic tree by distance matrix
analysis using the neighbor-joining method with MEGA 7.0
software^[161]48. OrthoANIu was used to analyze the ANI of the SCUT-3
genome relative to the genomes of 1,222 other Streptomyces
strains^[162]49.
Transcriptomes of SCUT-3 cultured in LB and feather medium
SCUT-3 was cultured for 24 h in LB and 1% CFM in exponential growth
phase. Total RNA was extracted using the RNAprep Pure Cell/Bacteria
Kit, according to manufacturer’s specifications (TIANGEN Biotech Co.
LTD) and sequenced by LongseeMed (Guangzhou, China). Assembled
transcriptome data were screened for differentially expressed genes
using edgeR33 with a threshold of FDR < 0.05 and |log[2] FC | > 1,
resulting in identification of 1,459 genes with significantly different
expression levels. Gene Ontology (GO) and Kyoto Encyclopedia of Genes
and Genomes (KEGG) pathway enrichment analysis of significant
differentially expressed genes (DEGs) was conducted using
clusterProfiler^[163]50. Selected DEGs (n = 23) were subjected to
further verification by real-time qRT-PCR using the primers detailed in
Supplementary Table [164]4, with 16 S rRNA levels used as an endogenous
control to normalize gene expression levels. The 2^−ΔΔCT method was
used to estimate relative target gene expression levels, which are
expressed as relative fold-change values. All samples were analyzed in
triplicate. To determine the position of DEGs in the genome,
transcription data were mapped to the genome sequence data. FGENESB was
used for genomic operon prediction. DEGs and their associated operons
were visualized in the genome using the IGV genome browser^[165]51.
Based on the results of transcriptome analysis, nine protease genes
(log[2] FC > 2) and one disulfide reduction related gene were detected
at 0, 3, 6, 12, and 24 h by qRT-PCR.
Overexpression of protease Sep39 in SCUT-3
To further validate the key genes in feather degradation, an
overexpression system was constructed in SCUT-3. The plasmid
constructed in this study was introduced into Streptomyces sp. SCUT-3
according to the Escherichia coli–Streptomyces conjugation method
reported previously^[166]52. The protease gene sep39, which has the
highest up-regulation fold in transcriptome data, was used as the
target gene. The overexpression plasmid pSET-sep39 was obtained by
inserting this gene into the NdeI/EcorI sites of pSET152. Integrated
plasmid pSET-sep39 was transformed into the donor strain E. coli
ET12567/PUZ8002, and then further transferred into SCUT-3 by
conjugation. In order to detect whether the target gene was
successfully overexpressed in SCUT-3, the relative expression of sep39
was determined in the wild strain SCUT-3 and the overexpression strain
SCUT-3-sep39 by qRT-PCR. Keratinase activity, degradation rate, and
soluble protein and amino acid content were also determined in SCUT-3
and SCUT-3-sep39 by the method mentioned above.
Analysis of SCUT-3 reducing power
To test the reducing power of SCUT-3, SCUT-3 was inoculated in 50 mL 1%
CFM for 24 h and centrifuged to collect the cell-free supernatant
(extracellular component) and cell pellets (living cells). Cells were
also disrupted by sonication and centrifuged to collect intracellular
components in the supernatant. Extracellular supernatant (10 mL),
intracellular component, and living cells (10^9 CFU) in 10 mL PBS were
added into 50 mL tubes containing 2 mM GSSG as a disulfide bond
substrate and incubated at 37 °C. Samples were collected at different
time points and released sulfhydryl groups tested using the method
described above.
Statistics and reproducibility
Mean from three independent biological experiments was presented in
each plot, in which error bars represented standard deviation. A number
n suggested biological replications indicated in the figure legends.
Statistical significance was assessed in GraphPad Prism 8.0. Unpaired
two-tailed Student’s t test was used for comparison of two experimental
groups. A P-value of 0.05 was deemed statistically significant and
statistical details are found in the figures and figure legends.
Reporting summary
Further information on research design is available in the [167]Nature
Research Reporting Summary linked to this article.
Supplementary information
[168]Supplementary Information^ (990.5KB, pdf)
[169]42003_2020_918_MOESM2_ESM.pdf^ (5.7KB, pdf)
Description of Additional Supplementary Files
[170]Reporting Summary^ (68.8KB, pdf)
[171]Peer Review File^ (251.5KB, pdf)
[172]Supplementary Data 1^ (12.4KB, xlsx)
[173]Supplementary Data 2^ (11.7KB, xlsx)
[174]Supplementary Data 3^ (13KB, xlsx)
[175]Supplementary Data 4^ (8.9KB, xlsx)
[176]Supplementary Data 5^ (13.6KB, xlsx)
[177]Supplementary Data 6^ (12.8KB, xlsx)
Acknowledgements