(2019) 18:158
Quesada‑Ganuza et al. Microb Cell Fact
https://doi.org/10.1186/s12934‑019‑1203‑0
Microbial Cell Factories
Open Access
RESEARCH
Identification and optimization of PrsA
in Bacillus subtilis for improved yield of amylase
Ane Quesada-Ganuza1, Minia Antelo-Varela2, Jeppe C. Mouritzen1, Jürgen Bartel2, Dörte Becher2,
Morten Gjermansen1, Peter F. Hallin1, Karen F. Appel1, Mogens Kilstrup3, Michael D. Rasmussen1
and Allan K. Nielsen1*
Abstract
Background: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native
PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion.
Results: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA coexpression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific
to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress
response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion
stress.
Conclusions: In this work we show how heterologous PrsA overexpression can give a better result on heterologous
amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alphaamylases. We also demonstrate that on top of increasing amylase yield, a good PrsA–amylase pairing can lower the
secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance
in both supporting amylase secretion and lowering secretion stress.
Keywords: Bacillus subtilis, Recombinant protein production, Secretion stress, PrsA
Introduction
Enzymes are used as catalysts to manufacture a variety
of commercial products—like sugar, beer, bread, and
ethanol. They are also used directly in products such
as household care detergents, where they help remove
stains and enable low-temperature and more sustainable laundry [1]. Many of these industrial relevant
enzymes are produced in Gram-positive bacteria such
as Bacillus licheniformis and Bacillus subtilis which
are well known for high-level protein secretion and are
generally regarded as safe [2, 3]. To achieve commercially relevant yields of enzymes, it is crucial to identify
potential bottlenecks in the protein production route
*Correspondence: aknn@novozymes.com
1
Research and Technology, Novozymes A/S, Krogshoejvej 36,
2880 Basgvaerd, Denmark
Full list of author information is available at the end of the article
from transcription, translation to folding and secretion. Even though several heterologous proteins can be
produced in these Bacillus species in very high yields,
some enzymes cannot be secreted into the extracellular medium in titres high enough for their production
to be economically cost-efficient. The bottlenecks that
the production of heterologous proteins encounter can
be found at different stages. In the cytoplasm, newly
synthesized proteins can form insoluble aggregates and
thus be degraded. On the membrane, they can be either
poorly targeted or rejected by the preprotein translocation system. If the proteins are not folded quickly and
correctly after translocation, they can be degraded by
the several proteases that exist around the membrane,
cell wall or extracellular medium. Two of the quality control proteases that degrade misfolded proteins
around the cell-wall interface are HtrA and HtrB [4].
The expression of these is controlled by the CssRS two
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Quesada‑Ganuza et al. Microb Cell Fact
(2019) 18:158
component system [5]. CssS is a sensor kinase that
responds to overexpression of secretory proteins and
heat stress by phosphorylating both itself and CssR, a
cytoplasmic response regulator. The phosphorylated
CssR activates the bicistronic CssRS operon and the
htrA and htrB genes [6]. This response is referred to as
secretion stress, which has been shown to be triggered
by the secretion of heterologous alpha amylases, such
as AmyQ from Bacillus amyloliquefaciens, or B. subtilis’ Lipase A [7].
Most exported bacterial proteins are translocated
through the highly conserved SecA-YEG pathway
[8]. Proteins are exported through this pathway in an
unfolded state and to avoid proteolysis they must fold
into their native conformation shortly after the signal peptide is cleaved, and leave the membrane. At this
post-translocational stage, various thiol-disulphide oxidoreductases, negatively charged cell wall polymers and
folding catalysts play a prominent role [9].
The major extra cytoplasmic folding factor in Bacillus subtilis is the PrsA protein [10]. PrsA belongs to
the parvulin family of prolyl cis/trans isomerases (PPIases), which are ubiquitous in all types of cells and cell
compartments, and catalyze rate-limiting protein folding steps at peptidyl bonds preceding proline residues
[11]. PrsA and PrsA like proteins are widely conserved
among gram-positive bacteria, and while it is essential
for viability in many non-pathogenic Bacillus species, it
is also involved in antibiotic resistance in gram-positives
[12] such as Staphylococcus aureus [13], and it is essential for virulence and survival within the host cell in the
pathogenic Listeria monocytogenes [14]. In addition, it
has been shown that heterologous PrsAs are able to functionally complement complex activities such as flagellum-mediated swimming motility and pH resistance [15].
PrsA forms dimers in vivo and has two domains: PPIase
domain and NC or chaperone domain [11]. The PPIase
domain, which has a structure homologous to human
Pin1, is the only one responsible for the PPIase activity
of PrsA and it contains the hydrophobic proline-binding
pocket [16]. The NC or chaperone domain is formed by
the N-terminal (Ser4-Gly114) and C-terminal (Arg208Ser260) regions and exhibits a motile clamp-like segment
conserved among chaperones.
The PPiase domain is located near the NC domain,
and both domains are joined by a short linker of 5 amino
acids. This linker confers the PPIase domain rotational
freedom from the chaperone domain. The dimeric form
of PrsA is achieved solely by the interactions of the NC
domains, and when dimeric, these form a highly hydrophobic bowl-like crevice surrounded by charged amino
acids. This crevice, together with the two clamp regions,
could sequester unfolded or unstable polypeptides of
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up to 20KDa, or even more considering the flexibility of
both the NC and the PPI domains [11].
In B. subtilis, PrsA is a lipoprotein attached to the outer
part of the membrane, and it is essential for cell viability
due to its function on assisting the folding of the Penicillin Binding Proteins responsible for the synthesis of the
cell wall [10]. PrsA may also take on other roles as there is
now good evidence that it acts as a very efficient folding
catalyst for exported amylases and various other enzymes
[17–19].
In microbial cell factories, the yield of different amylases varies from one amylase to another even when
industrial and biological settings are kept constant. PrsA
foldases, when co-expressed with heterologous amylases,
often enhances yield by supporting post-translocational
folding and secretion of the product [19]. Expression of
PrsA over wildtype level has in several studies proven
beneficial for secretion of amylases such as AmyS (from
Geobacillus stearothermophilus) [20], AmyL (from
Bacillus licheniformis) [17] and AmyQ (from Bacillus
amyloliquefaciens) [19]. Even though the native PrsA can
support the production of various heterologous enzymes
in B. subtilis, it may not be the optimal choice of PrsA
for enhancing secretion of heterologous amylases from
this bacterium. There are several examples that overexpression of the native PrsA may increase, decrease or
not affect the secretion of the heterologous protein being
produced, depending on the target exoprotein [20]. The
B. subtilis amylase (amyE) evolved in the same host as its
cognate PrsA and may, therefore, have a better fit to this
PrsA than heterologous amylases.
Nature offers a wide range of both amylases and PrsA
foldases and choosing the right match may increase the
frequency of productive interactions between the enzyme
and its foldase. For heterologous expression of amylases,
it is most obvious to co-express the cognate prsA gene.
The choice is more open in cases with engineered amylases, but a prsA gene from an organism close by in the
evolutionary tree would be a good starting point.
In this study we set out to co-express six heterologous PrsAs with their cognate amylases, using B. subtilis as host. The heterologous amylase–PrsA pairs were
equipped with their native signal peptides but were
expressed from identical, non-native promoters at chromosomal locations. Furthermore, we constructed a
matrix of strains containing all possible combinations of
the six amylase-PrsA pairs and studied the importance
of choice of PrsA for production of specific amylases.
We show how the different heterologous PrsAs show
diversity of specificity against the different amylases. In
addition, we found that the cognate PrsA results in the
highest increase in production in most cases. Moreover, we demonstrate how a good PrsA–amylase match is
Quesada‑Ganuza et al. Microb Cell Fact
(2019) 18:158
not only capable of increasing the amylase yield, but also
relieves secretion stress. Lastly, we present a new recombinant PrsA that shows better performance on both
improving AmyL secretion and on relieving secretion
stress.
Results
The evolutionary relationship of amylase and PrsA variants
The evolutionary relationships of the PrsA and alphaamylase homologs used in this study are shown in Fig. 1.
The sequence divergence within our set of alpha-amylase
homologs is in general higher than what is found within
the PrsA homologs which appear to be more conserved.
Among bacterial proteins, essential genes appear to be
more conserved than non-essential genes [21]. The B.
subtilis AmyE protein is however atypical since it is quite
distantly related to the AmyE homologs from the other
closely related Bacillus species used in the study. This is
also evident from the classification of GH13 amylases
in the Carbohydrate Active Enzyme Database (CAZY)
which has subdivided the GH13 family into subfamilies
and mapped the B. subtilis AmyE to family GH13_28, and
the rest of the AmyE homologs to the GH13_5 amylase
family [22, 23]. The two amylase families are primarily distinguished by the presence of a large C-terminal
carbohydrate binding domain in the GH13_28 members, which is completely absent in the members of the
GH13_5 family. Of phylogenetic significance is also the
presence of a large number (15 in the aligned area, shown
in Additional file 1: Figure S1) of deletions spanning from
1 to 60 amino acids, in the amino acid sequence of the
common domains of the GH13_5 family compared to the
GH13_28.
Indels, defined as insertions or deletions are strong
phylogenetic markers [25] and show that the division of
the two amylase families did not happen late in the amylase evolution. Accordingly, a deep branching is found
between AmyE and the GH13_5 family members (Fig. 1,
right panel). Aside from B. subtilis, some isolates of B.
amyloliquefaciens (not AmyQ), and B. atrophaeus contain amylase genes belonging to the GH13_28 family. The
closest homologues to this atypical Bacillus amylase gene
cluster are in Streptococcus isolates, while the genes in
Clostridium isolates are somewhat more distant relatives.
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This atypical relationship is not observed for the PrsA
proteins in the same organisms listed in Table 1. Here all
PrsA proteins share a high degree of similarity, with only
a few indels present (Additional file 1: Figure S2). Here
Geobacillus stearothermophilus harbors the PrsA protein
that diverts the most, as expected from the general phylogenetic relationship of the six bacterial species shown
in Fig. 1 and Table 1.
This analysis shows that the evolutionary relationship
between secreted amylases and their native extracellular PrsA foldases is different for the AmyE/PrsA pair in
B. subtilis than for the AmyL/PrsABl, AmyQ/PrsABa, and
AmyS/PrsAGs pairs in B. licheniformis, B. amyloliquefaciens], and G. stearothermophilus, respectively. It also
raises doubts whether the B. subtilis AmyE would be
more evolutionary adapted to the B. subtilis PrsA than
AmyL, AmyQ, or AmyS. Therefore, the effect of different
PrsA homologues on heterologous amylase production
was investigated.
Chromosomal organization and expression of amyE/prsA
homologous in B. subtilis
Expression cassettes were constructed and integrated
into the chromosome of B. subtilis AN2 as described in
Experimental Procedures. PrsA expression cassettes were
integrated into the pel locus and consisted of the synthetic promoter PconsSD followed by a prsA gene and
the B. subtilis prsA native terminator. Expression cassettes for amylases were integrated into the amyE locus
and consisted of the synthetic promoter PconsSD followed by an amy gene and the B. amyloliquefaciens amyQ
terminator. All the elements remained constant between
strains except for the open reading frame of the gene of
interest in the expression cassettes (Fig. 2).
Initial experiments using strains which express gfp
from the expression cassettes in either amy- or pel- loci
verified the activity of PconsSD throughout the culturing
period (Fig. 3). All strains used in this study also harbor
the native prsA locus at its original location. Inactivation of the native prsA gene was only possible if preceded
by the expression of either B. subtilis, prsABl or prsABa.
However, we observed no significant differences in amylase activities when these three strains were compared
to their respective isogenic strains harboring the native
Fig. 1 The interrelationship between members of the PrsA family proteins used in the study (left) and between the members of the AmyE family
used (right). The phylogenetic tree was based on the mature protein sequence excluding signal peptides and calculated with the Phylogeny.fr tools
(http://www.phylogeny.fr/) [24]. Branch lengths are proportional to the divergence of the amino acid sequences within each tree
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Fig. 2 Expression cassettes for amylase and prsA expression. a The expression cassette for amylase expression containing the amyE locus 5′ and 3′
regions for homologous recombination, the PconsSD promoter followed by the amylase gene and the amyQ terminator. b Expression cassete for
prsA expression containing the pel locus 5′ and 3′ regions for homologous recombination, the PconsSD promoter followed by the prsA gene and
the native terminator of B. subtilis prsA
Table 1 PrsA (A) and amylase (B) identity matrices
B. subtilis
B. licheniformis
B. amyloliquefaciens
G. stearothermophilus
B. sonorensis
L12
B. sp. NSP9.1
(A) PrsA
B. subtilis
B. licheniformis
100
66
85
55
67
70
66
100
66
50
85
86
B. amyloliquefaciens
85
66
100
54
66
69
G. stearothermophilus
55
50
54
100
51
55
B. sonorensis L12
67
85
66
51
100
84
B. sp. NSP9.1
70
86
69
55
84
100
(B) Amylase
B. subtilis
B. licheniformis
100
35
25
31
35
30
35
100
65
81
84
84
B. amyloliquefaciens
25
65
100
66
67
67
G. stearothermophilus
31
81
66
100
83
81
B. sonorensis L12
35
84
67
83
100
85
B. sp. NSP9.1
30
84
67
81
85
100
Percent sequence identity between (A) PrsA homologs and (B) amylase homologs of different species calculated based on the pairwise Smith and Waterman
alignment of the mature protein sequence
prsA locus. This suggests that the activity arising from the
native locus in these strains is overshadowed by the abundance of their homologous expressed from the strong
PconsSD promoter in pel locus. A matrix of strains coexpressing all the combinations of the previously listed
prsA’s and amylases, one to one, was constructed.
Relative quantification of membrane PrsA protein levels
by quantitative proteomics analysis
The strains expressing amyL from B. licheniformis from
the amy locus and co-expressing each one of the previously listed prsA’s from the pel locus were selected for
the measurement of the PrsA levels in the membrane
fraction.
A quantitative proteomics analysis of the membrane
fraction of all the AmyL expressing strains and the
parental B. subtilis 168 sigF strain was performed by
label-free quantification by LC-MS/MS. The Hi3 relative quantification method determines protein abundancies by adding up the signal intensity of the three
most abundant peptides of each protein. The relative
amount of PrsA is calculated by computing the Hi3
data of all strains and calculating the ratio of the PrsA
intensities divided by the sum of the whole detected
proteome intensities. This way the amount of the heterologous PrsAs relative to the total amount (or intensity) of all the proteins detected, can be compared
with each other. In Fig. 4 the relative amounts of the
heterologous PrsAs are shown, whereas the relative
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Fig. 3 Gfp expression through growth. A gfp gene was inserted in the amy or pel locus in the same context as the amylase or prsA genes
respectively to assess its expression profile. The GFP signal and the cell density were measured on-line in a Biolector plate reader
amounts of the native B. subtilis PrsA in each strain
are shown in Fig. 5.
As can be seen in Fig. 4, all heterologous PrsAs are
detected in the membrane. Nevertheless, the relative
amount of each of them is not the same in all strains.
This could be due to variations in either mRNA degradation, degradation in the cytoplasm, incorrect
destination sorting or degradation after translocation
through the Sec pathway. Regarding the native B. subtilis PrsA (Fig. 5), it is an interesting observation that
the strain that overexpress AmyL but no heterologous
PrsA has a twofold increase of native PrsA in comparison to the wildtype B. subtilis 168 strain. The amount
of the native PrsA is not increased in the strain overexpressing the B. subtilis PrsA, and it is surprisingly
reduced in the strain co-expressing the cognate PrsABl
with AmyL. The same twofold increase in the amount
of native PrsA can be seen in the strain co-expressing
PrsABN with AmyL.
Heterologous co‑expression of prsA and amylase cognates
and effect on amylase activity
Table 2 shows amylase activities measured in supernatants from each series of strains co-expressing a specific amylase and the various heterologous prsA genes
included in this study. Values in each series are set relative to the strain that expresses the amylase from the
amyE locus but which do not co-express any prsA from
the pel locus. The table reveals that the highest amylase
activities in most cases were obtained when the heterologous amylase was co-expressed with its cognate prsA
gene in B. subtilis. Some non-cognate combinations of
amylase and prsA genes were also observed to increase
amylase activity other than the cognate ones, but none
of these were superior to a cognate pair (Table 2). The
only exception to this general observation was when the
G. stearothermophilus amylase (AmyS) and PrsA were
co-expressed in B. subtilis. In this case the extracellular amylase activity was lower than when the amylase
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Fig. 4 Relative abundance of heterologous PrsA in the cell membrane fraction. The membrane proteome of B. subtilis strains was analysed by
LC-MS/MS and label-free Hi3 quantification. The relative amount of heterologous PrsA is given as the PrsA intensities divided by the sum of the total
proteome intensities. All strains except B. subtilis 168 are expressing amyL. Levels of heterologous PrsA are shown except for B. subtilis 168 and No
added PrsA strains, in which only the native PrsA is present. For the strain co-expressing a second copy of B. subtilis PrsA from the pel locus, the total
amount of B. subtilis PrsA is shown
Fig. 5 Relative abundance of native PrsA in the membrane fraction. The membrane proteome of B. subtilis strains was analysed by LC-MS/MS and
label-free Hi3 quantification. The relative amount of native PrsA is given as the PrsA intensities divided by the sum of the total proteome intensities.
All strains except B. subtilis 168 are expressing amyL
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Table 2 Relative extracellular amylase activity in growth medium of Bacillus subtilis 168 sigF co-expressing
heterologous amylases and heterologous prsA genes
Origin of heterologous prsA gene inserted into the pel locus
Origin
of heterologous
amyE
homologue
inserted
into the amyE
locus
None (only
wild type prsA
gene)
+ B. subtilis
prsA
+ B.
licheniformis
prsABl
+ B.
amyloliquefaciens
prsABa
+ G.
stearothermophilus
prsAGs
+ B.
+ B. NSP9.1
sonorensis L12 prsABN
prsABson
Bacillus subtilis
(amyE)
1 (0.09)
1.25 ( 0.11)
0.91 (0.06)
1.18 (0.13)
1.17 (0.18)
0.84 (0.07)
0.56 (0.16)
Bacillus licheniformis (amyL)
1 (0.08)
1.20 (0.15)
1.40 (0.08)
1.48 (0.06)
1.44 (0.08)
1.46 (0.10)
1.06 (0.17)
Bacillus
amyloliquefaciens (amyQ)
1 (0.08)
1.18 (0.13)*
1.27 (0.18)
1.19 (0.05)*
0.29 (0.01)
0.94 (0.007)
0.89 (0.02)
Geobacillus stearo- 1 (0.13)
thermophilus
(amyS)
1.40 (0.22)
2.19 (0.15)
2.41 (0.21)
1.01 (0.05)
0.93 (0.03)
1.74 (0.03)
Bacillus sonorensis
L12
1 (0.1)
0.95 (0.28)
2.35 (0.41)
2.07 (0.25)
1.72 (0.18)
2.54 (0.19)
1.94 (0.44)
Bacillus NSP9.1
1 (0.17)
1.12 (0.26)
1.77 (0.15)
1.64 (0.16)
1.38 (0.1)
1.26 (0.13)
1.50 (0.10)
Values are calculated as the mean of six determinations, normalized to the level of amylase activity in the strain expressing each amylase with no added prsA gene.
Results highlighted in italics are statistically significant ( p < 0.05) for a pairwise t‑test Bonferroni corrected regarding the strain overexpressing the amylase with no
added prsA expression. Results marked with an asterisk have a p value 0.10 < p > 0.05
was co-expressed with a prsA gene from either of B.
subtilis, B. licheniformis ( prsABl ), or B. amyloliquefaciens ( prsABa). Only in the case where AmyQ was coexpressed with the G. stearothermophilus ( prsAGs) PrsA
did we observe a severe negative effect of extra PrsA on
amylase activity.
The development of biomass in cultures was monitored
on-line and all grew alike except those expressing prsABN .
These cultures ended up with optical densities approximately 40 percent lower than the cultures expressing
other PrsAs likely due to increased cell lysis in their stationary phases (Additional file 1: Figure S3). The level of
amylase activity in the supernatant was still comparable
to other cultures indicating that PrsABN may be particularly good at supporting amylase secretion as compared
to the other expressed homologs.
It is interesting to notice that the amount of PrsA found
in the membrane and the effect of that PrsA on AmyL
secretion does not seem to be directly connected. PrsABl,
PrsABa, PrsAGs and PrsABson, despite appearing in different amounts in the membrane (Fig. 4), have the same
positive effect on AmyL secretion (Table 2).
Effect of PrsA in the secretion stress response
Overproduction of amylases in B. subtilis has previously
been reported to cause secretion stress. The cell responds
by increasing the production of the quality control proteases HtrA and HtrB of the CssRS regulon which then
remove misfolded proteins that would otherwise block
the essential secretion machinery [5, 26]. To measure
how co-expression of the various heterologous prsA
genes with AmyL affects activity of the CssRS regulon, we
employed a promoter fusion between the secretion stress
inducible htrA promoter (PhtrA) and the lacZ gene. The
PhtrA-lacZ cassette was integrated into the xylose locus
of the chromosome and the level of beta galactosidase
activity was measured as an indicator for the degree of
secretion stress (see Fig. 6 and experimental procedures).
Figure 7 shows the beta-galactosidase activities measured in 24-h old cultures of the various prsA expressing
strains, all expressing AmyL from the PconsSD-amyL
fusion used above. The figure reveals that co-expression of prsABl or prsABson with AmyL not only result in
increased amylase activities in the supernatants (Table 2)
but also leads to a significant decrease in activity of the
htrA promoter. This observation may indicate that PrsABl
(the cognate PrsA) and PrsABson both are able to support proper heterologous secretion of AmyL in B. subtilis
and by doing so also decreases cellular secretion stress.
The remaining four PrsAs, even those influencing AmyL
secretion, do not seem to have any significant effect on
the PhtrA activity when co-expressed with AmyL. Figure 7 also reveals the interesting observation that a strain
co-expressing amyL and prsABN does not appear more
secretion stressed than the reference strain with no extra
prsA, suggesting that the increased cell lysis during the
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Fig. 6 Expression cassette for measuring htrA induction. The expression cassette contains the xyl locus 5′ and 3′ regions for homologous
recombination, the PhtrA promoter followed by the lacZ gene
Fig. 7 Specific β-galatosidase activity after 24 h. Two independent experiments with biological triplicates in each one were conducted and β
-galatosidase activity was measured in the culture pellets. P-values are calculated by a pairwise T-test Bonferroni corrected
stationary phase is not a direct consequence of secretion
stress.
Engineering of PrsAs chaperone domain for improved
amylase secretion
As noted previously we observed increased cell lysis
in stationary phase cultures of all strains expressing
prsABN and this increased cell lysis was not correlated
with a significant decrease in final total amylase activity.
One potential and very interesting explanation to this
could be the presence of specific structural or biochemical features in PrsABN that somehow facilitates superior
amylase secretion, despite having a detrimental effect on
cell growth. This observation led to the following question: would it be possible to modify PrsABl to facilitate a
superior specific amylase secretion, like the one observed
with PrsABN, while at the same time maintaining normal
growth without cell lysis?
PrsA has two domains: the PPIase domain, responsible for the peptidyl prolyl cis-trans isomerase activity,
and the NC or chaperone domain [11]. While the PPIase
domains of the PrsA homologues are highly conserved,
the NC domains are more variable [15] (Additional file 1:
Figure S2). The latter differs greatly between species, both
in sequence and molecular surface, which varies from
very hydrophobic, as in B. subtilis, to very polar, as in L.
monocytogenes’ PrsA1 [14]. This diversity on sequence
and charge may reflect the diverse substrate specificities [11] and hence also explain the variations in yield of
amylase we obtain dependent on choice of PrsA. It has
been speculated that the bowl-like crevice formed by the
NC domains of dimeric PrsA is involved in sequestration of unfolded or unstable polypeptides [11]. Dynamic
interactions between the chaperone and its substrate
would be highly dependent upon the charge distribution/
electronegativity in the surface landscape in this region,
and could influence the frequency of productive substrate
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Fig. 8 Modelled structures of PrsABl and PrsABN. The models were constructed by alignment to the known structure of B. subtilis PrsA (MUSCLE) and
the calculation of the distribution of charges was done by PDB3PQR [27] and PROPKA for pKa calculations [28]. Positive charges are shown in blue
and negative charges in red. a PrsA from B. licheniformis. b PrsA from B. NSP9.1. c Recombinant PrsABl-BN
Fig. 9 The six residues selected for substitution. The figure shows the NC domain of a PrsABl and b PrsABN. Positive charges are shown in blue,
negative charges in pink. Red shows hydrophobicity
interactions [14]. PrsABl and PrsABN are very similar, differing in only 34 out of 286 positions (Additional file 1:
Figure S2). Nevertheless, we observed a substantial difference in electronegativity when the NC domains of the
two proteins were compared (Fig. 8). To bring the NC
chaperone region of PrsABl closer in structure to PrsABN,
six amino acids were substituted (Fig. 9). This new
recombinant PrsA was named PrsABl-BN.
The recombinant prsABl-BN mutant was co-expressed
with amyL and the yield of AmyL was compared to strains
co-expressing amyL together with the prsABl , prsABN ,
or just the wildtype levels of prsABs gene (Fig. 10a). The
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Page 10 of 16
Fig. 10 Total amylase activity and β-galactosidase activity after 24h of growth. Two independent experiments with three biological replicates were
conducted in each case. P-values were calculated with a pairwise t-test Bonferroni corrected. a Total amylase activity in the supernatant. Results are
normalized to the level of amylase activity in the strain expressing each amylase with no added prsA gene. b Specific β-galactosidase activity in the
cell cultures after treatment with lysozyme. P-values are calculated by a pairwise T-test Bonferroni corrected
surface re-modeled PrsABl-BN foldase increased total
amylase activity compared to that of the native PrsABl
and PrsABN foldases. Furthermore, the strain expressing AmyL with this new recombinant PrsABl-BN foldase
appeared less secretion stressed than when AmyL was
expressed with the native foldases, with an almost inverse
correlation between AmyL yield and Secretion Stress
level (Fig. 10). In addition, the strain expressing PrsABl-BN
showed no increased lysis in the stationary phase compared to the wildtype strain (Additional file 1: Figure S3).
Regarding the amount of PrsA present in the membrane, there is less PrsABl-BN present than PrsABl,
(Fig. 11a). There is also less heterologous PrsA in the
membrane than native PrsA in both cases (Fig. 11a).
As can be seen in Fig. 11b, in both cases the amount of
native PrsA that can be found in the membrane is lower
than in the strain expressing AmyL and no extra PrsA
and there is no significant difference between the native
amounts of PrsA between the two heterologous PrsA
expressing strains.
Discussion
In this study we set out to co-express six heterologous
PrsAs with each of six heterologous amylases, using
B. subtilis as a host. We constructed a matrix of strains
containing all combinations of the six amylase–PrsA
pairs and studied the importance of choice of PrsA for
yield of amylases. Essential genes tend to be more conserved among bacteria than non-essential genes [21].
Even though correct folding of amylases is dependent on
PrsA, the essential function of this foldase is however not
related to the folding of amylases. The penicillin binding protein 2B (PBP2B), which in B. subtilis is an essential protein required for the synthesis of the cell wall, is
dependent on PrsA for proper folding [10]. Thus, it may
have been essential for PBP2B to evolve with a the cognate PrsA but there is no strict dependency of the amylase to evolve with the cognate PrsA.
Inactivation of the native prsA gene was only possible
in strains expressing either B. subtilis, B. licheniformis or
B. amyloliquefaciens prsA. This suggests that although
a heterologous PrsA can complement B. subtilis’ native
one, not all PrsAs can do so. B. anthracis PrsA has previously been shown to complement B. subtilis PrsA for
both cell viability and heterologous protein secretion
[29]. In L. monocytogenes, several different heterologous
PrsAs can complement some of the native PrsA2 functions, like swimming motility, pH tolerance, and secretion of virulence factors, but not for others, like osmotic
stress and cell wall biosynthesis [15]. So, even if some
PrsA functions are very conserved among Gram positives, this foldase also appears to be very diverse in its
substrate specificity. This may explain why some PrsAs
can complement B. subtilis’ own, while others cannot,
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Page 11 of 16
Fig. 11 Relative abundance of PrsA in the membrane fraction. The membrane proteome of B. subtilis strains was analysed by LC-MS/MS and
label-free Hi3 quantification. The relative amount of native PrsA is given as the PrsA intensities divided by the sum of the total proteome intensities.
a Measured relative amounts of heterologous PrsA. b Measured relative amounts of native PrsA
possibly because they don’t facilitate proper folding of the
essential PBPs.
We expressed heterologous prsA genes in B. subtilis
and measured their abundancies by relative quantification of the membrane proteomes. Different relative
abundancies of the heterologous PrsAs were found in the
membrane despite the fact that the different PrsAs were
expressed from an identical genetic context. This could
be due to several reasons: different levels of transcription, mRNA degradation, PrsA degradation in the cytoplasm, incorrect destination sorting, or degradation after
translocation through the Sec pathway. Despite these differences, several PrsAs had identical effects on the yield
of AmyL, suggesting that either the amount of PrsA is
not relevant at this level or the different foldases fold the
amylase with different efficiency.
If the reason for the differences in PrsA levels is incorrect sorting, some PrsA could accumulate in the cytosolic or extracellular fractions. Nevertheless, it has been
shown that this foldase is only active in its dimeric form,
and dimerization is only thought to occur when anchored
to the membrane or present in the medium in very high
concentrations (750 µM) [11]. Therefore, it is unlikely
that soluble PrsA present in the media could affect the
folding of the amylases.
To our knowledge, there are no reports on the regulation of the prsA gene expression in B. subtilis. However, an interesting observation of this work was that
the level of native PrsA was affected in some of the
strains expressing amyL. The native PrsA level was twofold higher in the strain expressing amyL compared
to the wild type B. subtilis 168 strain. We also noticed
that the native PrsA level went back to normal when
some heterologous PrsAs were co-expressed, but not
others. Remarkably, the level of the native PrsA was
back to normal when the cognate PrsA from B. licheniformis was expressed. We also observed that this cognate PrsA is superior to the other tested heterologous
PrsAs when it comes to relieving secretion stress in
AmyL producing strains. When looking at the genetic
context in which the native PrsA gene is located, we
identified two putative σ A promoters centered 60 (P1
promoter) and 92 bp (P2 promoter) upstream of the
reading frame. Both promoters match 5 of the 6 canonical bases in their −10 and −35 boxes, and two transcriptional start sites (TSS’s) were mapped at exactly
+1 in accordance with these locations. Superimposed
on the −35 region of P1, we see a putative CssR binding box (TTT TTACA) which share 7 of 8 bases with
the canonical sequence (TTT TCACA) [5]. Another
putative CssR box (TTT TCAAA) is found between
the start codon and the Shine–Dalgarno sequence but
located on the other strand. The location and orientation of these putative CssR operator sites opens for
speculations about CssRS being involved in regulation
of prsA expression. A sensible notion is that CssRS calls
Quesada‑Ganuza et al. Microb Cell Fact
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for increased expression of the prsA foldase gene when
poorly folded proteins are detected in the membrane/
wall interface. In support of this theory we observed
a twofold increase of native PrsA in the membranes
of cells which over-express AmyL as compared to a
B. subtilis reference strain. Overexpression of AmyL
triggers the CssRS to two-component system to autophosphorylate CssR which then becomes able to activate transcription from the htrA- and htrB- promoters.
While CssR is thought to act as a classical activator
of the htrA/B promoters by binding to operators just
upstream of their −35 boxes, the location of the putative CssR operators in the prsA P1 promoter region
suggests that the regulator protein in the case of prsA
regulation functions as a classical repressor. This could
be by means of DNA looping facilitated by dimeric
CssR or simply by monomeric CssR preventing access
of the RNA polymerase to P1 when bound to its operators in the same promoter.
De-repression of a transcription site by CssR has
already been reported in the case of the anti-adaptor
protein YirB. The repressor YuxN forms a DNA loop
by binding two boxes upstream and downstream of the
yirB promoter. CssR P then binds a box that overlaps the
upstream YuxN box, derepressing the yirB promoter [30].
Unless in prsA, in the case of yirB, the CssR P binding box
is located upstream of the −35 site. In the gram-positive
bacterium Staphylococcus aureus, PrsA is regulated by
the VraRS two component system. Two TSS’s have also
been identified in this case, one located 42 bp upstream
of the ATG start codon, and another 139 bp upstream of
ATG. The ATG proximal TSS has the VraR binding box
on and upstream of the −35 site [12].
Overexpression of the native PrsA from B. subtilis
increased the activity of AmyE, AmyS and AmyQ in the
media in accordance with previous studies [19, 20], but
had no significant effect on the other amylases tested
in this work. While PrsA enhances productive secretion of enzymes like alpha amylases or subtilisin, it has
no effect or even a detrimental one in the secretion of
other proteins [20]. Thus, PrsA seems to have specificity for its substrates. As we have shown, the specificity of
the PrsAs for its cognate amylase seems to be maintained
in most cases even when expressed in a different host.
In all cases, except in the case with the G. stearothermophilus pair, the cognate pairs gave the highest amylase
yields or at least just as high as other non-cognate pairs.
In the case of G. stearothermophilus amylase, though,
co-expressing its cognate PrsA had no measurable effect
on its secretion, but other PrsAs (B. subtilis, B. licheniformis and B. amyloliquefaciens) had. This observation
illustrates the great potential of strain optimization prior
to commercial exploration of microbial cell factories. G.
Page 12 of 16
stearothermophilus PrsA is phylogenetically the furthest
away from the hosts PrsA. It is possible that although
the G. stearothermophilus cognate amylase–PrsA pair
would perform best in their original host, their interaction could be affected when moved to such a different
host like Bacillus subtilis. The physicochemical properties around the membrane-cell wall interface could affect
the nature of those interactions.
Overexpression of most heterologous PrsA proteins did
not affect the growth profiles of the strains, except in the
case of strains expressing PrsABN. This could indicate that
this particular PrsA might be very good at supporting
amylase secretion but have a detrimental effect on other
cell functions. As all strains were expressing both nativeand heterologous prsA genes, two different homodimers
and one heterodimer could potentially form. One of these
combinations could increase folding efficiency for certain
alpha-amylases, but reduce proper folding of other physiologically relevant proteins. The increase in cell lysis, a
phenotype that is seen when PrsA is depleted or defective
[19], might be a symptom of this.
In this work we measured the effect of heterologous
PrsA over-expression on the secretion stress response of
cells with forced alpha-amylase production. The intensity of the secretion stress response, defined as the level
of activity of the CssRS regulon, was previously shown
to be correlated with the level of AmyQ production [6].
Also, the introduction of the prsA3 mutation, which
reduces the level of PrsA more than tenfold, was shown
to induce the secretion stress response in B. subtilis. This
response was further increased if the prsA3 mutation
was combined with AmyQ production [26]. Thus, overexpression of amylase may impose stress to the cell and
the intensity of this stress may be affected by the abundance, and perhaps also the nature of the co-expressed
PrsA. In this study we show that heterologous expression of a PrsA in an alpha-amylase secreting strain could
increase amylase secretion and at the same time decrease
secretion stress. In an AmyL producing strain, the coexpression of B. licheniformis PrsA, B. sonorensis PrsA,
and a mutated B. licheniformis PrsA decreased the secretion stress response while increasing amylase activity in
the supernatant. A previous study showed that the secretion stress response, measured by a htrB-lacZ reporter
gene fusion, decreased with the decrease in heterologous
AmyQ secretion [31]. To our understanding, these two
results are not contradictory: In the previous work, the
decrease on AmyQ production was due to the lack of stability of the plasmid coding for amyQ, which would most
probably result in a lower amount of AmyQ being produced, translocated though the Sec system, misfolded at
the membrane-cell wall interface, leading to a decrease
on CssS activation.
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In our work, the chromosomal AmyL expression cassettes were the same in all strains, so the production
and translocated amount of AmyL through the Sec system is expected to be the same. If the prsA co-expressed
has a positive effect on the post-translocational folding of AmyL, more amylases would be rescued with
less accumulation of misfolded proteins as a result. This
would end in both an increase in the amount of secreted
proteins and less stimulation of CssS. The recombinant PrsABl-BN, whose design was based on differences
between the NC domains of the wild type foldases PrsABl
and PrsABN, not only increased the measured activity of
AmyL in the supernatant and decreased secretion stress,
but also maintained a growth profile during the stationary phase similar to the parental B. subtilis 168 strain.
This suggests that the cause of the detrimental interactions of PrsABN was not transferred to the recombinant
PrsABl-BN. The substitution of only 6 amino acids around
the NC domain affected significantly the efficiency of
the folding and secretion of AmyL. The changed amino
acids increased slightly the overall charge of this domain,
which is very hydrophobic in the case of PrsABl. It is possible that a more charged NC domain affects the specific
interaction between the unfolded amylase stretches and
the PrsA positively. Since the primary physiological role
of PrsA in the cell most likely is to fold PBPs and not
amylases, it appears that there is room to improve this
foldase for biotechnological purposes.
The positive effect that point mutations had on the
effect of PrsABl-BN compared to PrsABl not only adds to
the idea that the NC domain of PrsA could be important
for substrate specificity, as suggested for the PrsA proteins of L. monocytogenes [14], but also opens a door for
the design and improvement of PrsA.
Experimental procedures
Strains and growth conditions
Bacillus subtilis strains used in this study are listed in
Additional file 1: Table S1. They are sporulation deficient derivatives of 168 (trpC2, sigF). AN2 is the parental
strain which was used as a host for heterologous expression of proteins. Strains were cultivated at 37C in LB
medium supplemented with chloramphenicol (6 µg/mL)
or erythromycin (1 µg/mL) when appropriate. Competent cells and transformation of B. subtilis was obtained
as described in Yasbin et al. [32]
Construction of B. subtilis strains containing heterologous
prsA‑ and/or amy‑ alleles
Gene Splicing by the Overlapping Extension (SOE)
method [33] was used to generate linear recombinant DNA for transformation. Recombinant DNA
Page 13 of 16
was directed to a specific locus by addition of flanking
regions containing sequences homologous to that locus.
An antibiotic resistance marker gene was also included.
Chromosomal integration was facilitated by homologous recombination and cells in where double cross over
events occurred were selected for on LB agar plates containing the appropriate antibiotic. A PrsA expression cassette targeting the pel locus was assembled by use of the
following DNA components: pel 5′ region (2.5 kb PCR
product) + ermC (1.45 kb PCR product) + synthetic
consensus promoter with SD sequence (pconsSD, 212 bp
synthetic DNA) + prsA open reading frame with terminator (970 bp PCR product) + pel 3′ region (3.3 kb PCR
product). Chromosomal DNA from AN2 was used as a
template for PCR amplifications except for ermC where
a Novozymes in-house plasmid served as a template. The
resulting 8.73 kb SOE-PCR product thus targets the pel
locus and contains the prsA expression cassette linked to
an erythromycin resistance marker gene. This linear DNA
was used directly for transformation of B. subtilis AN2
resulting in strain AQ34 which contains the PrsA(bs)
expression cassette in pel locus. AQ34 chromosomal
DNA then served as a master template for amplification
of flanking regions used to direct ermC and heterologous
prsA genes (orf exchange) to the pel locus of AN2. Templates for amplification of heterologous genes were either
chromosomal DNA isolated from the indicated organism or synthetic DNA. Similarly, an expression cassette
for the Ban-amylase (amyQ) targeting the amyE locus
was assembled by use of the following DNA components: amyE 5′ region (2.8 kb PCR product) + synthetic
consensus promoter with SD sequence (PconsSD, 172 bp
synthetic DNA) + amyQ open reading frame with terminator (1.69 kp PCR product) + cat (1.2 kb PCR product)
+ amyE 3′ region (3.6 kb PCR product). Chromosomal
DNA from AN2 was used as a template for PCR amplifications except for cat where a Novozymes in-house
plasmid served as a template. The resulting 9522 bp SOEPCR product thus targets the amyE locus and contains
the amyQ expression cassette linked to a chloramphenicol resistance marker gene. This linear DNA was used
directly for transformation of B. subtilis AN2 resulting in
strain AQ1 which contains the AmyQ expression cassette
in amyE locus. AQ1 chromosomal DNA then served as
a master template for amplification of flanking regions
used to direct cat and heterologous amylase genes (orf
exchange) to the amyE locus of AN2. The native amyE
becomes inactivated in this process. Templates for amplification of heterologous genes were either chromosomal
DNA isolated from the indicated organism or synthetic
DNA.
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Growth conditions, protein sample preparation, and mass
spectrometry (MS)
Bacillus subtilis cells were grown aerobically at 37 °C
in the presence of 20% glycerol in a synthetic minimal
medium [34]. For the analysis of the membrane fraction,
cells were harvested by centrifugation at early stationary
phase. The cells were disrupted mechanically in a Precellys 24 homogenisator (PeqLab; 3 x 30 s at 6.5 ms− 1).
Glass beads (0.1–0.11 mm diameter) and cell debris were
removed by centrifugation (20,000xg, 10 min, 4 °C). Subsequently, the protein concentration of the samples was
determined. Membrane proteins were enriched according to the protocol published in Eymann et al. [35]. All
samples were analysed by the GeLC-MS workflow. After
electrophoretic fractionation of each mixed sample
by one-dimensional SDS-PAGE, gel lanes were sliced
into 10 equidistant gel pieces followed by tryptic digestion as described by Eymann et al. [35]. For LC-MS/MS
analyses of 1D gel samples, in-house self-packed columns
were prepared and used with an EASY-nLC II system
(Thermo). The peptides were loaded onto the column by
the LC system with 10 µL of buffer A (0.1% (v/v) acetic
acid) at a constant flow rate of 500 nL/min without trapping. The peptides were subsequently eluted using a nonlinear 100 min gradient from 1 to 99% buffer B (0.1% (v/v)
acetic acid in acetonitrile) with a constant flow rate of
300 nL/min and injected online into the mass spectrometer. MS and MS/MS data were acquired with an LTQ
Orbitrap XL (Thermo). After a survey scan at a resolution of 30 000 in the Orbitrap using lockmass correction,
the five most abundant precursor ions were selected for
fragmentation. Singly charged ions, as well as ions without detected charge states, were not selected for MS/MS
analysis. Collision-induced dissociation (CID) fragmentation was performed for 30 ms with a normalized collision energy of 35, and the fragment ions were recorded in
the linear ion trap.
Comparable protein amounts were calculated by the
Hi3 method as described by Silva et al. [36]. For data
processing and protein identification, raw data were
imported into MaxQuant (1.6.3.3) where database search
was carried against the respective B. subtilis strain with
added contaminants from MaxQuant contaminant
list with the following parameters: peptide tolerance:
default, min fragment ions matches per peptide: 1, match
between runs was enabled with the default settings, primary digest reagent: trypsin, missed cleavages: 2, variable modifications: oxidation M (+15.9949), acetylation
N, K (+42.0106). Results were filtered on 1% FDR on
spectrum, peptide and proteins level PSM. The peptide.
txt file obtained from the MaxQuant (1.6.3.3, or above)
search was loaded into R (v1.1.463); modified peptides
Page 14 of 16
were filtered out; peptide intensities of each biological
replicate were separately normalized by division through
the median, and the intensity sum of the three peptides
of the corresponding protein with the highest normalized
intensity were calculated, if at least three peptide intensities were reported in the corresponding biological replicate. A table containing these normalized intensities was
exported to .xlsx format for each strain. A protein was
only considered valid for quantification if values were
existent in two out of 3 biological replicates [37]. In order
to determine the relative amount of PrsA, we divided its
intensity by the summed intensity of all quantified proteins. This value was then compared between all strains
to see what are the changes between the expression of
heterologous PrsAs.
Construction of strains containing the phtrA-lacZ fusion
A LacZ expression cassette under the control of the htrA
promoter and targeting the xyl locus was assembled by
SOE PCR using the following DNA components (Fig. 6)
: 5′xyl region (3.4kb PCR product) + spc (1.2 kb PCR
product) + string A containing phtrA and the first 12 of
the lacZ gene (1.6kb PCR product) + string B containing
the second 12 of the lacZ gene (1.6 PCR product) + 3′xyl
region (4.2 kb PCR product). Chromosomal DNA of AN2
was used as a template for PCR amplification of the 5′
and 3′ xyl regions and an in house plasmid was used as a
template for the amplification of the spc resistance gene.
The resulting PCR product was used direcly for transformation of strains AQG640, AQG492, AQG98, AQG77,
AQG97, AQG174, AQG126 and AQG647 resulting in
strains AQG735, AQG736, AQG737, AQG379, AQG741,
AQG742, AQG745, AQG746 respectively, which express
the lacZ gene under the htrA promoter.
Microplate fermentation
The BioLector is a microfermentation system that monitors online common fermentation parameters such as
biomass, pH, oxygen saturation and fluorescence. It
contains a temperature and humidity controlled incubation chamber that carries a single microplate. The fermentation can be monitored continuously by an optical
fiber that moves below the plate. In this work, a BioLector® (m2p-Labs, Baesweiler, Germany) was used for the
measurement of scattered light and GFP fluorescence.
Cultivations were performed in LB media, at a shaking frequency of 1000 rpm, 37 °C and 85% humidity in
48-well Flowerplates (M2p-labs), covered with a Sealing
Foil with Reduced Evaporation (M2p-Labs). The fermentation was carried out in biological triplicates for 24 h,
and the supernatant was harvested for subsequent amylase activity measurements.
Quesada‑Ganuza et al. Microb Cell Fact
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Amylase activity assay
Culture samples were measured for amylase activity in
technical duplicates in 96 well plates. A calibration curve
with increasing concentrations of BAN amylase (0–500
UCF/µL, Novozymes in-house product) was added to
each 96 well plate. AmyL (Roche/Hitachi) Reagent 1 (66
mL) and Reagent 2 (16 mL) were mixed, and 180 µL of
the mixture was added to the plate. The colorimetric
reaction was measured in a Cytation5 plate reader at
A405 nm, 23 °C for 6 min, measuring absorbance each
minute. One unit of alpha amylase activity was defined as
the amount of enzyme required to increase one unit of
absorbance per minute under the assay conditions.
β‑Galactosidase assay
Cells were grown in 1.5 mL LB media in biological triplicates in Flowerplates at 37 °C and 1000rpm. After 24 h of
growth, the OD600 was measured in technical duplicates,
and the culture was transferred to 1.5 mL eppendorfs
and centrifuged for 5 min at 15,000xg. The supernatant was discarded and the pellets were resuspended in
1 mL Z-buffer (10 mM DTT, 60 mM Na2 HPO4 , 40 mM
NaH2 PO4 , 10 mM KCl, 1 mM MgSO4 , pH 7.0) and 10µL
lysozyme (25 mg/mL) and incubated for 60 min at 37 °C
and 700 rpm. After incubation, 2 technical replicates of
100µL each were transferred to new tubes, and 0.4 mL
of ONPG was added to initiate the reaction. This was
carried out at 30 °C and 700 rpm for 15 min when 1 mL
Na2 CO3 1M was added to stop the reaction. After 15 min
of stopping the reaction, the absorbance of the samples
at 420 nm and 550 nm was measured. This experiment
was done twice. One β-galactosidase activity units was
defined as (Miller units: nmol O.D.−1 min−1).
Supplementary information
Supplementary information accompanies this paper at https://doi.
org/10.1186/s12934-019-1203-0.
Additional file 1. Additional figures and table.
Acknowledgements
This work was made possible by the Horizon 2020 program under a Ph.D.
Grant from the Marie Skłodowska-Curie Grant Agreement No. 642836 “Protein
Factory”. We thank Esben P. Friis for the assistance with Python and Pymol.
Authors’ contributions
AQG performed strain constructions, cultivations, activity assays, preparation
of samples for MS analysis and data analysis. MAV, JB and DB performed the
MS analysis and Hi3 data analysis. KFUA performed strain construction. MGJM
and PFHa performed the phylogenetic analysis. JCM, MK and MDR contributed with scientific discussions. MDR, AQG and AKN designed the experimental work. AQG and AKN drafted the manuscript. MK helped writing and
revising the manuscript. All authors read and approved the final manuscript.
Page 15 of 16
Funding
This work was Funded by the Horizon 2020 program under a Ph.D. Grant to
AQG from the Marie Sk lodowska-Curie Grant Agreement No. 642836 Protein
Factory”.
Availability of data and materials
All data generated or analysed during this study are included in this published
article (and its additional files).
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Research and Technology, Novozymes A/S, Krogshoejvej 36, 2880 Basgvaerd,
Denmark. 2 Institute for Microbiology, Department of Microbial Proteomics,
Ernst-Moritz-Arndt-University Greifswald, F.- Hausdorff-Str. 8, 17489 Greifswald,
Germany. 3 Technical University of Denmark, Søltofts Plads, Building 221, Room
204, 2800 Lyngby, Denmark.
Received: 23 May 2019 Accepted: 3 September 2019
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