Abstract
Background
Methanol, a promising non-food fermentation substrate, has gained
increasing interest as an alternative feedstock to sugars for the
bio-based production of value-added chemicals. Butyribacterium
methylotrophicum, one of methylotrophic-acetogenic bacterium, is a
promising host to assimilate methanol coupled with CO[2] fixation for
the production of organic acids, such as butyric acid. Although the
methanol utilization pathway has been identified in B.
methylotrophicum, little knowledge was currently known about its
regulatory targets, limiting the rational engineering to improve
methanol utilization.
Results
In this study, we found that methanol assimilation of B.
methylotrophicum could be significantly improved when using corn steep
liquor (CSL) as the co-substrate. The further investigation revealed
that high level of lysine was responsible for enhanced methanol
utilization. Through the transcriptome analysis, we proposed a
potential mechanism by which lysine confers improved methylotrophy via
modulating NikABCDE and FhuBCD transporters, both of which are involved
in the uptake of cofactors essential for enzymes of methanol
assimilation. The improved methylotrophy was also confirmed by
overexpressing NikABCDE or FhuBCD operon. Finally, the de novo
synthetic pathway of lysine was further engineered and the methanol
utilization and butyric acid production of B. methylotrophicum were
improved by 63.2% and 79.7%, respectively. After an optimization of
cultivation medium, 3.69 g/L of butyric acid was finally achieved from
methanol with a yield of 76.3%, the highest level reported to date.
Conclusion
This study revealed a novel mechanism to regulate methanol assimilation
by lysine in B. methylotrophicum and engineered it to improve methanol
bioconversion to butyric acid, culminating in the synthesis of the
highest butyric acid titer reported so far in B. methylotrophicum.
What’s more, our work represents a further advancement in the
engineering of methylotrophic-acetogenic bacterium to improve
C1-compound utilization.
Graphical Abstract
[39]graphic file with name 13068_2023_2263_Figa_HTML.jpg
Supplementary Information
The online version contains supplementary material available at
10.1186/s13068-023-02263-w.
Keywords: Methylotrophy, B. methylotrophicum, Lysine, Butyric acid
Background
The microbial assimilation of one-carbon (C1) compounds has been argued
for as a promising technological approach to meet the growing
challenges of the global climate change and energy resource scarcity
[[40]1]. As one of C1 compounds, methanol has attracted particular
attention due to its abundance and liquid state. More importantly,
methanol represents the highest electron content in all liquid C1
compounds and can also be easily produced from the greenhouse gases
methane and CO[2] [[41]2–[42]4], which makes it more promising as a
non-food fermentation substrate to replace conventional sugars
[[43]5–[44]7]. In recent years, efforts on the bioconversion of
methanol into fuels and value-added chemicals have therefore
intensified by the engineering of the native methylotrophs for improved
methylotrophic phenotype, or development of entirely new methylotrophic
organisms [[45]8–[46]12].
Native methylotrophs are the ideal host for methanol-based
biomanufacturing as their ability to grow on methanol as the sole
carbon and energy source. Methylotrophic-acetogenic bacterium, such as
Acetobacterium woodii (A. woodii), B. methylotrophicum, Eubacterium
limosum (E. limosum), Sporomusa ovata, or Moorella thermoacetica, are a
group of obligate anaerobes that could utilize various C1 compounds
(CO[2], CO, methanol and formate) as growth substrates [[47]13–[48]15].
B. methylotrophicum was one of represented methylotrophic-acetogenic
bacterium with the ability to convert methanol into butyric acid, a
four-carbon carboxylic acid that could be widely used in the food,
pharmaceuticals, and polymer industries. The methanol utilization
pathway has been identified in B. methylotrophicum, where methanol
assimilation was coupled with CO[2] fixation through the
methyltransferase systems and the carbonyl branch of the Wood–Ljungdahl
pathway (WLP) [[49]16]. The characteristic of co-utilization of
methanol and CO[2] to produce butyric acid by B. methylotrophicum has
made it more potential to serve as a chassis for methanol
bioconversion. Therefore, the further improvement in the methanol
conversion efficiency of B. methylotrophicum is highly desired.
Recent efforts have been made to develop the genetic tools for
engineering methylotrophic-acetogenic bacterium [[50]17]. In the
previous work, the plasmids with the stable replication origins, the
functional promoters as well as the efficient electroporation protocols
have been also developed for the engineering of B. methylotrophicum
[[51]16]. Based on these genetic tools, the genes encoding the
methyltransferase systems (mtaA, mtaB and mtaC) in B. methylotrophicum
were overexpressed, and the biomass yield, methanol consumption, and
butyric acid production were increased by 1.25-fold, 1.69-fold, and
1.38-fold, respectively [[52]16]. What’s more, several valuable
chemicals, such as acetone and lactate have been produced through the
metabolic engineering of methylotrophic-acetogenic bacterium [[53]18,
[54]19]. However, little knowledge was currently known on the
understanding of regulation targets to improve methanol utilization of
the methylotrophic-acetogenic bacterium. Adaptive laboratory evolution
was thus employed for enhancing methylotrophy [[55]20]. The
understanding of metabolic regulation targets of
methylotrophic-acetogenic bacterium is urgently required in improving
acetogenic conversion of methanol.
As an important nutrient component, we hypothesized that the nitrogen
source might affect methanol assimilation of methylotrophic-acetogenic
bacterium as observed in other native methylotrophs [[56]21, [57]22].
With B. methylotrophicum as a representative, the effect of different
nitrogen source on methanol utilization was evaluated, and corn steep
liquor (CSL) was found to be more superior in enhancing methanol
utilization. Subsequently, high levels of lysine in CSL medium were
identified to be responsible for the improved methylotrophy of B.
methylotrophicum. Transcriptome analysis revealed that the addition of
lysine up-regulated eight ABC transporters (NikABCDE and FhuBCD),
involving in the transport of the essential cofactors for the enzymes
in the methanol assimilation pathway. The improved methylotrophy of B.
methylotrophicum was further confirmed by the overexpression of
NikABCDE or FhuBCD operon, suggesting a potential molecular mechanism
by which lysine improves methanol utilization of B. methylotrophicum.
Finally, the de novo synthetic pathway of lysine was engineered to
enhance the methanol bioconversion, and the highest reported titer of
butyric acid from methanol was achieved.
Results
Corn steep liquor is a superior nitrogen source compared to yeast extract in
enhancing methanol assimilation
In addition to carbon source, nitrogen source is another key nutrient
that affects cell growth and product formation. In native
methylotrophic P. pastoris, nitrogen source was reported capable of
regulating the gene expression in methanol utilization pathway
[[58]21]. In synthetic methylotrophic E. coli, the addition of yeast
extract could also enhance methanol assimilation [[59]23]. Thus, we
hypothesized that the types of nitrogen sources contributed differently
to the methanol assimilation of B. methylotrophicum. To test it,
different kinds of nitrogen sources including peptone, corn steep
liquor, beef extract, ammonium chloride and ammonium sulphate were
supplemented in the modified DSM 135 medium with 200 mM methanol to
replace the yeast extract with the equal nitrogen content.
As shown in Fig. [60]1, when the yeast extract was replaced by peptone,
beef extract or inorganic nitrogen source of ammonium chloride and
ammonium sulphate, the cell growth and methanol consumption were
significantly decreased. With ammonium sulphate as an example, the
final biomass and methanol consumption rate were decreased by 43% and
32%, respectively (Fig. [61]1). On the contrary, the use of corn steep
liquor increases the methanol utilization rate of B. methylotrophicum
by 35.9% (Fig. [62]1b). The final biomass yield in corn steep liquor
medium was increased by 56.5% with a yield of 0.248 g[DCW]/g[MeOH]
(Fig. [63]1a). The butyric acid formation in different nitrogen source
was consistent with the methanol consumption. In corn steep liquor
medium, 2.56 g/L butyric acid was produced with a yield of 56.0%, a
1.07-fold higher than that in yeast extract medium, while 2.16 g/L
acetic acid was accumulated (Fig. [64]1c, d). The results indicated
that corn steep liquor was a superior nitrogen source for B.
methylotrophicum to grow in methanol.
Fig. 1.
[65]Fig. 1
[66]Open in a new tab
Evaluating the effect of nitrogen source on the methanol fermentation
phenotype of B. methylotrophicum. A The growth of B. methylotrophicum
in medium supplemented with different nitrogen sources. B The methanol
consumption of B. methylotrophicum in medium supplemented with
different nitrogen sources. C The butyric acid production of B.
methylotrophicum in medium supplemented with different nitrogen
sources. D The acetic acid production of B. methylotrophicum in medium
supplemented with different nitrogen sources
Lysine was identified to be responsible for enhanced methylotrophy in B.
methylotrophicum
Free amino acid is the main content of organic nitrogen source. To
elucidate the potential molecular mechanism by which the corn steep
liquor (CSL) was benefit for methanol assimilation of B.
methylotrophicum compared to yeast extract (YE), the amino acid
composition in CSL medium and YE medium was comparatively investigated
during the fermentation process. The culture broth was sampled at 0 h,
36 h (middle exponential phase), 60 h (later exponential phase), and
84 h (stationary phase), respectively. Through the GC/MS analysis, 15
kinds of amino acids were detected including Gly, Ala, Lys, Asn, Thr,
Glu, Asp, Val, Ile, Ser, Tyr, Pro, and Phe. The levels of the 15 amino
acids were all gradually decreased with the fermentation both in the
CSL medium and YE medium (Additional file [67]1: Fig. S1).
Among them, the content of 11 amino acids in CLS medium was initially
higher than those in YE medium, which are Gly, Lys, Glu, Asn, Asp, Ile,
Thr, and Met respectively (Fig. [68]2a). These results indicated the
advantage of CLS in supplying free amino acids. During the whole
fermentation process, the contents of Glu, Asn, Asp, Ile, Thr and Met
in CLS medium were always higher than those in YE medium, while the
contents of Phe, Pro, Ser and Leu in CLS medium were always lower than
those in YE medium (Fig. [69]2a). For the amino acids with the higher
level in CLS medium, Gly, Lys, and Asn represented the most significant
difference. In addition, the difference of Gly, Lys, Ala and Val in CLS
medium and YE medium changed most significantly during the whole
fermentation process (Fig. [70]2a). We speculated that these
significantly different amino acids with higher content in CSL medium
might be related to enhanced methanol assimilation in B.
methylotrophicum.
Fig. 2.
[71]Fig. 2
[72]Open in a new tab
The identification of the key factors by which corn steep liquor is
superior to yeast extract in improving methylotrophy of B.
methylotrophicum. A The difference of amino acid component between corn
steep liquor medium and yeast extract medium in different fermentation
stage. All ratio values were log-transformed (base 2) for the fold
change of the relative abundance of each amino acid in CSL medium
relative to that in YE medium in different fermentation stage. B 15
kinds of amino acids (5 mM) were supplemented to the medium for their
effects on methylotrophy of B. methylotrophicum, respectively
To further identify the certain amino acids important for enhanced
methanol metabolism in B. methylotrophicum, Gly, Lys, Ala, Val, and Asn
were added into the medium to evaluate their effects, while the other 9
amino acids were also comparatively analyzed. From the results shown in
Fig. [73]2b, when methanol was used as a sole carbon source, the
specific growth rate and the final biomass of B. methylotrophicum were
improved by 12.1% and 29.2% with the addition of Lys, while the
addition of Gly, Val and Ala showed a moderately increase in final
biomass. Based on these results, lysine was determined as an important
factor for regulating methanol assimilation of B. methylotrophicum.
As high lysine level was supposed to be beneficial for improving
methanol assimilation of B. methylotrophicum, the effect of different
concentrations of lysine was further evaluated. The results are shown
in Fig. [74]3. The lysine concentration ranged from 5 to 30 mM. When a
small amount of 5 mM lysine was supplemented, the cell growth and
methanol consumption of B. methylotrophicum were slightly affected
(Fig. [75]3a, b). With a higher lysine concentration from 10 to 30 mM,
the cell growth, methanol consumption and butyric acid production of B.
methylotrophicum were significantly improved (Fig. [76]3a–c), further
confirming our conclusion that increasing the availability of lysine
level could enhance methanol assimilation of B. methylotrophicum. At
the condition of 20 mM lysine, the cell growth, methanol consumption
and butyric acid production of B. methylotrophicum reached the maximum
level, which were increased by 38.44%, 21.52%, and 53.07%, respectively
(Fig. [77]3a–c). In addition, we found that the addition of lysine
could also improve the butyric acid yield from methanol (Fig. [78]3d),
while the production of acetic acid was decreased (Additional file
[79]1: Fig. S2). In acetogenic bacteria, several amino acids have been
reported to be oxidized and degraded to support cell growth. For
example, alanine could serve as growth substrate for Sporomusa
aerivorans (S. aerivorans) and A. woodii [[80]24, [81]25], and E.
limosum was able to use isoleucine and valine as growth substrate
[[82]26]. We also evaluated whether lysine could be used as a growth
substrate for B. methylotrophicum and therefore stimulated growth on
methanol. As the result shows in Fig. [83]3a, in the cultivation medium
containing 3 g/L yeast extract, methanol was removed and 20 mM lysine
was supplemented to determine the effect on cell growth. The results
showed that the cells of B. methylotrophicum could not grow when
methanol was removed in presence of lysine, and none of lysine
consumption was observed (Fig. [84]3a and Additional file [85]1:
Fig. S3). Under the condition of 100 mM methanol supplemented with
different lysine concentration, lysine was also barely consumed with
methanol consumption and cell growth (Additional file [86]1: Fig. S3).
These results indicated that lysine was not served as a growth
co-substrate to improve methanol assimilation in B. methylotrophicum.
Fig. 3.
[87]Fig. 3
[88]Open in a new tab
The effect of lysine supplementation on the methanol utilization and
butyric acid production of B. methylotrophicum. The strain B.
methylotrophicum was cultivated in modified DSM135 medium supplemented
with extra concentrations of lysine (0 mM, 5 mM, 10 mM, 20 mM and
30 mM) anaerobically. The growth profiles (A), the methanol consumption
(B), butyric acid production (C), and butyric acid yield (D) were
determined
Transcriptional analysis revealed that up-regulated expression of ABC
transporters was triggered by lysine addition
To elucidate the potential mechanism of lysine in regulating methanol
assimilation of B. methylotrophicum, the transcriptional response to
lysine addition was determined. Through the RNA-seq experiments,
differentially expressed gene (DEG) analysis identified 58 up-regulated
and 920 down-regulated genes (Fig. [89]4a). The significantly changed
genes are illustrated in Additional file [90]1: Tables S2 and S3. KEGG
pathway analysis was subsequently conducted to identify the pathways
for these DEGs. With the corrected p-value < 0.05, the up-regulated
genes were significantly enriched in 8 pathways, while the
down-regulated genes were enriched in 7 pathways (Fig. [91]4b and
Additional file [92]1: Fig. S4). In the enriched pathways for
up-regulated genes, ABC transporters represented the most significant
one (Fig. [93]4a). In the enriched pathways for down-regulated genes,
the pathways involved in ribosome, fatty acid synthesis,
phosphotransferase system, fructose and mannose metabolism and lysine
biosynthesis changed most significantly (Additional file [94]1:
Fig. S4).
Fig. 4.
[95]Fig. 4
[96]Open in a new tab
The transcriptional response to B. methylotrophicum to lysine addition.
A Volcano map of differential genes in cells cultivated with and
without extra lysine addition. Significantly differentially expressed
genes are represented by red dots (up-regulated) and blue dots
(down-regulated). The abscissa represents the fold change of gene
expression in different samples, log[2](Fold Change) > 1; the ordinate
represents the statistical significance of the difference in gene
expression, padjust < 0.05. B Pathway enrichment analysis of the
significantly up-regulated genes with the corrected p-value < 0.05. C
The transcriptional response of genes in NikABCDE tansporter system to
lysine addition in B. methylotrophicum. D The transcriptional response
of genes in FhuBCD transporter system to lysine addition in B.
methylotrophicum
Here, the genes in the up-regulated pathways attracted our interested.
Eight significant up-regulated ABC transporters including NikA, NikB,
NikC, NikD, NikE, FhuB, FhuC, and FhuD was clustered into two classes.
The first cluster including genes of NikA, NikB, NikC, NikD and NikE
was defined as the nickel transport system [[97]27], while another gene
cluster of FhuB, FhuC, and FhuD was defined as an iron complex
transport system involved in the uptake of siderophores, heme and
cobalamin (vitamin B12) [[98]28]. In B. methylotrophicum, methanol was
assimilated to acetyl-CoA with CO[2] as an electron acceptor through
the methyltransferase system along with the carbonyl branch of the WLP
pathway [[99]16]. In the methyltransferase system, the
corrinoid-dependent methyltransferase (MtaB) first transfers the methyl
group of methanol to a corrinoid protein (MtaC), and then
methyltetrahydrofolate-methyltransferase (MtaA) transfers the methyl
group from methyl-MtaC to tetrahydrofolate (THF), where cobalamin is an
important cofactor for the activity of MtaB and MtaC [[100]29],
up-regulation of the FhuBCD transporter may enable MtaB and MtaC
catalytic activity, thereby affecting methanol assimilation in B.
methylotrophicum. In the carbonyl branch of the WLP pathway, both
carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS) are
nickel-containing enzymes [[101]30], up-regulation of the NikABCDE
transporter may enhance the uptake of nickel and affect the catalytic
activity of CODH and ACS. The gene expression of NikA, NikB, NikC,
NikD, and NikE was significantly up-regulated by lysine addition with
fold changes of 7.9, 9.9, 8.8, 3.6, 10.8, respectively (Fig. [102]4c),
and the expression of FhuB, FhuC, and FhuD was increased by 3.3-fold,
3.0-fold, and 2.7-fold, respectively (Fig. [103]4d). To further
demonstrate whether lysine stimulated the up-regulation of NikABCDE and
FhuBCD transporters, we carried out RT-qPCR experiments. Following the
results shown in Additional file [104]1: Fig. S5, the relative
expression level of NikABCDE and FhuBCD transporters was significantly
up-regulated by 2.19 and 1.98 times, respectively, when 20 mM lysine
was added into the methanol medium. We thus speculated that the
up-regulated cofactor uptake system might be involved in the improved
methanol metabolism triggered by lysine in B. methylotrophicum.
Overexpression of NikABCDE or FhuBCD improved methanol assimilation of B.
methylotrophicum
To further identify whether the up-regulation of these two-transport
system was responsible for the improved methanol utilization of B.
methylotrophicum, NikABCDE or FhuBCD was engineered to evaluate their
effects on methanol metabolism of B. methylotrophicum. As shown in
Fig. [105]5a, the overexpression of FhuBCD or NikABCDE resulted in a
significant improvement in the methylotrophic phenotype over the empty
plasmid control. With methanol as the sole carbon source, the specific
growth rate of NikABCDE overexpressing strain reached 0.01393 h^−1,
1.4-fold higher than the empty plasmid control, and the overexpression
of FhuBCD increased the specific growth rate to 0.01641 h^−1, 1.2-fold
higher than the control (Fig. [106]5a). Higher final biomass titer was
also achieved by the overexpression of FhuBCD or NikABCDE
(Fig. [107]5a). At the meantime, the methanol consumption rate of the
recombinant B. methylotrophicum/pXY1-FhuBCD and B.
methylotrophicum/pXY1-NikABCDE increased by 24.5% and 34.7%,
respectively (Fig. [108]5b). The titer and yield of butyric acid from
the methanol were also significantly improved when these two systems
were overexpressed (Fig. [109]5c). The butyric acid titer in the
recombinant B. methylotrophicum/pXY1-FhuBCD and B.
methylotrophicum/pXY1-NikABCDE was increased by 38.9% and 52.5%,
respectively, and the butyric acid yield in the recombinant B.
methylotrophicum/pXY1-FhuBCD and B. methylotrophicum/pXY1-NikABCDE was
increased by 9.9% and 16.1%, respectively. Based on these results, we
confirmed that the overexpression of NikABCDE or FhuBCD could improve
methylotrophy of B. methylotrophicum (Fig. [110]5c). We thus proposed a
possible regulatory mechanism that increasing lysine level triggered
the expression of NikABCDE and FhuBCD transport system responsible for
improved methanol utilization in B. methylotrophicum (Fig. [111]5d).
Fig. 5.
[112]Fig. 5
[113]Open in a new tab
The overexpression of NikABCDE or FhuBCD transporter system confers the
improved methylotrophy for B. methylotrophicum. A The effects of
overexpressing NikABCDE or FhuBCD operon on the growth of B.
methylotrophicum. B The effects of overexpressing NikABCDE or FhuBCD
operon on the methanol consumption of B. methylotrophicum. C The
effects of overexpressing NikABCDE or FhuBCD operon on the butyric acid
of B. methylotrophicum. D The schematic depicting the potential
mechanism of lysine regulation in methylotrophy for B. methylotrophicum
The engineering of lysine synthetic pathway enhanced methanol utilization and
butyric acid production in B. methylotrophicum
As exogenous lysine addition could improve methanol utilization of B.
methylotrophicum, we hypothesized that increasing lysine synthesis
through the genetic modification of the de novo lysine synthetic
pathway was able to improve methanol assimilation. The lysine
biosynthetic pathway in B. methylotrophicum was identified to be
similar with that in E. coli, where L-aspartate was converted to
generate L-lysine by the catalysis of lysC, asd, dapA, dapB, dapC,
dapD, dapE, dapF, and lysA (Fig. [114]6a). The overexpression of lysA,
dapA, or dapB has been confirmed to be an efficient approach for
enhancing flux through the lysine synthetic pathway [[115]31–[116]34].
Here, two plasmids of pXY1-P[thl]-dapA-dapB for the overexpression of
dapA and dapB, and pXY1-P[thl]-lysA for the overexpression of lysA were
constructed and electro-transformed into B. methylotrophicum. As the
results illustrated in Fig. [117]6b, both the overexpression of lysA or
co-overexpression of dapA and dapB could significantly enhance the
methylotrophic phenotype of B. methylotrophicum. The biomass
concentration and methanol consumption of B.
methylotrophicum/pXY1-P[thl]-lysA was increased by 38.8%, and 23%,
respectively (Fig. [118]6b, c). The recombinant strain B.
methylotrophicum/pXY1-P[thl]-dapA-dapB exhibited the best growth
advantage in methanol medium, in which the final biomass concentration
was increased by 51%, and the specific growth rate reached 0.0259 h^−1
(Fig. [119]6b). The methanol consumption of B.
methylotrophicum/pXY1-P[thl]-dapA-dapB was increased by 63.2%
(Fig. [120]6c). In addition, the enhancement of lysine could also
improve butyric acid production titer and yield from methanol
(Fig. [121]6d, e). The overexpression of lysA increased the butyric
acid production by 33.8% with a final titer of 0.99 g/L, while the
accumulation of acetic acid was significantly decreased (Additional
file [122]1: Fig. S6). For the engineered B.
methylotrophicum/pXY1-P[thl]-dapA-dapB, a titer of 1.33 g/L butyric
acid was achieved with a yield of 83.1%, which was increased by 79.7%
and 10.4% compared to that in the control strain, respectively
(Fig. [123]6c, d). These results confirmed that increasing the flux
through the lysine biosynthetic pathway could efficiently improve
methanol utilization and butyric acid production of B.
methylotrophicum, further identifying that lysine is an important
target for the regulation of methanol utilization in B.
methylotrophicum.
Fig. 6.
[124]Fig. 6
[125]Open in a new tab
The overexpression of de novo lysine synthetic pathway enhances
methanol utilization and butyric acid production for B.
methylotrophicum. A The lysine synthetic pathway in B.
methylotrophicum. B The effects of overexpressing lysA or dapAB operon
on the growth of B. methylotrophicum. C The effects of overexpressing
lysA or dapAB operon on the methanol consumption of B.
methylotrophicum. D The effects of overexpressing lysA or dapAB operon
on butyric acid production of B. methylotrophicum. E The effects of
overexpressing lysA or dapAB operon on butyric acid yield of B.
methylotrophicum
The presence of CO[2] as an electron acceptor could affect methanol
consumption and product distribution in B. methylotrophicum. Different
concentrations of bicarbonate were therefore supplemented and the cell
growth on methanol was largely improved (Fig. [126]7a). The percentage
of methanol consumption was increased to 97% in conditions of 20 mM or
40 mM bicarbonate from 55% in conditions of 0 mM bicarbonate
(Fig. [127]7b). The titer of butyric acid reached 1.45 g/L when the
methanol to bicarbonate ratio was 100 mM:20 mM (Fig. [128]7c). With the
further increase of bicarbonate concentration to 40 mM, butyric acid
titer was just increased to 1.6 g/L, and meanwhile large amount of
acetic acid (2.0 g/L) was produced (Fig. [129]7d). To further improve
the butyric acid production of the engineered B.
methylotrophicum/pXY1-P[thl]-dapA-dapB from methanol, the fermentation
was performed in CSL medium. As shown in Fig. [130]8, under the
condition of 200 mM methanol and 40 mM bicarbonate, the cell grew into
the stationary phase after the fermentation 96 h. With the prolonged
fermentation time to 158 h, the production of butyric acid reached
3.69 g/L, while 1.92 g/L acetic acid was accumulated. Finally, 5.7 g/L
methanol and 15.97 mM bicarbonate were totally consumed, and the yield
of butyric acid from methanol could reach 76.3%.
Fig. 7.
[131]Fig. 7
[132]Open in a new tab
The presence of CO[2] to improve methanol utilization and butyric acid
production of B. methylotrophicum. The growth profiles (A), methanol
consumption (B), butyric acid production (C), and acetic acid
production (D) of B. methylotrophicum under 100 mM methanol condition
with the addition of 0 mM, 20 mM, and 40 mM sodium bicarbonate
Fig. 8.
Fig. 8
[133]Open in a new tab
The fermentation of the engineered B.
methylotrophicum/pXY1-P[thl]-dapA-dapB in CSL medium supplemented with
200 mM methanol and 40 mM bicarbonate
Discussion
Methanol is an abundant and attractive fermentation substrate as an
alternative to sugars with the advantage of high electron and energy
content. Recent attempts to develop methylotrophic cell factories have
been made by using native or synthetic methylotrophs [[134]35,
[135]36]. Nowadays, most of studies have focused on the aerobic
methylotrophs. For example, through the engineering of aerobic
methylotrophic P. pastoris, Zhou et al. enable high-level production of
free fatty acid or fatty alcohols from sole methanol [[136]37,
[137]38]. Meanwhile, anaerobic methanol utilization is also an
interesting area as anaerobic conditions are also desirable for
production of many metabolites, such as organic acids or alcohols
[[138]39]. B. methylotrophicum is one of representative anaerobic
methylotrophic bacteria. Specially, CO[2] could be used as the sole
electron acceptor during methanol assimilation, making B.
methylotrophicum as an ideal platform for C1-compound bioconversion.
Compared to the aerobic methylotrophs, anaerobic methylotrophs are
relatively slow growing and achieve relatively low cell densities. Rare
knowledge on the understanding of regulating methylotrophic ability of
anaerobic methylotrophic bacteria also limited their rational
engineering for efficient C1-compound bioconversion.
Nitrogen source, one of nutrient component essential for cell growth,
has been shown to affect methanol metabolism ability in various native
methylotrophs, such as P. pastoris [[139]21], Methylomicrobium album
[[140]40] or Methylocystis sp [[141]22]. In a previous study, the use
of yeast extract could also improve methanol utilization in a synthetic
methylotrophic E. coli, Subsequent work determined that this
stimulatory effect was responsible threonine [[142]41]. In another
study, it was found that triggering the stringent response to increase
the synthesis of several amino acids could also improve cell growth on
methanol [[143]42], indicating the important role of amino acid on
methanol metabolism in methylotrophs. Here, we found that the use of
corn steep liquor significantly improved methylotrophic ability of B.
methylotrophicum. Further investigation revealed that high level of
lysine contributed to the improved methylotrophic ability of B.
methylotrophicum. Several amino acids could be oxidized to support cell
growth of methylotrophic-acetogenic bacterium. For example, E. limosum
is able to use isoleucine and valine as substrates [[144]26], and
alanine could serve as substrate for S. aerivorans and A. woodii
[[145]24, [146]25]. However, lysine could not support cell growth of B.
methylotrophicum as observed in our results (Fig. [147]3a), indicating
that the improved methylotrophic ability by lysine addition was not due
to the extra presence of growth substrate.
The transcriptional response of B. methylotrophicum to lysine addition
revealed that eight ABC transporters clustered into two classes of
NikABCDE and FhuBCD are specifically up-regulated. NikABCDE transporter
system is the main importer of nickel in microorganisms [[148]43,
[149]44]. Nickel is an essential micronutrient for a wide variety of
microorganisms, and involves in numerous of cellular processes, serving
as a cofactor for nickel-dependent enzymes [[150]27, [151]45]. In the
methylotrophic-acetogenic bacterium, nickel is essential
metallocofactor for the activity of CODH and ACS, which are responsible
for CO[2] reduction and acetyl-CoA synthesis in methanol assimilation
pathway [[152]46]. The up-regulation of NikABCDE by lysine addition in
B. methylotrophicum might enhance the uptake of nickel, increase the
catalytic activity of CODH and ACS, and thus impact methylotrophy.
Another FhuBCD transporter system up-regulated by lysine addition was
involved in the uptake of cobalamin, an essential cofactor for the
activity of MtaB and MtaC, which catalyzes the initial methyltransfer
step in methanol assimilation pathway of B. methylotrophicum. The
improved methylotrophy through the overexpression of NikABCDE or FhuBCD
further suggested the potential mechanism by which lysine addition
could regulate methylotrophic ability of B. methylotrophicum
(Fig. [153]5).
Based on the results in this study, we confirm that lysine plays an
important role in regulating methylotrophy activity of B.
methylotrophicum, which referred to ABC transporter involved in the
uptake of essential cofactors for enzymes in methanol assimilation
pathway. Amino acid metabolism has been reported to be an important
factor in regulating methanol metabolism in several methylotrophs
[[154]41, [155]42]. When we performed amino acid addition experiments,
we also found that the addition of Gly, Val and Ala could show a
moderate increase in final biomass. The different amino acids might
have a synergistic effect on methanol utilization, which would be
further investigated in our following works. To our knowledge, this is
the first report to uncover the regulatory role of amino acid in
methylotrophy activity of methylotrophic-acetogenic bacterium. Based
the proposed targets, B. methylotrophicum was further engineered by
enhancing the endogenous lysine synthesis (Fig. [156]8), 3.69 g/L
butyric acid was finally produced from methanol, which was the highest
titer reported so far by B. methylotrophicum with methanol and
bicarbonate as the sole carbon source [[157]19, [158]47, [159]48]. Our
study not only identified new gene targets for understanding methanol
metabolism mechanism in methylotrophic-acetogenic bacterium, but also
constructed several engineered strains with improved methylotrophy,
which could serve as customized host strains for methanol bioconversion
to produce variate value-added chemicals.
Conclusions
Here, we revealed a potential regulation mechanism for improved
methanol metabolism in B. methylotrophicum. Higher level of lysine was
identified to be responsible for enhanced methanol utilization of B.
methylotrophicum. With the transcriptome analysis, we found that lysine
could up-regulate cofactor transporters essential for enzymes of
methanol assimilation. The regulation mechanism of lysine was further
confirmed by improved methylotrophy of B. methylotrophicum when
overexpressing NikABCDE or FhuBCD operon. Based on our proposed gene
targets, the de novo synthetic pathway of lysine was enhanced, and the
bioconversion of methanol to butyric acid was successfully improved.
Materials and methods
Microbial strains and growth conditions
All strains used in this work are listed in Table [160]1. Escherichia
coli strains were cultivated at 37 °C in Luria–Bertani medium (tryptone
10 g/L, yeast extract 5 g/L, NaCl 10 g/L) supplemented with appropriate
antibiotics at the following concentrations: 100 μg/mL spectinomycin
(Spe), or 100 μg/mL ampicillin (Amp).
Table 1.
Strains and plasmids used in this study
Strains or plasmids Description of genotype Source
Strains
E. coli Trans T1 F¯φ80(lacZ)ΔM15ΔlacX74 hsdR(rK^−, mK^+) ΔrecA 1398
endA1 tonA TransGen
E. coli Top10 F¯mcrA Δ (mrr-hsd RMS-mcrBC) φ80 lacZΔM15Δ lacX74 recA
araΔ139(ara-leu)7697 galU galK rpsL(Str^R) endA1 nupG TransGen
B. methylotrophicum Wild type ATCC
B. methylotrophicum/pXY1-NikABCDE B. methylotrophicum overexpressing
the operon of NikABCDE under the control of P[thl] This study
B. methylotrophicum/pXY1-FhuBCD B. methylotrophicum overexpressing the
operon of FhuBCD under the control of P[thl] This study
B. methylotrophicum/pXY1-LysA B. methylotrophicum overexpressing the
gene of LysA under the control of P[thl] This study
B. methylotrophicum/pXY1-DapAB B. methylotrophicum overexpressing the
gene of DapA and DapB under the control of P[thl] This study
Plasmids
pMCljS pACYC184, methyltransferase gene of C. ljungdahlii (CLJU
c03310), Spe^R [[161]16]
pXY1 pCB102 ORI, ColE1 origin; Amp^R, Em^R [[162]16]
pXY1-P[thl]-NikABCDE Derived from pXY1, with the operon of nikA, nikB,
nikC, nikD and nikE overexpression This study
pXY1-P[thl]-FhuBCD Derived from pXY1, with the operon of FhuB, FhuC and
FhuD overexpression This study
pXY1-LysA Derived from pXY1, with the gene of LysA overexpression This
study
pXY1-DapAB Derived from pXY1, with the gene of DapA and DapB
overexpression This study
[163]Open in a new tab
Spe^R, spectinomycin resistance; Amp^R, ampicillin resistance; Em^R,
erythromycin resistance; pCB102 ORI, Gram-positive origin of
replication from Clostridium butyricum
Butyribacterium methylotrophicum ATCC 33266 was cultivated
anaerobically at 37 °C in modified DSM 135 medium described previously
in an anaerobic chamber (AW500TG, Electro-Tech, Co., Ltd., UK)
[[164]16]. 30 μg/mL of erythromycin (Em) was supplemented when
recombinant B. methylotrophicum strains were cultivated. The certain
concentration of methanol (100 mM-200 mM) was added as the carbon
source. To determine the effect of nitrogen source on methanol
utilization, 2.64 g/L peptone, 4.13 g/L beef extract, 8.25 g/L corn
milk, 0.17 g/L urea, 0.17 g/L ammonium sulfate, or 0.34 g/L ammonium
chloride, which contained the equal nitrogen content as 3 g/L yeast
extract, was supplemented to replace the yeast extract in the modified
DSM 135 medium. To determine the effect of amino acid on methanol
utilization, 5 mM of glycine (Gly), alanine (Ala), lysine (Lys),
asparagine (Asn), threonine (Thr), glutamate (Glu), aspartate (Asp),
valine (Val), isoleucine (Ile), serine (Ser), tyrosine (Tyr), proline
(Pro), or phenylalanine (Phe) were added into the modified DSM 135
medium with 100 mM or 200 mM methanol as the sole carbon source. To
evaluate the effect of lysine on methanol metabolism, different
concentrations of sodium bicarbonate (5 mM, 10 mM, 20 mM, and 30 mM)
were added into the modified DSM 135 medium with a fixed methanol
concentration of 100 mM.
Analysis of the amino acid composition
Culture broth of B. methylotrophicum in medium supplemented with corn
steep liquor (CSL) or yeast extract (YE) was sampled at 0 h, 36 h
(middle exponential phase), 60 h (later exponential phase), and 84 h
(stationary phase), respectively. After the centrifugation at 13,000 g
for 10 min, the supernatant was taken and filtered. 200 μL of
supernatant was lyophilized under low temperature under low temperature
(− 60 °C). The dried samples were derivatized at 40 °C for 160 min with
50 μL methoxamine hydrochloride (20 mg/mL in pyridine) and 80 μL
N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA). Derivatized
samples were subsequently analyzed using a TRACE 1310 GC system (Thermo
Fisher Scientific, USA) combined with ISQ 7000 MS system (Thermo Fisher
Scientific, USA), which was equipped with a fused-silica capillary
column (30 m × 0.25 mm i.d., 0.25 μm DB-5MS stationary phase, J&W
Scientific, Folsom, CA). Four replicates were performed for each
sample.
Transcriptome analysis
Cells of B. methylotrophicum cultivated in modified DSM 135 medium
with/without 20 mM of lysine was sampled at the middle exponential
phase, respectively, for transcriptome analysis. Total RNA was
extracted using TRIzol^® Reagent according to the manufacturer’s
instructions (Invitrogen). The genomic DNA was removed using DNase I
(TaKaRa). RNA quality was then determined with a 2100 Bioanalyzer
(Agilent) and quantified with an ND-2000 (NanoDrop Technologies). An
RNA-seq transcriptome library was prepared following instructions in
the TruSeq™ RNA sample preparation Kit from Illumina (San Diego, CA).
The data generated from the Illumina platform were used for
bioinformatics analysis. All of the analyses were performed using the
free online Majorbio Cloud Platform ([165]www.majorbio.com) from
Shanghai Majorbio Bio-pharm Technology Co., Ltd.
RNA isolation and reverse transcription quantitative PCR
B. methylotrophicum was cultivated in modified DSM 135 medium with the
addition of 100 mM methanol or 100 mM methanol and 20 mM lysine. Cells
at the exponential growth stage were harvested by centrifugation
(4000 × g, 30 min), washed three times using ultra-pure water, and
immediately frozen in liquid nitrogen. The total RNA was purified with
the RNAprep Pure Cell/Bacteria kit (Tiangen Biotech, China), and the
cDNA was obtained by reverse transcription of the total RNA and used as
the template for quantitative real-time PCR (qPCR).
The qPCR was performed with a Light Cycler Instrument (Thermo Fisher
Scientific, USA). The PCR reaction was performed with SYBR Premix Ex
Taq (Takara) and the primers for NikABCDE and NikABCDE are listed in
Additional file [166]1: Table S1.
Plasmid construction and transformation
All plasmids used in this work are shown in Table [167]1. The genes
lysA, dapA, and dapB were amplified from the genome of B.
methylotrophicum using the primer pairs of lysA-F/lysA-R and
dapAB-F/dapAB-R, respectively, and then ligated into the plasmid pXY3
(linearized by the BamHI and XbaI) through the homologous recombination
obtaining the plasmid pXY3-P[thl]-lysA and pXY3-P[thl]-dapAB. To
overexpress the genes related to the ion channels, the operon of
NikABCDE and FhuBCD were amplified from the genome of B.
methylotrophicum and ligated into the plasmid pXY3 (linearized by the
BamHI and XbaI) by homologous recombination, respectively. The primers
used in the recombinant plasmid construction are shown in Additional
file [168]1: Table S1. Before transformation into B. methylotrophicum,
plasmids were transformed into E. coli Top10 containing methylation
plasmid pMCljS for in vivo methylation. Subsequently, the methylated
plasmids were extracted from E. coli and electro-transformed into B.
methylotrophicum.
Analytical techniques
During the fermentation, the cells were harvested by centrifugation at
12,000 rpm for 5 min and were resuspended with deionized water for the
measurement of optical density at 600 nm with a UV–VIS
spectrophotometer. The liquid supernatant of fermentation broth was
used to measure the concentration of methanol, acetate and butyric acid
by a high-performance liquid chromatography equipped with a refractive
index detector and a column (Bio-Rad HPX-87H). The column temperature
was 60 °C. The mobile phase was 8 mM H[2]SO[4] solution with a flow
rate was 0.6 mL/min. The concentration of lysine was analyzed by an
SBA-40C biosensor analyzer (Shandong Province Academy of Sciences,
China).
A SP-7890Plus gas chromatograph (Shandong Lunan Ruihong, Chinese)
equipped with a TCD detector was used to calculate the net CO[2]
consumption rate (n = 3). To calculate the bicarbonate consumption rate
by B. methylotrophicum, the headspace CO[2] concentration (V/V) was
measured by collecting headspace gas in different time periods. Then
using calculation Eq. ([169]1) to convert CO[2] concentration (mM). The
concentration conversion of sodium bicarbonate and CO[2] was carried
out as in Eq. [170]2:
[MATH:
CmM,CO2=100022.4
∗Cv,CO2∗273∗pa273+t<
/mrow>∗101.3, :MATH]
1
[MATH:
CmM,NaHCO3∗VNaHCO3=CmM,CO2∗VCO2. :MATH]
2
Supplementary Information
[171]13068_2023_2263_MOESM1_ESM.docx^ (318.9KB, docx)
Additional file 1. Fig. S1: The changes of amino acids in YE medium (A)
and CSL medium. B during the fermentation process of B.
methylotrophicum. Fig. S2: The acetic acid production of B.
methylotrophicum in medium supplemented with different concentrations
of lysine. Fig. S3: The lysine consumption of B. methylotrophicum in
medium supplemented with different concentrations of lysine. Fig. S4:
Pathway enrichment analysis of the significantly down-regulated genes
of B. methylotrophicum in response to lysine addition with the
corrected p-value < 0.05. Fig. S5: Transcriptional levels of the
NikABCDE and FhuBCD transporters in response to lysine in B.
methylotrophicum. Fig. S6: The effects of overexpressing lysA or dapAB
operon on acetic acid production of B. methylotrophicum. Table S1: The
primers used in this study. Table S2: Significantly up-regulated genes
of B. methylotrophicum in response to lysine addition. Table S3:
Significantly down-regulated genes of B. methylotrophicum in response
to lysine addition.
Abbreviations
C1
One-carbon
CSL
Corn steep liquor
WLP
Wood–Ljungdahl pathway
Spe
Spectinomycin
Amp
Ampicillin
Em
Erythromycin
Gly
Glycine
Ala
Alanine
Lys
Lysine
Asn
Asparagine
Thr
Threonine
e Glu
Glutamate
Asp
Aspartate
Val
Valine
Ile
Isoleucine
Ser
Serine
Tyr
Tyrosine
Pro
Proline
Phe
Phenylalanine
YE
Yeast extract
DEG
Differentially expressed gene
MtaB
The corrinoid-dependent methyltransferase
MtaC
A corrinoid protein
MtaA
Methyltetrahydrofolate-methyltransferase
THF
Tetrahydrofolate
CODH
Carbon monoxide dehydrogenase
ACS
Acetyl-CoA synthase
Author contributions
WJ and WX designed the research; WJ performed the experiments; WJ, LY,
QJL, MC and JYQ analyzed the data; WJ and WX wrote and revised the
manuscript, and JZ, JL and GD supervised the project. All the authors
read and approved the finally manuscript.
Funding
This work was supported by the National Key Research and Development
Program of China (2018YFA0901500), the Jiangsu Synergetic Innovation
Center for Advanced Bio-Manufacture (XTD2218).
Availability of data and materials
The datasets used and analyzed during the current study are available
from the corresponding author on reasonable request.
Declarations
Ethical approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
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References