Transcriptomic comparison of E3 and NRRL23338
Most physiological events originate at the transcriptional level. In
order to characterize the impacts of genomic variations on gene
expression profiles, both samples of NRRL23338 and E3 taken from the
minimal liquid medium (Hopwood, 1985) were subjected to the high
throughput RNAseq. Cell samples were harvested in exponential phase (10
h) and stationary phase (50 h) to extract RNA. 205 million reads or 107
million reads were generated for NRRL23338 or E3, respectively. After
mapping all the reads to the respective genome, the coverage showed that
the general transcription of core region was higher than that of the
noncore region both in NRRL23338 and E3 (Figure S2).
A total of 822 genes were identified to be differentially expressed in
the exponential phase. In order to obtain an overview of the general
expression difference, we conducted functional enrichment analysis based
on KEGG and GO annotations (Figure 3). KEGG enrichment analysis of
differentially expressed genes (DEGs) showed that most affected pathways
were [a] biosynthesis of 12-, 14- and 16-membered macrolides,
[b] synthesis of type I polyketides, [c] polyketide sugar unit
biosynthesis, [d] phosphotransferase system (PTS), [e]
sesquiterpenoid
and triterpenoid biosynthesis (Fig. 3a). The up-regulation of genes
involved in [a] biosynthesis of 12-, 14- and 16-membered macrolides,
[b] synthesis of type I polyketides, and [c] polyketide sugar
unit biosynthesis contribute directly to the biosynthesis of
erythromycin. The significant change in expression of PTS genes
suggested the unneglectable role of phosphate metabolism in regulating
erythromycin synthesis (Y. Xu, You, Yao, Chu, & Ye, 2019). On the
contrary, purine/pyrimidine metabolism, biosynthesis of
ribosome/peptidoglycan, translation and DNA replication/repair were
down-regulated significantly in E3. This was in accordance with the
repressed growth rates of E3. Besides the above pathways/functions, GO
enrichment analysis implied that in E3 activities of oxidoreductase and
NADH dehydrogenase were altered significantly (Fig. 3b), which hinted
alterations regarding the intracellular level of cofactors.
Next, a genome-scale metabolic model (GSMM) was used to identify
reporter metabolites, around which the most significant changes in
expression occurred (Licona-Cassani, Marcellin, Quek, Jacob, & Nielsen,
2012). L-glutamate/L-glutamine and
2-oxoglutarate
were observed among the high scoring reporter metabolites both in the
exponential and the stationary phase, along with some precursors of
erythromycin and molecules essential for electron transport (Figure 4).
The production onset of erythromycin in S. erythraea has proven
associated with nitrogen starvation (Zou, Hang, Chu, Zhuang, & Zhang,
2009a), while L-glutamate/L-glutamine and 2-oxoglutarate are tightly
interconnected with nitrogen metabolism. Therefore, results of the
reporter metabolites stressed the importance of nitrogen metabolism for
the production of erythromycin.
Hierarchical clustering of DEGs was conducted to show alterations
regarding specific subset of genes (Figure 5 & Table S4). It is
noteworthy that all genes in erythromycin BGC and genes related to the
biosynthesis of dTDP-sugar were drastically up-regulated in E3 in the
exponential phase. Transcription of erythromycin BGC depends on the
positive regulation by BldD and PhoP (Chng et al., 2008; Y. Xu et al.,
2019). However, transcription of neither bldD nor phoP was
stimulated in E3. This indicated that the regulatory networks affecting
the transcription of erythromycin BGC changed in E3.
Transcription of 40 genes encoding (putative) transcriptional regulators
were significantly changed in E3, 32 of which were up-regulated at both
sampling points (Table S5). The transcription of SACE_4906 in E3 was
stimulated most by 41.6 folds. The gene product of SACE_4906 comprises
a cyclic N-Methyl pyrrolidone (cNMP) binding domain and is predicted as
a gene involved in carbon catabolite repression. This implied that the
substrate uptake system in E3 may vary from that of NRRL23338. In
addition, two WhiB-like regulators encoded by SACE_5583 and SACE_6426
were stimulated significantly. In streptomycetes, WhiB-like regulators
usually affect stress response and sporulation processes, which are
interconnected with biosynthesis of secondary metabolites (Molle,
Palframan, Findlay, & Buttner, 2000; Zheng, Long, & Xie, 2012).
Although functions of the remaining transcriptional regulators are
unknown, this analysis still narrowed the range of transcriptional
regulator candidates, which probably have impact on the biosynthesis of
erythromycin.
Transcriptional divergence was observed for genes involved in glycolysis
and the TCA cycle (Figure S3).
Expression
of genes encoding the pyruvate dehydrogenase complex, which catalyzes
the key step from glycolysis to the TCA cycle, and pycresponsible for the reaction from pyruvate to oxaloacetate were
repressed. Genes encoding aconitate hydratase were down-regulated
slightly. Expression of icd , sucAB , korAB andsucCD were up-regulated, whereas genes coding for succinate
dehydrogenase and fumarate hydratase were repressed more significantly
in E3. Given the transcriptional up-regulation of mutAB , carbon
flux from isocitrate in E3 might flow into methylmalonyl-CoA metabolism
via 2-oxoglutarate and then succinyl-CoA rather than to the
dehydrogenation of succinate. Furthermore, gabT coding for the
enzyme to catalyze conversion from 2-oxoglutarate to L-glutamate was
down-regulated. Most of genes involved in the glutamate and glutamine
metabolism were repressed, which could exert a nitrogen-starvation
signal and triggered the biosynthesis commence of erythromycin (Z. Xu et
al., 2019). As mentioned, nonsynonymous mutations were also identified
in icd and sucAB (Table S3). Therefore, the genomic and
transcriptomic variations regarding reactions from isocitrate to
succinyl-CoA addressed the importance of these pathways in accumulating
precursors of erythromycin.
Expression of genes involved in propanoate metabolism were generally
much lower than those in TCA cycle both for NRRL23338 and E3 (Table S4),
which indicated an inactive propanoate metabolism because the
cultivation in the minimal medium had no propanol addition. However,mmsA and mmsB encoding enzymes to catalyze the degradation
of branched amino acids into propionyl-CoA were significantly
stimulated. This was beneficial for the biosynthesis of erythromycin in
E3 (Z. Xu et al., 2018). All genes coding for acetyl-CoA carboxylase
were repressed, and this implied that the lower accumulation of
malonyl-CoA led to the reduced production of pigment by E3.
Since the degradation of fatty acids provides NADPH and coenzyme A, the
up-regulation of genes related to the degradation of fatty acids
benefits the production of erythromycin (Karnicar et al., 2016).
SACE_3084/6363 encoding acetyl-CoA acyltransferase and SACE_5380/5383
encoding acyl-CoA dehydrogenase were up-regulated significantly in E3
(Table S4). Another important source of NADPH is the oxidative pentose
phosphate pathway (PPP), which partly connected with Entner-Doudoroff
(ED) pathway. It is noteworthy that all genes involved in ED pathway
were stimulated to different extents in E3. This expression change in
the ED pathway was consistent with a previous conclusion drawn by13C labeling experiments that the ED pathway was the
main glucose utilization pathway of E3 cultivated in minimal or
chemically defined meidum (Hong et al., 2016). Relative to
Embden-Meyerhof-Parnas pathway (EMP), the utilization of glucose
predominantly through ED pathway not only increased the supply of NADPH
for erythromycin production, but also decreased the generation of NADH,
that exerts a negative effect on expression of erythromycin BGC via
c-di-GMP (X. Li, Chu, & Jensen, 2020; Z. Xu et al., 2019). prsAwhich controls the conversion from PPP to purine/pyrimidine metabolism
was down-regulated notably. Genes (purF /purH /pyrC )
engaged in purine/pyrimidine metabolism, which is linked to the signal
transduction through secondary messengers such as (p)ppGpp and c-di-GMP
(Sivapragasam & Grove, 2019), were also transcriptionally
down-regulated in E3 starting from the exponential phase (Table S4).