Transcriptional changes regarding specific genes with genomic
variations
Interestingly, most of the doubled genes in Table S1 exhibited
relatively low transcriptional levels both in NRRL23338 and E3 (Table
3). As an exception, SACE_0337 was expressed 4 to 6 times higher in the
exponential phase in E3 compared to NRRL23338. Since in principle the
accumulation of acetyl-CoA benefits the formation of precursors of
erythromycin through the TCA cycle, the upregulation of SACE_0337 in E3
emphasized the importance of synthesis of acetyl-CoA for erythromycin
production.
Apart from CDSs with large-fragment deletions (Table S2), 214 CDSs
showed extremely low transcriptional levels close to zero, both in
exponential and stationary phase (Table S6). 25 gene clusters inS. erythraea genome were responsible for biosynthesis of
secondary metabolites (Oliynyk et al., 2007). However, we observed
low-transcribed genes in 9 out of the 25 clusters, including rpp ,nrps1 , nrps4 , nrps7 , pks1 , pks2 ,pks3 , pks4 and hop clusters in E3 (Table S6).
Particularly in the rpp cluster, that is responsible for the
reddish pigment synthesis, several genes coding for acetyl-CoA
carboxylase were transcribed lowly. This transcriptional repression ofacc cluster along with the intergenic SNP around SACE_3400 turn
less acetyl-CoA into malonyl-CoA and finally reduce the biosynthesis of
the reddish pigment in E3 (Cortés et al., 2002). This was in line with
the reduced production of the reddish pigment by E3. Therefore, it
appeared that biosynthesis of secondary products other than erythromycin
in E3 was more likely to decrease rather than to disappear. Secondary
products, particularly Pks-related products, are assembled from some
common starter units (Yuzawa, Keasling, & Katz, 2017). The low
transcriptions of several pks clusters manifested themselves as
engineering targets to enhance the production of erythromycin, as their
synthesis may competent with erythromycin for precursors. Furthermore,
low transcriptional levels were also observed in several genes of acyl-,
methyl- or glycosyl-transferase. Biosynthesis of most secondary products
requires post-PKS modification, e.g. methylation or glycosylation of the
molecule backbone (Dhakal, Sohng, & Pandey, 2019). Thus, the
transferase genes with low transcription were likely associated with
modifications of the secondary products of the 9 clusters in Table S6.
Taken together, we observed the genome variation and the low
transcription of gene clusters responsible for biosynthesis of secondary
metabolites other than erythromycin, which addressed the importance of
construction of super-producer through genome minimization i.e., removal
of gene clusters associated with other secondary metabolites (Weber,
Charusanti, et al., 2015). Another 30 CDSs with low transcriptional
levels were associated with transport processes, such as the transport
of mannose, mannitol, arabinose, 1,4-digalacturonate or
C4-dicarboxylate. Accordingly, two more genes (SACE_0179/0186) encoding
arabinosyltransferases were also transcribed lowly. Transcriptional
repression in these transporter genes of E3 implied distinguished
preferences upon substrate, which corresponded to the repression on the
biosynthesis of erythromycin by mannose, mannitol or arabinose with
respect to glucose as the carbon source (El-Enshasy, Mohamed, Farid, &
El-Diwany, 2008). SACE_2955-2956 encodes the bd type terminal
oxidase of the electron transport chain (ETC), which exhibited a lower
energetic efficiency compared tobc1-aa3 complex (Scott, Salmon,
& Poole, 1992). The low transcription of SACE_2955-2956 implied that
alternative operons (SACE_0142-0143) encoding the bd complex
were operative or E3 preferred an alternative electron transport
channel, such as through bc1-aa3complex. The low transcription of SACE_3011, which encodes a copper
resistance gene, fitted the conclusion drawn in our recent research that
appropriate exogenous addition of copper ions would increase the
production of erythromycin in E3 (Qiao, Li, Ke, & Chu, 2020).
Transcriptional levels of genes with nonsense mutations were all
repressed significantly in E3, although their transcription in NRRL23338
were also pretty low (Table 4). Particularly, transcription of
SACE_0019/2875/3073/7243 were as low as they seemed to be deleted
completely in E3 genome. The results confirmed again that nonsense
mutations can be considered as gene deletion. It seemed that nonsense
mutations in SACE_1076 and SACE_1257 played ambiguous effects on
stopping expression by some unknown mechanisms. SACE_0019 encodes ano -succinylbenzoate-CoA ligase, which catalyzes a reaction
consuming ATP and coenzyme A. SACE_3073 encodes a hydrogenase
expression/formation protein HypD and SACE_7243 encodes a FAD-dependent
oxygenase. Therefore, nonsense mutations in SACE_0019/3073/7243
indicated that the intracellular redox status might be changed in E3.
Putative
mechanisms and promising molecular targets/strategies by which E3
enhances its biosynthesis of erythromycin
With the comparative omics analysis of S. erythraea , it is
possible to propose putative mechanisms by which E3 enhances its
production of erythromycin. In other words, these mechanisms provide
promising molecular targets to engineer for the further enhancement of
erythromycin production. Here, we concluded the cellular change of E3
compared to NRRL23338 at three aspects related to [a] intracellular
biochemical environment, [b] signal transduction along with
transcriptional regulation, and [c] the supply of precursors for
erythromycin (Fig. 6).
Firstly, it is very likely that E3 created an intracellular
pseudo-limited condition by genomic variations, which triggered the
onset of secondary metabolism much earlier compared to NRRL23338. For
instance, the transcriptional repression on glutamine/glutamate
metabolism in E3 could exert a nitrogen-starvation signal (Figure 4 &
Table S4), which benefits the earlier onset of the biosynthesis of
erythromycin (Liao et al., 2015; Z. Xu et al., 2019). The extremely low
transcription of transporter genes and the transcriptional change of PTS
genes in E3 implied distinguished preferences of substrate utilization
in E3 (Figure 3 & Table S6), which probably resulted in that E3 tended
to have a different intracellular biochemical environment relative to
NRRL23338 (Liao et al., 2015). In addition, one pair of bd type
oxidase genes were lowly transcribed (Table S6), which can have a
significant effect on the intracellular energy level (Figure S4) or
redox status (Fischer, Falke, Naujoks, & Sawers, 2018). To engineer E3
for a better production of erythromycin, several strategies regarding
the intracellular biochemical environment are worth trying, e.g., to
repress the nitrogen metabolism by down-regulating the flux from
2-oxoglutarate to L-glutamate/glutamin, to optimize the components of
industrial medium, or to directly regulate the intracellular energy or
redox status. Based on the present mechanism, in our recent research we
improved the production of erythromycin in E3 28% by enhancing the
intracellular energy level (X. Li, Chen, Andersen, Chu, & Jensen, 2020)
and 60% by reducing the intracellular redox status with more oxygen
supply (X. Li, Chu, et al., 2020).
Secondly, E3 presented differently but unclearly altered regulatory
networks which also contributed to the stimulation of erythromycin BGC
transcription (Figure 5). E3 did not stimulate the transcription of the
direct activator BldD or PhoP of erythromycin BGC (Chng et al., 2008; Y.
Xu et al., 2019), which indicated a more complex regulatory network
influencing the transcription of erythromycin BGC in E3. Regulator genes
with significantly different transcriptional levels in Table S5 are
candidates which can affect the transcription of erythromycin BGC.
Besides, a two component system (TCS) regulating the copper homeostasis
in S. erythraea was identified in our recent study based on the
present comparative genomics (Qiao et al., 2020). Copper functioned as a
cofactor of bc1-aa3 terminal
oxidase (Fujimoto, Chijiwa, Nishiyama, Takano, & Ueda, 2016). The
addition of copper in the medium may increase the efficiency of ATP
synthesis and then benifits the biosynthesis of erythromycin. The
genomic mutation in this TCS gene i.e. SACE_0101 enhanced the copper
tolerance of E3 compared to NRRL23338. Gene deletion of SACE_0101 in
NRRL23338 increased its erythromycin titer by 110% and the addition of
copper enhanced the erythromycin titer of E3 by 17% (Qiao et al.,
2020).
Thirdly, the supply of precursors for erythromycin appeared higher in
E3. In general, the decreased biomass formation ensures that more carbon
flows to secondary metabolism (Figure 1). The stimulated transcription
of the ED pathway, enhancing the dehydrogenation of 2-oxoglutarate to
succinyl-CoA and the degradation of branched amino acids, increased the
supply of NADPH, propionyl-CoA and methylmalonyl-CoA, respectively, for
the synthesis of erythromycin (Figure S3). In addition, repressed
synthesis of other secondary metabolites in E3, such as pigment and
other polyketides, drains more carbon flux to the biosynthesis of
erythromycin (Table S6). The repressed synthesis of other secondary
metabolites was also observed in another erythromycin overproducer Px
(Peano et al., 2012).