Further improvement of erythromycin production in E3 by
engineering the molecular targets
Specific genes functioning to supply precursors for erythromycin were
chosen as targets in the present study for metabolic engineering to
further enhance the production of erythromycin by the overproducer E3.
2-oxoglutarate was considered one of the reporter metabolites identified
by transcriptional analysis between E3 and NRRL23338 (Fig. 4).
Additionally, nonsynonymous or intergenic variations were also found in
genes (icd , sucA and sucB ) related to
2-oxoglutarate metabolism (Table S3). Several genes surrounding
2-oxoglutarate, i.e., icd , sucA and sucB , were
stimulated in E3 with respect to NRRL23338 (Fig. 7a). As such, the
metabolic flux from isocitrate to succinyl-CoA was supposed to be
increased in E3. Although this still requires metabolomic data to
support, metabolic engineering regarding 2-oxoglutarate was worthwhile.
Metabolic manipulations upon pathways around 2-oxoglutarate were
performed by straightforwardly overexpressing icd , sucAand/or sucB in E3 (Fig. 7b). The overexpression of icd in
E3 decreased the production of erythromycin by 45% and the seperate
overexpression of sucA or sucB exhibited no significant
effects on the production. Inspiringly,the simultaneous overexpression
of sucB and sucA boosted the erythromycin titer by 40% in
shake flask culture. Based on the result, bacth experiments were
porformed in 5-L bioreactors with minimum essential medium (Fig. 8).
Growth curves revealed that overexpression of sucBA led to a significant
delaying on growth. However, the growth of E3::sucBA expedited with a
faster rate after 36 h, and reached into the stationary phase at a
similar time to E3. Similar trends were observed on the carbon dioxide
evolution rate (CER) too. Fig.8c reveals that a rapid increase in CER
occurred with both strains, with a lag of approximately 12h. After 60 h
when both strains exhibited CER peaks, the CER of E3::sucBA droped
rapidly, and maintained at lower levels than E3 during the stational
phase. The change of CER indicated that the combinational expression of
sucB and sucA accelerate the metabolic transition between exponential
phase and stational phase of S. erythraea . The erythromycin titer
of E3::sucBA was increased by 71% compated to E3, reaching to 967 mg/L.
In consideration of glucose consumption rates, the overexpression of
sucB and sucA led to a higher yield of erythromycin of E3::sucBA
compared to E3.
The overexpression of icd is expected to enhance the
intracellular 2-oxoglutarate pool
generated from isocitrate . 2-oxoglutarate plays a role of indicating
the cellular nitrogen status, and the anticipated larger 2-oxoglutarate
pool in E3::icd relative to E3 signaled nitrogen deficiency that would
lead to a stimulated nitrogen metabolism (Radchenko, Thornton, &
Merrick, 2013), which exerts negative effects on the biosynthesis of
erythromycin (Liao et al., 2015; Zhang et al., 2014). However, the
overexpression of sucBA could avoid the accumulation of
2-oxoglutarate. 2-oxoglutarate dehydrogenase contributes to the
degeneration of 2-oxoglutarate to succinyl-CoA. Via the catalysis by the
methylmalonyl-CoA mutase, succinyl-CoA is then converted to
methylmalonyl-CoA and subsequently to the biosynthesis of erythromycin.
The anticipated increase of CoA-related precursors for erythromycin and
decrease of 2-oxoglutarate for nitrogen metabolism might account for the
titer enhancement and growth retardation by sucBA overexpression,
respectively. It suggests that the overexpression of sucBAeffectively reproduced the evolution between the wild type NRRL23338 and
industrial overproducer E3.