Comparative RNA-sequencing of the acarbose producer Actinoplanes sp. SE50/110 cultivated in different growth media

Schwientek, Patrick; Wendler, Sergej; Neshat, Armin; Eirich, Christina; Rückert, Christian; Klein, Andreas; Wehmeier, Udo F.; Kalinowski, Jörn; Stoye, Jens; Pühler, Alfred

doi:10.1016/j.jbiotec.2012.10.019

Cited by 22 publications

(34 citation statements)

References 56 publications

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“…Through purification and structural elucidation by nuclear magnetic resonance (NMR) spectroscopy, 1-epi-valienol (8) and valienol (9) were confirmed to be present in P-1 (Supplementary Figs. 1-10; Supplementary Table 2; Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, the low transcription of acbB seemed to be a general phenomenon, which was also found in the transcriptome analysis of Actinoplanes sp. SE50/110 cultured in different fermentation media 9 . In our work, two synthetic biology strategies were performed to adjust the activity of 4,6-DH: (1) balancing the transcription levels of acbB and other genes in the acb cluster by titrating gene copy numbers; (2) improving the 4,6-DH activity by recruiting heterologous enzymes with higher catalytic activities from M. smegmatis, E. coli, or P. aeruginosa [28][29][30] .…”

Section: Discussionmentioning

confidence: 99%

“…The derivatization reagent BSTFA:TMCS (99:1) was purchased from Sigma-Aldrich. The standards of valienol (9) (Supplementary Fig. 48a) and valienone (13) were synthesized by WuXi Apptec Co., Ltd.…”

Section: Methodsmentioning

confidence: 99%

“…1b). Furthermore, the product was purified and confirmed as (9). c HPLC-QQQ/MS analysis of 1 after feeding with 3 (as positive control), 8 or 9 to the ΔacbC mutant QQ-4, and fermentation of QQ-4 without feeding was set as negative control.…”

Section: Articlementioning

confidence: 99%

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A severe leakage of intermediates to shunt products in acarbose biosynthesis

et al. 2020

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The α-glucosidase inhibitor acarbose, produced by Actinoplanes sp. SE50/110, is a well-known drug for the treatment of type 2 diabetes mellitus. However, the largely unexplored biosynthetic mechanism of this compound has impeded further titer improvement. Herein, we uncover that 1-epi-valienol and valienol, accumulated in the fermentation broth at a strikingly high molar ratio to acarbose, are shunt products that are not directly involved in acarbose biosynthesis. Additionally, we find that inefficient biosynthesis of the amino-deoxyhexose moiety plays a role in the formation of these shunt products. Therefore, strategies to minimize the flux to the shunt products and to maximize the supply of the amino-deoxyhexose moiety are implemented, which increase the acarbose titer by 1.2-fold to 7.4 g L −1 . This work provides insights into the biosynthesis of the C 7 -cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Articlementioning

confidence: 99%

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A severe leakage of intermediates to shunt products in acarbose biosynthesis

et al. 2020

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“…Next, 200 µL of the supernatant was mixed with 800 µL methanol by vortexing and centrifuged again (20,000 g, 2 min) to remove precipitate. Afterwards, the samples were transferred to HPLC vials and analyzed in an Agilent 1100 HPLC system (G1312A Binary Pump, G1329A [90], resulting in 90.3% to 98.9% mapped reads. The coverage of each annotated feature (protein-coding genes and RNA genes) was determined using featureCounts [91].…”

Section: Acarbose Quanti Cation Using Hplc Measurementmentioning

confidence: 99%

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

Droste

Ortseifen

Schaffert

et al. 2020

Preprint

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Background: Actinoplanes sp. SE50/110 is the natural producer of the diabetes mellitus drug acarbose, which is highly produced during the growth phase and ceases during the stationary phase. In previous works, the growth-dependency of acarbose formation was assumed to be caused by a decreasing transcription of the acarbose biosynthesis genes during transition and stationary growth phase.Results: In this study, transcriptomic data using RNA-seq and state-of-the-art proteomic data from seven time points of controlled bioreactor cultivations were used to analyze expression dynamics during growth of Actinoplanes sp. SE50/110. A hierarchical cluster analysis revealed co-regulated genes, which display similar transcription dynamics over the cultivation time. Aside from the expected metabolic switch from primary to secondary metabolism during transition phase, we observed a significantly decreasing transcript abundance of all acarbose biosynthetic genes, with the strongest decrease for the monocistronically transcribed genes acbA, acbB, acbD and acbE. Our data confirm a similar trend for acb gene transcription and acarbose formation rate.Surprisingly, the proteome dynamics does not follow the respective transcription for all acb genes. This suggests different protein stabilities or post-transcriptional regulation of the Acb proteins, which in turn could indicate bottlenecks in the acarbose biosynthesis. Furthermore, several genes are co-expressed with the acb gene cluster over the course of the cultivation, including eleven transcriptional regulators (e.g. ACSP50_0424), two sigma factors (ACSP50_0644, ACSP50_6006) and further genes, which have not previously been in focus of acarbose research in Actinoplanes sp. SE50/110.Conclusion: In conclusion, we have demonstrated, that a genome wide transcriptome and proteome analysis in a high temporal resolution is well suited to study the acarbose biosynthesis and the transcriptional and post-transcriptional regulation thereof.

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Analysis of the effects of different nitrogen sources and calcium on the production of amphotericin by Streptomyces nodosus based on comparative transcriptome

Huang

Zhang

Chen

et al. 2021

Biotech and App Biochem

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Streptomyces nodosus is known as the main manufacturer of amphotericin B (AmB), which is an effective antifungal drug. It is verified that the optimization of fermentation conditions and key growth factors have a great impact on the yield of AmB. The AmB production of S. nodosus in cotton-seed meal (CM) medium was 1.6 times than that of beef-paste medium. The transcriptome analysis was used to analyze the effects of different nitrogen media and calcium on S. nodosus. Related genes of the EMP and TCA pathways, such as phosphofructokinase, pyruvate dehydrogenase, and citrate synthase, were upregulated in CM medium. The expression level of the PKS modules of the amphotericin synthesis gene cluster in beef-paste medium was higher. Other functional genes, such as amphGH and amphRIV, have the advantage of expressing in CM medium. Ca 2+ promoted the upregulation of genes in metabolic pathways such as EMP pathway (pyruvate dehydrogenase), TCA pathway (citrate synthase), and amphotericin synthesis genes (PKS modules). The expression of WhiB family genes SNOD_RS 13310 and SNOD_RS 17625 was positively correlated with Ca 2+ concentration. In addition, in the presence of calcium, the expression level of Sec transport system genes of S. nodosus was lower.

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Comparative RNA-sequencing of the acarbose producer Actinoplanes sp. SE50/110 cultivated in different growth media

Cited by 22 publications

References 56 publications

A severe leakage of intermediates to shunt products in acarbose biosynthesis

A severe leakage of intermediates to shunt products in acarbose biosynthesis

The expression of the acarbose biosynthesis gene cluster in Actinoplanes sp. SE50/110 is dependent on the growth phase

Analysis of the effects of different nitrogen sources and calcium on the production of amphotericin by Streptomyces nodosus based on comparative transcriptome

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