Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Found 1 result for A severe leakage of intermediates to shunt products in acarbose biosynthesis
Filters applied. Clear filters
. 2020 Mar 19;11(1):1468.
doi: 10.1038/s41467-020-15234-8.

A severe leakage of intermediates to shunt products in acarbose biosynthesis

Qinqin Zhao  1   2 , Yuchang Luo  1   2 , Xin Zhang  1   2 , Qianjin Kang  1   2 , Dan Zhang  1   2 , Lili Zhang  3 , Linquan Bai  4   5   6 , Zixin Deng  1   2
Affiliations

A severe leakage of intermediates to shunt products in acarbose biosynthesis

Qinqin Zhao et al. Nat Commun. .

Abstract

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 C7-cyclitol moiety and highlights the importance of assessing shunt product accumulation when seeking to improve the titer of microbial pharmaceutical products.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The biosynthesis of acarbose in Actinoplanes species.
a The biosynthetic gene cluster of acarbose (1) (acb cluster, GenBank accession no. Y18523.4). b The biosynthetic pathway to 1, including previously determined biosynthetic steps (blue arrow), possible conversion without experimental confirmation (gray dashed arrow), previously proposed biosynthetic steps (gray arrow), confirmed biosynthetic steps in this work (green arrow), and update of the previously proposed biosynthetic steps according to this work (purple arrow). The biosynthetic pathways to the C7-cyclitol moiety, the amino-deoxyhexose moiety, and the shunt products are highlighted in blue, yellow, and plum, respectively, and the previously proposed biosynthetic pathway to the C7-cyclitol moiety is highlighted in gray.
Fig. 2
Fig. 2. Discovery and identification of shunt products accumulated in the fermentation broth of Actinoplanes sp. SE50/110.
a HPLC profiles of the parent strain Actinoplanes sp. SE50/110 (abbreviated as SE50/110), the ∆acb mutant QQ-3 and the complemented mutant QQ-3::pLQ666. b Structures of 1-epi-valienol (8) and valienol (9). c HPLC-QQQ/MS analysis of 1 after feeding with 3 (as positive control), 8 or 9 to the ∆acbC mutant QQ-4, and fermentation of QQ-4 without feeding was set as negative control. The standard (abbreviated as std) of 1 was also analyzed. Source data underlying Fig. 2c are provided as a Source Data file.
Fig. 3
Fig. 3. Identification of hydrolases involved in the dephosphorylation of 10 and 11.
a, b HPLC-TOF/MS analysis of the dephosphorylated products of 10 and 11 catalyzed by AcbJ, ACPL_8310, ACPL_2834, or ACPL_7709 in vitro, and the reaction without enzyme was set as a negative control. The standards (std) of 8 and 9 were also analyzed. All of the chromatograms show the simultaneous extraction of calculated ions m/z = 211.0379 [M + Cl]− for 8 and 9 and m/z = 255.0275 [M-H]− for 10 and 11. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Further clarification of the C7-cyclitol moiety biosynthetic pathway by characterizing AcbL- and AcbN-catalyzed conversions.
a HPLC-TOF/MS analysis of the standard (std) of 12 and the reaction products of AcbL with 4, boiled AcbL with 4, AcbN with 12, boiled AcbN with 12, AcbN with 11, and boiled AcbN with 11. All the chromatograms show the extraction of corresponding calculated ions. b GC-QMS confirmation of the reaction products after dephosphorylation by AcbJ. AcbJ was added to the reaction products of AcbL & 4, boiled AcbL & 4, AcbN & 12, boiled AcbN & 12, AcbN & 11, and boiled AcbN & 11. The standards (std) 13, 8, and 9 were also analyzed by GC-QMS after derivatization by BSTFA. All the chromatograms show the extraction of corresponding unique product ions. TMS is the abbreviation of trimethyl silicyl. For more details, see also Supplementary Figs. 11, 23. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. Elucidation of the biosynthetic pathway to the shunt product 8.
a HPLC-TOF/MS analysis of the reduction products of 12 by the crude cell-free extract of Actinoplanes sp. SE50/110 (abbreviated as SE50/110), SE50/110ΔacbN and QQ-3, purified ACPL_7966, ACPL_7511 and ACPL_5736 proteins, and the reaction with boiled crude cell-free extract (BCF) was set as a negative control. All the chromatograms show the extraction of calculated ion m/z = 255.0275 [M-H]−. b GC-QMS confirmation of the configuration of the reaction products after dephosphorylation by AcbJ. AcbJ was added to the reaction products of crude cell-free extract of Actinoplanes sp. SE50/110, SE50/110ΔacbN, and QQ-3, purified ACPL_7966, ACPL_7511, ACPL_5736 proteins, and control reaction. The standards (std) 8 and 9 were also analyzed by GC-QMS after derivatization by BSTFA. For more details, see also Supplementary Fig. 11. All the chromatograms show the extraction of unique product ion m/z = 332.2. TMS is the abbreviation of trimethyl silicyl. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Combined metablic engineering strategies for minimizing the flux toward the shunt products and maximizing the supply of the amino-deoxyhexose moiety.
a Schematic illustration of the strategies for diverting the metabolic flux of shunt products toward 1. b, c The titers of 1, 8, and 9 of Actinoplanes sp. SE50/110 (abbreviated as SE50/110), QQ-8 (down-regulation of the acbJ expression by introducing kasOp*-acbJ in the chromosome of QQ-7), QQ-9 (deletion of ACPL_7966 in QQ-8), QQ-10 (deletion of ACPL_7511 in QQ-9), QQ-11 (deletion of ACPL_5736 in QQ-10), QQ-12 (deletion of ACPL_7667 in QQ-11), QQ-12::pSET152 (as control), and QQ-12::AN (overexpression of acbN in QQ-12) after fermentation for 4 days. For more details, see also Supplementary Figs. 42–44. d Schematic illustration of increasing the biosynthetic capacity of amino-deoxyhexose moiety by stepwise optimization of biosynthetic reactions at gene dosage or enzymatic activity. e, f The titers of 1, 8, and 9 of mutants carrying pSET152 (as control) and its derived plasmids containing cassettes AP, AB, AP-AB1, AP-AB2, AP-AB3, AP-AB4, AP-AB5, AP-MB, AP-PB, AP-EB, AP-MB-AA, AP-MB-MA, AP-MB-PA, or AP-MB-EA after fermentation for 4 days. Two-tailed paired t tests. Error bars, mean ± SD (n = 3 biological replicates). For more details, see also Supplementary Figs. 45, 46. Source data underlying Figs. 6b, c, e, f are provided as a Source Data file.
Fig. 7
Fig. 7. Integration of the effective engineering strategies.
a, b The titers of 1, 8, and 9 of SE50/110::pSET152 (as a control), QQ-12::pSET152 (as a control), and QQ-12::AP-MB-EA and QQ-12::AP-MB-EA-AN after fermentation for 4 days. Two-tailed paired t tests. c, d Fed-batch fermentation of SE50/110::pSET152 (rhombus) and QQ-12::AP-MB-EA-AN (circle). Time courses of 1 (orange), 8 (gray), and 9 (green) titers were monitored. Error bars, mean ± SD (n = 3 biological replicates). For more details, see also Supplementary Fig. 47. Source data are provided as a Source Data file.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wehmeier UF, Piepersberg W. Biotechnology and molecular biology of the α-glucosidase inhibitor acarbose. Appl. Microbiol. Biotechnol. 2004;63:613–625. doi: 10.1007/s00253-003-1477-2. - DOI - PubMed
    1. Asamizu S. Biosynthesis of nitrogen-containing natural products, C7N aminocyclitols and bis-indoles, from actinomycetes. J. Agric. Chem. Soc. Jpn. 2017;81:871–881. - PubMed
    1. Ahr HJ, et al. Pharmacokinetics of acarbose. Part I: absorption, concentration in plasma, metabolism and excretion after single administration of [14C]acarbose to rats, dogs and man. Arzneimittelforschung. 1989;39:1254–1260. - PubMed
    1. Juergen, B., Michael, S. & Ulrich, S. Osmotically controlled fermentation process for the preparation of acarbose. US patent 6,130,072 (2000).
    1. Feng ZH, Wang YS, Zheng YG. A new microtiter plate-based screening method for microorganisms producing α-amylase inhibitors. Biotechnol. Bioproc. E. 2011;16:894–900. doi: 10.1007/s12257-011-0033-7. - DOI

Publication types

开云球赛中国有限公司 | 万搏官方网站 | c7官网登录入口 | 九州平台-九州(中国) | 万搏官方网站 | 千亿球友会·(中国)官方网站 | 米兰体育网址 | 开云手机官方网站 | 江南手机网页版登录入口 |