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. 2017 Nov 27;2(4):302-309.
doi: 10.1016/j.synbio.2017.11.005. eCollection 2017 Dec.

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

Qinqin Zhao  1 , Huixin Xie  1 , Yao Peng  1 , Xinran Wang  1 , Linquan Bai  1
Affiliations

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp. SE50/110

Qinqin Zhao et al. Synth Syst Biotechnol. .

Abstract

The α-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp. and used as a potent drug in the treatment of type-2 diabetes. In order to improve the yield of acarbose, an efficient genetic manipulation system for Actinoplanes sp. was established. The conjugation system between E. coli carrying ØC31-derived integrative plasmids and the mycelia of Actinoplanes sp. SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture (mycelia and E. coli), quantity of recipient cells, donor-to-recipient ratio and the concentration of MgCl2, which resulted in a high conjugation efficiency of 29.4%. Using this integrative system, a cloned acarbose biosynthetic gene cluster was introduced into SE50/110, resulting in a 35% increase of acarbose titer from 2.35 to 3.18 g/L. Alternatively, a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm) was constructed for gene inactivation, which has a conjugation frequency as high as 0.52%. Meanwhile, almost all 5-flucytosine-resistant colonies were sensitive to apramycin, among which 75% harbored the successful deletion of targeted genes. Using this replicating vector, the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated, and component C was eliminated as detected by LC-MS. Based on an efficient genetic manipulation system, improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

Keywords: Acarbose; Actinoplanes sp.; Component C; Conjugation; Genetic manipulation.

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Figures

Fig. 1
Fig. 1
Acarbose and its biosynthetic gene cluster. a Structures of acarbose and related metabolites. b Acarbose biosynthetic gene cluster (acb cluster) of Actinoplanes sp. SE50/110 (accession: Y18523).
Fig. 2
Fig. 2
Introduction of an extra copy of acb cluster into SE50/110. a pLQ666 with whole acb cluster and cassette of int-attP-oriT-aac(3)IV from pSET152. b Acarbose production of QQ-1 (with an extra copy of acb cluster introduced by integration of pLQ666) and SE50/110::pSET152 (control strain with integrated pSET152). **, p < 0.05. c The transcription pattern of acbW, acbV, acbC, acbB, acbA in acb cluster of QQ-1 and SE50/110::pSET152 at 48 h during the fermentation process. The Y-axis scale represents the expression value of genes relative to that of hrdB. The average transcription of genes in SE50/110::pSET152 were set to 1 as standard, the transcription of genes in QQ-1 were accordingly calculated. Graphs depict means ± SD. Values represent average results from three independent experiments.
Fig. 3
Fig. 3
Elimination of component C by deletion of treY. a Schematic representation of the gene deletion of treY. b Confirmation of the mutant QQ-2 by PCR amplification. Using primers TV-F and TV-R, approximately a 0.70-kb fragment was amplified using the total DNA of QQ-2 or the recombinant plasmid pLQ756 as templates, whereas SE50/110 gave a 1.80-kb product. c HPLC profiles of SE50/110, treY mutant QQ-2, QQ-2::pLQ758 (complementation of treY gene in QQ-2) and SE50/110::pLQ758 (overexpression of treY gene in SE50/110). d Acarbose and component C production of SE50/110, QQ-2, QQ-2::pSET152 (control strain with the integration of pSET152 in QQ-2), QQ-2::pLQ758, SE50/110::pSET152 and SE50/110::pLQ758. Graphs depict means ± SD. Values represent average results from three independent experiments.
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