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

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.

A severe leakage of intermediates to shunt products in acarbose biosynthesis

“…In line with the results of previous feeding studies with 13 Clabeled 8 and 13, in which none of these compounds were incorporated into 1 19 , our work showed that both compounds are shunt products branched from the biosynthetic intermediate 12 ( Fig. 1b; Supplementary Fig.…”

“…SE50/110, and the mutant was named QQ-3. Both P-1 and 1 disappeared in the fermentation broth of mutant QQ-3, and were restored by trans-complementation of the acb cluster cloned on fosmid pLQ666 13 (Fig. 2a).…”

“…The standards of valienol (9) (Supplementary Fig. 48a) and valienone (13) were synthesized by WuXi Apptec Co., Ltd. The standard of 1-epi-valienol (8) was purified from the fermentation broth of SE50/110ΔacbN and confirmed by NMR spectroscopy ( Supplementary Fig.…”

“…Purified compounds were dissolved in D 2 O, and the one-dimensional (1D) ( 1 H, 13 C, and distortionless enhancement by polarization transfer) and 2D ( 1 H-1 H correlation spectroscopy [COSY], heteronuclear single-quantum correlation spectroscopy [HSQC], hetero-nuclear multiple-bond correlation spectroscopy [HMBC], and nuclear overhauser effect [NOESY]) NMR spectra were collected in D 2 O at 600 MHz ( 1 H NMR) and 150 MHz ( 13 C NMR) on Bruker Avance III 600 spectrometer (magnetic field strength with 14.09 T). The NMR data processing was performed using the software of MestReNova 9.0.1.…”

“…In addition, highly efficient genetic manipulation systems have been established for Actinoplanes sp. SE50/110 and are successfully used to delete melC2 (a tyrosinase gene) and treY (a maltooligosyltrehalose synthase gene), which eliminate the formation of eumelanin and the by-product component C, respectively [11][12][13] . These advances in the omics analysis and genetic toolbox development have paved the way to genetically engineer Actinoplanes sp.…”