Phylogeny‐guided characterization of glycosyltransferases for epothilone glycosylation

Zhang, Peng; Zheng, Zhen; Li, Zhi‐feng; Chen, Qi; Li, Yao‐yao; Gong, Ya; X, Yue; Sheng, Duohong; Zhang, You‐ming; Wu, Changsheng; Li, Yue‐zhong

doi:10.1111/1751-7915.13421

Cited by 14 publications

(18 citation statements)

References 43 publications

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“…1 H and 13 C-NMR data assigned 1a as midecamycin 2′- O -glucopyranoside ( Table 1 , Figures S2–S6 ), suggesting OleD preferred to attack 2′-OH in mycaminosyl moiety of midecamycin. In addition, OleD was able to glucosylate 2′-OH of oleandomycin [ 25 , 26 , 27 , 28 , 38 , 39 , 40 ]. These facts collectively indicated that 2′-OH might be the preferred site of glycosylation for macrolides.…”

Section: Resultsmentioning

confidence: 99%

“…Diverse macrolide-inactivating GTs had been isolated and functionally identified. The exemplifying macrolide-inactivating GTs were OleI and OleD from S. antibioticus [ 31 ], MGT from S. lividans [ 25 ], BaGT from Bacillus atrophaeus , BamGT from B. amyloliquefaciens , BcGT-1 from B.cereus , BgGT from B. glycinifermentans , BpGT from B. paralicheniformis , as well as BsGT-1 and BssGT from B. subtilis [ 38 , 39 ]. These GTs have been demonstrated to be acceptor promiscuous, recognizing multiple macrolides.…”

Section: Introductionmentioning

confidence: 99%

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Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Hong

Jiang

et al. 2021

IJMS

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Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2′-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2′-O-glucopyranoside, midecamycin 2′-O-GlcNAc and midecamycin 2′-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2′-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Hong

Jiang

et al. 2021

IJMS

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“…Bacillus subtilis SCK6 was used as the heterologous expression host. 21 Bacillus sp. LCB12 was previously isolated from alkaline soil.…”

Section: Bacterial Strains Plasmids and Mediamentioning

confidence: 99%

Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis

et al. 2019

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Keratinase has a great commercial value owing to its applications in the enzymatic dehairing of goatskins. In this study, we adopted a combined strategy to enhance the extracellular recombinant keratinase activity in Bacillus subtilis SCK6. First, nine signal peptides were screened to enhance the expression of extracellular keratinase. The recombinant strain with SP LipA exhibited the highest extracellular keratinase activity of 739.03 U per mL, which was two-fold higher activity of the wild type. Second, based on the multiple sequence alignment with the bacterial alkaline proteases, the mutant (M123L/V149I/A242N) was introduced into the keratinase. Comparing with the wild type of keratinase, the mutant M123L/V149I/ A242N showed an increase in the extracellular keratinase activity, which was about 1.2-fold higher activity of the wild type. Finally, the keratinase expression vector with SP LipA and mutant M123L/V149I/ A242N was constructed, and the extracellular keratinase activity reported at 830.91 U per mL was a 2.2fold activity of the wild type. Then, the mutant keratinase was purified and characterized. The mutant exhibited properties similar to those of the wild type at an optimal temperature of 60 C and pH 10.0.Conclusively, the extracellular expression of keratinase was enhanced via a combined strategy, and the mutant keratinase demonstrated properties similar to that of the wild type of keratinase. Fig. 3 Protein sequence alignment of keratinase and other proteases by ESPript 3.0. "a" represents a helix; "b" represents b sheet; the substitution sites (M123, V149 and A242) are indicated by "+". 33340 | RSC Adv., 2019,9,[33337][33338][33339][33340][33341][33342][33343][33344] This journal is

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“…[28] The recombination fragment was obtained by overlap-extension PCR and transformed into B. subtilis strains by a previously described method. [29] The following antibiotics were used for selection: kanamycin (30 μg mL −1 ) and zeocin (30 μg mL −1 ). The positive clones were identified by PCR amplification by primers AlsSDLF and AlsSDRR and further confirmed by sequence analysis.…”

Section: Gene Deletionmentioning

confidence: 99%

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

Zhao

Tian

Shen

et al. 2019

Biotechnology Journal

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N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food‐grade NeuAc production via whole‐cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N‐acetylglucosamine 2‐epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L−1 NeuAc production with a molar conversion rate of 26.55% from N‐acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L−1, which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N‐terminal coding sequences of genes with high expression levels are fused to the 5′‐end of the ShNanA gene, resulting in 56.82 g L−1 NeuAc production. Finally, formation of the by‐product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L−1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole‐cell biocatalyst for efficient NeuAc production.

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Phylogeny‐guided characterization of glycosyltransferases for epothilone glycosylation

Cited by 14 publications

References 43 publications

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site

Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis

Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

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