Unexpected enzyme-catalysed [4+2] cycloaddition and rearrangement in polyether antibiotic biosynthesis

Rory Little, Fernanda C. R. Paiva, Robert Jenkins, HuiHong, Yuhui Sun, Yuliya Demydchuk, MarkiyanSamborskyy, Manuela Tosin, Finian J. Leeper, Marcio V. B. Dias, and Peter F.Leadlay*

Nature Catalysis 2019, 2:1045-1054

Epub Date: 14 October 2019

DOI: 10.1038/s41929-019-0351-2


Enzymes that catalyse remarkable Diels–Alder-like [4+2] cyclizationshave been previously implicated in the biosynthesis of spirotetronate andspirotetramate antibiotics. Biosynthesis of the polyether antibiotic tetronasinis not expected to require such steps, yet the tetronasin gene cluster encodesenzymes Tsn11 and Tsn15, which are homologous to authentic [4+2] cyclases. Here,we show that deletion of Tsn11 led to accumulation of a late-stageintermediate, in which the two central rings of tetronasin and four of itstwelve asymmetric centres remain unformed. In vitro reconstitution showed thatTsn11 catalyses an apparent inverse-electron-demand hetero-Diels–Alder-like [4+2] cyclization ofthis species to form an unexpected oxadecalin compound that is then rearrangedby Tsn15 to form tetronasin. To gain structural and mechanistic insight intothe activity of Tsn15, the crystal structure of a Tsn15-substrate complex hasbeen solved at 1.7 A resolution.

In vitro packaging mediated one-step targeted cloning of natural product pathway

Weixin Tao*, Li Chen, Chunhua Zhao, Jing Wu, DazhongYan, Zixin Deng, and Yuhui Sun*

ACS Synthetic Biology 2019, 8(9):1991-1997

Epub Date: 11 September 2019

DOI: 10.1021/acssynbio.9b00248


Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways inconjunction with various gene editing systems. This approach features an expedited, convenient and generic manner, and it is conceivable it may be widely adopted for targeted cloning of the natural product pathways.

Base editing in Streptomyces with Cas9-deaminase fusions

Zhiyu Zhong#, Junhong Guo#, Liang Deng, Li Chen, Jian Wang, Sicong Li,Wei Xu, Zixin Deng, and Yuhui Sun*


Epub Date: 7 May 2019

DOI: 10.1101/630137


Conventional CRISPR/Cas genetic manipulation has beenprofitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desiredrecombinants. We have adapted for Streptomyces recently-introduced cytidinebaseeditors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, by passing DSB and the need for arepair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defectiveCas9-guided base editors and up to 100% by using nicked Cas9-guided baseeditors. Furthermore, we demonstrate the multiplex genome editing potential of thenicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improvedediting specificity. Collectively, this work provides a powerful new tool for genomeediting in Streptomyces.


Research advances in aminoglycoside biosynthesis


Sicong Li and Yuhui Sun*

中国抗生素杂志 2019, 44(11):1261-1274

Chinese Journal of Antibiotics 2019, 44(11):1261-1274 (Chinese)

Published online: 25 November 2019



As a kind of first-line antibiotics clinically used for treating bacterial infections, aminoglycosides have greatly contributed to human health against pathogenic microbes and its brilliance has also been realized. Despite its side effects such as ototoxicity and nephrotoxicity and growing challenges of drug resistance, aminoglycosides are still an indispensable and important member of the treasure house of medicine, since many new bioactivities of aminoglycosides that have not been known are constantly enriching and expanding their new potential with the development of science and technology and understanding. In this review, the research progress in genetics, chemical biology, and structural biology of natural and semi-synthesized aminoglycoside are summarized and briefly discussed.