Mechanistic insights into dideoxygenation in gentamicin biosynthesis

Sicong Li#, Priscila Dos Santos Bury#, Fanglu Huang#, Junhong Guo, Guo Sun, Anna Reva, Chuan Huang, Xinyun Jian, Yuan Li, Jiahai Zhou, Zixin Deng, Finian J. Leeper*, Peter F. Leadlay*, Marcio V. B. Dias*, and Yuhui Sun*

ACS Catalysis 2021, 11:12274-12283

Epub Date: 20 September 2021

DOI: 10.1021/acscatal.1c03508


Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathways of gentamicin have been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are successively modified by concerted actions of two PLP(pyridoxal 5′-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then reaminated by GenB3 using an amino donor. Importantly, crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes’ functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

The mechanism of dehydrating bimodules in trans-acyltransferase polyketide biosynthesis: A showcase study on hepatoprotective hangtaimycin

Minghe Luo#, Houchao Xu#, Yulu Dong, Kun Shen,Junlei Lu, Zhiyong Yin, Miaomiao Qi, Guo Sun, Lingjie Tang, Jin Xiang, Zixin Deng, Jeroen S. Dickschat*, and Yuhui Sun*

Angewandte Chemie International Edition 2021, 60:19139-19143

Epub Date: 28 July 2021

DOI: 10.1002/anie.202106250


A bioassay-guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis. The biosynthetic gene cluster was identified and resembles a hybrid trans-AT PKS/NRPS biosynthetic machinery whose first PKS enzyme contains an internal dehydrating bimodule, which is usually found split in other trans-AT PKSs. The mechanisms of such dehydrating bimodules have often been proposed, but have never been deeply investigated. Here we present in vivo mutations and in vitro enzymatic experiments that give first and detailed mechanistic insights into catalysis by dehydrating bimodules.

Improving the precision of base editing by bubble hairpin single guide RNA

Zhiwei Hu#, Yanna Wang#, Qian Liu, Yan Qiu, Zhiyu Zhong, Kangdi Li, Wenhua Li, Zixin Deng, and Yuhui Sun*

mBio 2021, 12(2)6:e 00342-21

Epub Date: 20 April 2021

DOI: 10.1128/mBio.00342-21


Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5′ end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency. Meanwhile, such a design also improves the purity of C-to-T conversions induced by base editor 3 (BE3) at on-target sites. Our results present a distinctive and effective strategy to improve the specificity of base editing.

A cold shock protein promotes high-temperature microbial growth through binding to diverse RNA species

Zikang Zhou#, Hongzhi Tang#,*,Weiwei Wang, Lige Zhang, Fei Su, Yuanting Wu, Linquan Bai, Sicong Li, Yuhui Sun, Fei Tao, and Ping Xu*

Cell Discovery 2021, 7:15

Epub Date: 16 March 2021

DOI: 10.1038/s41421-021-00246-5


Endowing mesophilic microorganisms with high-temperature resistance is highly desirable for industrial microbial fermentation. Here, we report a cold-shock protein (CspL) that is an RNA chaperone protein from a lactate producing thermophile strain (Bacillus coagulans 2-6), which is able to recombinantly confer strong high-temperature resistance to other microorganisms. Transgenic cspL expression massively enhanced high-temperature growth of Escherichia coli (a 2.4-fold biomass increase at 45 °C) and eukaryote Saccharomyces cerevisiae (a 2.6-fold biomass increase at 36 °C). Importantly, we also found that CspL promotes growth rates at normal temperatures. Mechanistically, bio-layer interferometry characterized CspL's nucleotide-binding functions in vitro, while in vivo we used RNA-Seq and RIP-Seq to reveal CspL's global effects on mRNA accumulation and CspL's direct RNA binding targets, respectively. Thus, beyond establishing how a cold-shock protein chaperone provides high-temperature resistance, our study introduces a strategy that may facilitate industrial thermal fermentation.

Two new streptovaricin derivatives from mutants of Streptomyces spectabilis CCTCC M2017417

Minghe Luo, Yulu Dong, Lingjie Tang, Xu Chen, Zhiwei Hu, Wenxinyu Xie, Zixin Deng, and Yuhui Sun*

Natural Product Research 2021, 1-6

Epub Date: 8 February 2021

DOI: 10.1080/14786419.2021.1881517


Two new ansamycin derivatives, damavaricin H (1) and protostreptovaricin VI (2) were isolated from the Streptomyces spectabilis CCTCC M2017417 derived mutants of ΔstvP5 and ΔstvA2, respectively. The structures of 1 and 2 were established by analysis of the HRESIMS as well as 1D and 2DNMR data sets. The minimum inhibitory concentration (MIC) results showed that compounds 1 and 2 possessed the corresponding anti-MRSA bioactivities of 4~8 μg/ml and 8~16 μg/ml, which confirmed the structure-activity relationships of streptovaricins reported previously andalso revealed that addition of the hydroxyl group at C-8 increased the anti-MRSA activity.