Education: 

1991-1995, Postdoc., Purdue University, USA, Microbiology and Molecular Biology 

1982-1988, Ph.D., Nanjing Agricultural University, Microbial Biochemistry  

1978-1982, B.S., Nanjing University, Biochemistry  

Working experience: 

1999-present,  Professor, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences 

1995-1999,   Associate Professor, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences 

1988-1991,   Lecturer, Nanjing Agricultural University, Biochemistry 

a) Metabolic regulation and engineering of important industrial microorganisms, focusing on solvent-producing Clostridium and antibiotic-producing Streptomyces strains. 

b) Development of efficient and advanced genetic manipulation platforms for these bacteria, aiming to explore and identify key functional genes, metabolic pathways and regulatory systems closely associated with target phenotypes (such as substrate utilization and chemical synthesis), and to elucidate the underlying molecular mechanisms. 

c) Design and reconstruction of microbial cell-factories for effectively producing medicines, biofuels and useful chemicals by natural or heterogeneous synthesis. 

1) Study of the antibiotic-producing streptomycetes  

a. Development of novel enabling technologies  Streptomyces has strong capability to produce a multitude and diversity of bioactive natural products (NPs) and remains invaluable sources for the discovery of novel drug leads. Convenient and efficient Streptomyces genome engineering will significantly advance functional genomics research, bioactive NPs mining as well as strain improvement. In the past five years, a panel of novel enabling technologies has been developed in our group. 

i) CRISPR/Cas9 (or Cpf1)-based high-efficiency genome editing tools, enabling efficient deletions of single or two genes/gene clusters and precise point mutations (Li L et al., Appl Environ Microbiol, 2018);  

ii) Recombinase-based multiplex site-specific genome editing tools, including MSGE (via the concept of “one integrase, multiple attB sites”) and aMSGE (via the concept of “multiple integrase, multiple attB sites”), allowing for multi-copy chromosomal integration of NP biosynthetic gene clusters (BGCs). Using these two methods, we achieved drastically increased production of several important antibiotics, such as pristinamycin II and 5-oxomilbemycin (Li L et al., Metab Eng, 2015，2019).  

iii) dCas9 (or ddCpf1)-based integrative CRISPRi tools, enabling simultaneous repression of up to four genes with high efficiencies (60-98%) (Zhao Y et al., Biotechol J, 2018; Li L et al., Appl Environ Microbial, 2018). Meanwhile, an in vitro DNA manipulation method (CGE) for NP BGCs editing by combining the endonuclease Cas9 and Gibson assembly were also established (Li L et al., Metab Eng, 2017). 

Overall, the newly developed enabling technologies represent advanced technology platforms, which will greatly benefit fundamental and applied research of actinomycetes. These tools have been widely used in many labs at home and abroad. 

b. Dissection of regulation mechanisms of pathway-specific or global regulators involved in antibiotic biosynthesis  Natural products (NPs) biosynthesis in Streptomyces is under strict regulation, involving pathway-specific and global regulators. A deep understanding of the regulatory mechanisms will be of great importance for strain improvement as well as novel NPs discovery. In the past five years, a number of important transcription regulators involved in antibiotic biosynthesis have been characterized in our lab. In the model strain Streptomyces coelicolor, we demonstrated for the first time that GlnR not only plays as a master nitrogen regulator, but also is directly involved in regulation of antibiotic biosynthesis via pathway-specific regulators (He J et al., J Biol Chem, 2016). Additionally, a novel two component system GluR/K involved in glutamate sensing and uptake was identified and its function model was proposed (Li L et al., J Bacterial, 2017). In the pristinamycin-producing strain Streptomyces pristinaespiralis, three regulators, including the cluster-situated regulator PapR6, two TetR family regulators AtrA-p and PaaR, were identified being involved in regulation of pristinamycin biosynthesis and the mechanisms were elucidated (Dun J et al., J Bacterial, 2015; Zhao Y et al., J Bacterial, 2015).   

c. Construction of Streptomyces cell factories and chassis for high-yield antibiotic production  Using the newly established technologies, we generated two Streptomyces cell factories with high-yield production of two important drugs, pristinamycin II (PII) and 5-oxomilbemycin A3/A4, respectively. An engineered S. pristinaespiralis strain with enhanced production of PII (against antibiotic-resistant bacteria) by 10.7-fold was obtained through combinatorial metabolic engineering, involving pathway-specific regulatory network refactoring and MSGE-assisted PII BGC amplification (Li L et al., Metab Eng, 2015, 2017). A 5-oxomilbemycin-producing cell factory (Streptomyces milbemyinicus) with a high yield of 6.37 g/L was constructed by aMSGE-assisted 5-oxomilbemycin BGC amplification. These efforts have significantly promoted the commercialization process of these antibiotics in China and the cooperation with pharmaceutical companies has been established.  

In addition, using the MSGE method, we constructed a series of powerful S. coelicolor heterologous hosts, in which up to four copies of the BGC responsible for biosynthesis of the anti-tumour compound YM-216391 was efficiently integrated, leading to drastically elevated productivity (Li L et al., Metab Eng, 2017). In collaboration with Prof. Gongli Tang from Shanghai Institute of Organic Chemistry, two new YM-216391 derivatives with higher activities were obtained by the combination of CGE-assisted BGC editing and heterologous expression in our developed Streptomyces superhosts. We believe these robust hosts will significantly facilitate pathway identification as well as the discovery of novel drug leads by refactoring silent BGCs. Until now, these superhosts have already been widely used in many laboratories. 

2) Study of solventogenic clostridia 

a. Establishment of advanced technology platform  Solventogenic clostridia are good producers for commercial chemicals and fuels. In recent years, we have established an efficient technology platform to deeply understand and modify these industrially important bacteria: (1) synthetic promoter library that is universal in both saccharolytic and gas-fermenting Clostridium species for  precise control of gene expression (Yang G et al., ACS Synthetic Biology, 2017); (2) mariner transposon-mediated random mutagenesis of clostridia, yielding a high quality mutant library for phenotypic screens; (3) genome editing tools based on CRISPR-Cas9/Cpf1 system (Huang H et al., ACS Synthetic Biology, 2016); (4) chromosomal integtration of large biosynthetic pathways in clostridia mediated by phage attachment/integration systems (Huang H et al., Metab Eng, 2019).  

b. Dissection the mechanism of the regulatory systems related to substrate utilization and solvent synthesis in clostridia  

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. We identified a novel three-component regulatory system (XylFII-LytS-YesN) in Firmicutes bacteria, which can percepts environmental d-xylose and activates an ABC transporter for d-xylose uptake (Sun Z et al., Mol Microbiol, 2015). To further reveal the mechanism underlying signal perception and integration of the processes, structural study of the XylFII-LytS complex was conducted in collaboration with Dr. Peng Zhang. A detailed working model of the complex was proposed, which provides a molecular basis for d-xylose utilization and metabolic modification in bacteria (Li J et al., PNAS, 2017).  

The global regulation mediated by CcpA (catabolite control protein A) is essential for gram-positive bacteria. We identified a novel flexible CcpA-binding site architecture (cre_var) that is distinct from all known cre sites and revealed the cre_var-based CcpA regulatory network in solventogenic C. acetobutylicum (Yang Y et al., MBio, 2017). In addition, we discovered a dual-cre motif-based autoregulation of CcpA in C. acetobutylicum, enabling the dynamic control of CcpA expression, which provides an useful strategy in genetic engineering of C. acetobutylicum (Zhang L et al., Appl Environ Microbiol, 2018).  

c. Engineering clostridial cell factories for the productive performance  Molecular modulation of pleiotropic regulator CcpA combined with sol operon overexpression alleviated the carbon catabolite repression (CCR) effect in C. acetobutylicum. This offered a CcpA mutant with optimized regulatory function, yielding a valuable platform host toward solvents production from lignocellulosic biomass (Wu Y et al., Metabolic Engineering, 2015). Besides, we identified biotin synthetic pathway in C. acetobtuylicum and use it as the target for improving the solvents production and cell growth by reinforcing this metabolic pathway in vivo (Yang Y et al., Metabolic Engineering, 2016). Moreover, a series of cell factories derived from gas-fermenting Clostridium species were also obtained, achieving the biological production of a variety of bulk chemical and biofuels (including 1-butanol, butyric acid, isopropanol and 3-hydroxybutyrate) by a gas mixture (CO/CO₂) fermentation.