Principal Investigator
Researcher
Email:zhouzhihua@sippe.ac.cn; zhzhou01@sibs.ac.cn
Personal Web:

Key Laboratory of Synthetic Biology, CAS

Dec., 2004------ present:  Principal investigator, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, CAS; 

April, 2003---Nov, 2004:  Associate Professor, Shanghai Jiaotong University, China; 

April, 2000---Mar, 2003:  Post-doc fellow at the University of Tokyo, Japan, sponsored by JSPS (Japanese Society for Promotion of Sciences);  

July, 1993---March, 1997:  Lecturer, Laboratory of Ecology, Department of Forestry 

Central-South Forestry University, China; 

July, 1988---Aug, 1990:  Assistant Professor, Department of Forestry,  

Hunan Forestry Technique College, China; 

The demand for energy, materials and drugs in the global is increasing. Plant natural products and their derivatives are the important reservoir for the development of medicines, health products and food additives. The synthetic biology technology bring new strategies to synthesize rare natural products with complicated structures by artificially constructing and optimizing the biosynthesis pathway of target compounds in microbial chassis cells. Thus, large quantities of rare natural products would be manufactured via microbial fermentation at low costs and large scale to meet the increasing pharmaceutical demand⁴. Our research interests in this field are focused on: (1) to elucidate the biosynthetic pathways of active nature products in P. ginseng, P. notoginseng, Epimedium spp and other traditional Chinese medicinal plants; (2) to uncover and characterize the key bioparts in the biosynthetic pathways of important natural products, such as terpene synthase (TS), UGT, MT, PT and P450; (3) to design, construct and optimize yeast chassis cells or yeast cell factories to produce terpenoids, flavonoids and other natural compounds at high efficiency. 

The high cost and low activities of lignocellulases are still major barriers to make use of lignocellulose efficiently to produce biofuel and chemical materials. Developing energy-efficient and cost-effective methods to decompose lignocellulose require scientific and technological breakthroughs of lignocellulase producing organisms. The filamentous fungus T. reesei with the strongest protein synthesis and secretion ability is currently the main producer of cellulase, the protein titer of which could reach to 100g/L in a fermentor. However, the regulation system which controls the induction, synthesis and secretion of lignocellulases in T. reesei is not yet completely characterized. Our research interests in this field are focused on: (1) to elucidate the regulation system for the induction, synthesis and secretion of lignocellulase in T. reesei, and discover and characterize related novel regulators; (2) to design and rebuild a new regulation system to improve the synthesis and modification of lignocellulases and other secreted proteins in T. reesei; (3) to design and build T. reesei chasses to manufacture different secreted protein products with high value. 

   Biopart, which is defined as a DNA or RNA sequence with certain function, is the basic unit to construct a device, a gene circuit, and a synthetic or regulating pathway to achieve more complex and diverse functions in synthetic biology. It is expected that synthetic biology provides a toolbox of reusable bioparts to be plugged into circuits or pathways at will. However, many knowledge gaps still exist in how life works, which also limit our information to uncover the key bioparts involved in specific systems. Our research interests in this field are focused on: (1) to build a registry and database of well characterized bioparts for synthetic biology;(2) to carry out studies about the phylogenetic relationship of key biopart groups as well as the relationship between the sequencing structure and function of the key bioparts, and the optimization of key bioparts to improve their compatibility in chassis cells. 

The newly rising synthetic biology is expected to solve many global challenges, such as converting cheap, renewable resources into biofuels or chemicals; producing high quality drugs and health products to fight diseases. Yeast and filamentous fungi have been widely applied in medicine and food industry. As Generally Recognized as Safe (GRAS) strains, Saccharomyces cerevisiae and Trichoderma reesei are the ideal fungal chassis cells to produce natural products and high-value protein products, respectively. The overall research goal in our group is to develop usable knowledge and technologies to design and rebuild fungal chassis as well as to uncover the synthetic pathways of rare natural products or valuable proteins and characterize related bioparts, which will advance synthetic biological strategies to improve the biosynthesis of rare natural products with potential therapeutic applications and protein products with high value, and finally lead to technologies deployable in industry.  

a) Specific hypotheses addressed by your research work. 

1) S. cerevisiae has an intact mevalonate (MVA) pathway, which provides common precursors for different terpenoid compounds. We hypothesize that systematically engineering of the energy and metabolic pathways and regulating systems in S. cerevisiae could provide a series of excellent chasses or cell factories for different terpenoids. 

2) The hyper-producers of lignocellulases were usually obtained by long-term strain improvements through repeated mutagenesis and screening. Based on the genome-editing technology and characterization of the regulation system for the induction, synthesis and secretion of lignocellulase inT. reesei, we could design and construct a new regulating system in T. reesei and thus improve its ability to produce lignocellulase or heterologous protein products with high value; 

3) Biopart is the basic unit to construct a novel synthetic pathway or a novel regulating system as designed. However, we still need to uncover the key bioparts involved in specific systems in nature or to engineering the natural ones and improve their compatibility with other bioparts in chasses. We hypothesize that we could build a designed bio-system by choosing bioparts from a registry of bioparts with more and more natural bioparts being accessed, characterized and modified. 

b) Major findings. 

1) Elucidation of the biosynthetic pathways of a series of triterpenoids (ginsenosides) of P. ginseng and P. notoginseng by cloning and characterizing more than 30UDP-glycosyltransferases and two NADPH-cytochrome P450 reductases 

P. ginseng and P. notoginseng have been traditionally used as herbal medicine in Asia for thousands of years. Its active constituents are ginsenosides, a group of triterpene saponins. More than 100 ginsenosides have been isolated from ginseng, which are mainly coming from two aglycones (protopanaxadiol (PPD) and protopanaxatriol (PPT)) by different type and elongated glycosylation at different positions (C-3, C-6 or C-20). During 2012 to 2013, we cloned and characterized the first UDP-glycosyltransferase UGTPg1 from P. ginseng, which catalyzes PPD to produce rare ginsenoside CK and PPT to produce rare ginsenoside F1, respectively (Yan et al 2014). Since 2013, we have cloned and functionally characterized more than 100 UGTs fromP. ginseng and P. notoginseng, more than 30 of which can catalyze the glycosylation of free hydroxyl at C-3, C-6 or C-20 of PPD or PPT or their ginsenosides (Wang et al. 2015; Wei et al. 2015)  or transfer glucose or xylose moiety to elongate the sugar chain of ginsenosides (Patent PCT/CN2013/088819; PCT/CN2015/081111; PCT/CN2018/087678). We also cloned and characterized two NADPH-cytochrome P450 reductases PgCPR1 and PgCPR2 involved in ginsenosides biosynthesis. Based on the discovery and functional identification of these bioparts, we uncovered the biosynthesis pathways of more than 30 ginsenosides, including Rh2, Rg3, Rh1, F1, Rg1, Rb1, Rd, Rb3, R1, R2, U, XIII, XVII, LXXV and etc. Our research results pave the ways to product these triterpene saponins with high value by approach of synthetic biology. We also identified several key amino acids of the above UGTs that may play important roles in determining their activities and substrate regio-specificities (Wei et al.2015). 

2) Systematically engineering of S. cerevisiae as a platform to produce different ginsenosides at high efficiency 

Taking usage of the bioparts (PgCPR1 and different UGTs) identified in our study as well as bioparts PgDDS, CYP716A47 and CYP716A53v2 identified by Korean scientists, we could reconstruct the biosynthesis pathway of different rare ginsenosides in yeast (Yan et al. 2014; Wang et al. 2015; Wei et al.2015). However, the ginsenosides yields at that time were very low. For example, the CK yield of the first version of CK yeast factories was only 0.8 mg/L (Yan et al. 2014).  

In order to increase the yield, we first tried to design and construct an efficient chassis strain for high-level PPD production, which is the common precursor of dammarane ginsenosides. By systemically boosting carbon flux to the MVA pathway via modular engineering of MVA pathway and improvement of P450 expression levels by optimization promoter and increasing copy number. We increase the PPD production by more than 1100 fold, from original 0.48 mg/L to 529.0 mg/L in shake flasks (Wang et al. in press), which is the highest PPD production ever reported.  

Based on this robust chassis strain, we established a series of cell factories to produce different PPD-type ginsenosides by introducing different UGTs bioparts. In order to further improve the glycosylation efficiency and the yield of ginsenosides. We employed several strategies to optimize the cell factories systemically, which included improving UGTs expression level by increasing its copy number, engineering its promoter and increasing its activity of UGTs via in vivo directed evolution or searching for novel alternative UGTs with higher glycosylation efficiencies from other plant species. We also engineered the UDP-glucose biosynthesis and regeneration pathway to enhancing in vivo sugar donor supply in yeast chassis. Combining all of these engineered strategies, we increased the yield of all PPD-type ginsenosides significantly. For example, the CK yield increased to 230.7 mg/L from 0.8 mg/L, and Rh2 yield increased to 179.3 mg/L from 16.9 mg/L in shake flasks (Wang et al in press) .  

3) Establishing a genome editing system for T. reesei 

Genetic engineering approaches have been well developed for filamentous fungi. However, these approaches are not as efficient as those available for yeast and bacteria due to the additional complexity of filamentous fungi. We established a CRISPR/Cas9 system in the T. reesei by specific codon optimization and in vitro RNA transcription (Liu et al. 2015). This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms, which provides a more efficient tool to investigate the regulatory network and achieve an optimized T. reesei chassis. 

4) The investigation of the regulation system for the induction, synthesis and secretion of lignocellulase in T. reesei 

Since 2013, we identified several novel potential regulators which might be involved in the induction, synthesis and secretion of lignocellulases in T. reesei. A putative methyltransferase (Sam) takes effect on the lignocellulase synthesis, which is further proved to modify a negative regulator Ace1 of lignocellulase transcription (unpublished data). The histone deacetylase Hda1 was confirmed to participate to form nucleosomes and alter the promoter structure of cellulase genes (unpublished data). In calcium signaling pathway, the transcription factor TrCrz1 could directly regulate cellulase and its activator Xyr1 through binding to the [T/G]GGCG region in their promoters. SxlR was found to repress the xylanase activity but not cellulase activity, and further testified as a specialized regulator of GH11 genes (Liu et al. 2017). Besides, a low expression level β-glucosidase Bgl3I has a high hydrolysis activity on the strongest cellulase inducer sophorose, and thus down-regulates the induction of lignocellulases in T. reesei (Zou et al. in press). Taken together with these reported regulators, it will refine the cellulase regulatory network and facilitate the construction of T. reesei chassis cells for different protein products with high value. 

5) Construction of a registry and database of bioparts fornature product biosynthesis 

Recently we set up a data mining platform by developing two authorized software to extract bioparts involved in nature product biosynthesis from omics data. We collected the information of more than 10⁵ predicted bioparts related to the skeleton formation and modification as well as transport of natural product synthesis. We set up the standard characterizing protocols for bioparts TS,  UGT,MT, PT and P450 related to natural products biosynthesis, and characterize a group of P450s from Ganoderma lucidum (yang et al. 2018), PTs from Epimedium spp. (PCT201710521825.1) and MTs from crop plants (PCT201810889216.6). Besides, we collected all of the published characterization information of TS, UGT, MT, PT and P450 from plants. Based on this registry and database of bioparts for nature product biosynthesis(http://npbiosys.scbit.org), we constructed a biosynthetic pathway of icariin from keampferol as design by choosing one PT, one MT and two UGTs bioparts from the registry. The artificial synthetic pathway from glucose to icariin could be constructed in microbial chassis to produce icariin. 

6) From laboratory to industry 

The ginsenoside yields could be further increased significantly by optimizing the fermentation conditions of yeast cell factories in fed-batch bioreactors. Now the titers of PPD, CK and Rh2 could reached to 11.02 g/L, >3g/L and 2.2g/L in a 10 L fed-batch bioreactor, respectively. And the developed techniques of the one-pot biosynthesis of ginsenosides CK, Rg3 and Rh2 have already transformed to two pharmaceutic companies. Besides, we have also been cooperating with an enzyme preparation company by supporting them with the genome editing technique of T. reesei.