Principal Investigator
Researcher
Email:syang@sibs.ac.cn
Personal Web: http://people.ucas.edu.cn/~yangsheng

Key Laboratory of Synthetic Biology, CAS

1995-2000 Ph.D., Biochemistry and Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences.  

1991-1995 B.E., Chemical Engineering, Zhejiang University, Hangzhou, China.  

Academic Experience 

2006-present Professor, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences 

2002-2006 Associate Professor, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences           

2000-2002 Research Associate, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences 

Green chemistry is the design of chemical products and processes that reduce or eliminate the use or generation of hazardous substances (Anne E. Marteel et al., 2003). The US Environmental Protection Agency presented five projects each year as Presidential Green Chemistry Challenge Awards since 1996. Biocatalysis has won more than a quarter of 114 awards [https://www.epa.gov/greenchemistry]. However, application of biocatalysis in chemical industry is still quite limited partially because the development and implementation of biocatalysts is rather slow compared to chemical synthesis (Keasling et al., 2012).  

Key advances in DNA sequencing and gene synthesis provide tremendous resource and possibility for building biocatalysts by functional expression and reorganization of enzymes into new biosynthetic pathways (Bornscheuer et al., 2012). Our overall research goal is the development of synthetic biology-based biocatalysts for industrial use by providing new enabling technologies and novel designs. Our scientific objectives include 1) a more efficient enzyme production protocol and facility for synthetic chemistry, 2) more efficient microbial genome editing tools for faster construction of designer cells, and 3) building genetically defined cells to understand the mechanism behind the efficient catalytic performance of evolved biocatalysts.  

A consensus biocatalysts enzymes production process was established on Escherichia coli T7 expression system with 2000 L fermentation capability. More than 200 biocatalysts enzymes have been made upon requests from synthetic chemistry. 19 enzymes have been used for producing 13 fine chemicals commercially in recent 5 years. CRISPR-Cas based genome editing tools have been setup for 10 microorganisms and are under adaptation in more microbial species with a diversified CRISPR-Cas nucleases collection. A rational designed and adaptive evolved xylose fermenting yeast has been used for commercially production of cellulosic ethanol in the United States and Brazil. The molecular mechanism behind its outstanding performance is currently under investigation through genotype reconstruction, as well as others evolved cells, such as an L-arginine hyperproducer Corynebacterium glutamicum strain. 

Cost-effective sustainable biomanufacturing systems are needed to enable the continued availability of products at low cost such as biopharmaceutical, nutraceutical, feed additive, bulk chemical and fuel. High-risk, high-reward research is necessary to achieve innovations for new biomanufacturing technologies. However, company fiduciary responsibilities often limit the levels of risk in research that companies can undertake on their own, with acceptable risk levels varying by different sectors. As a result, a critical need to support biomanufacturing is increasingly evident. Advanced DNA technologies, including next-generation DNA sequencing and low-cost DNA synthesis, provide massive genetic resources and fast access to genes, providing near-infinite possibilities for designing and creating novel biocatalysts (Bornscheuer et al., 2012). However, it is still in the early stage to predict the biocatalytic performance of synthetic biocatalysts (Mak et al., 2015). Therefore, a more powerful synthetic biology tool kit for faster building of designer biocatalysts is needed to provide the infrastructure necessary for translating laboratory prototypes into commercial products. Our laboratory is interested in the continuous expanding and improving the tool kit and uses it as a platform to develop biocatalysts together with industrial partners. The particular interests are biocatalysts for conversion of renewable resources to chemicals and biofuels with conventional chassis microorganisms, such as Escherichia coli, Corynebacterium glutamicum and Saccharomyces cerevisiae as well as non-conventional microbial hosts such as clostridia and Yarrowia lipolytica. 

Corresponding to our objectives, we proposed that 1) the Escherichia coli T7 expression system can efficiently produce most biocatalysts enzymes with a consensus protocol by screening “functional express-able” coding sequences according to a large number of recombinant expression practices previously; 2) CRISPR-Cas based genome editing tools can be established in any microbial species with a transformable plasmid in a reasonable time frame; 3) Unknown mechanism behinds evolved cells can be step-by-step understood by reconstruction of genotype from scratch providing highly efficient genome editing tool kit (Fig. 1). 

Figure 1. Build to understand the genetic basis and biochemical mechanism of improved variants for design of better biocatalysts. 

b) Major findings 

1) Bulk enzymes are mostly developed and produced by professional giants, including Novozymes, DuPont and DSM, etc. However, most of biocatalysts enzymes are not commercially available because of their limited uses. In this case, the synthetic chemists have to construct enzyme-producing strains and optimize enzyme-producing conditions in-house. Nevertheless, the patent and know-how barrier of this system is rather high, and the development duration is still too long. Consequently, most biocatalysts enzyme users in China, especially small- and medium-sized enterprises, cannot afford the corresponding development costs and technical risks.  

We shortened the developing time for enzyme producer strains by using only Escherichia coli BL21(DE3)/pET. 279 codon optimized synthetic genes were cloned into pET vector under control of T7lac promotor, and 258 genes were functionally over-expressed at 28℃ with 0.13 mM IPTG. The streamlined expression protocol skipped temperature optimization step in a consensus strategy based on collective analysis of 10,000 different proteins (Graslund, Nordlund et al. 2008) without loss in success rate. An optimized medium consisted of crude glycerol and corn steep liquor for culture of E.coli BL21(DE3) in high cell density further reduced the total cost of enzyme production by two thirds in our pilot plant at scale of 2,000 L. It takes only 2 weeks for construction of enzyme producer strains, and 1 week more for production of enzyme in kilograms (Jiang Y etal 2016, 陶荣盛等2016).  

While further optimization of each construct and expression condition should improve enzyme production, the consensus protocol is good enough for following catalytic process development. Thus, biocatalysts enzymes can be tailor-made at affordable cost and reasonable time frame in our pilot plant or by our industrial partners. 

2) Although the Streptococcus pyogenes CRISPR-Cas9 system has been adapted for genome editing of multiple organisms since 2013, it could not be introduced into Corynebacterium glutamicum which is an important industrial metabolite producer or Mycobacterium smegmatis which is the model organism for Mycobacterium species. We developed a Francisella novicida CRISPR-Cpf1-based genome-editing method for C. glutamicum and M. smegmatis (Jiang Y etal 2017, Sun B etal 2018). CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of bacteria that cannot utilize the S. pyogenes CRISPR-Cas9 system.  

We proposed a straight forward strategy for setup of CRISPR-Cas-based genome editing plasmids in bacteria cells (Fig. 2): i) Screen Cas Effector Protein (CEP) that have no toxicity to the receipt cell by transformation efficiency assay; ii) Select among non-toxic CEPs that cut receipt cell genomic DNA providing gRNA again by transformation efficiency assay; iii) Evaluate editing efficiency by providing editing templates with phage recombinases if necessary; iv) Optimize editing protocol for iterative genome editing. Thus, CRISPR-Cas-based genome editing was adapted in 10 microbial species in our lab(Jiang Y etal 2015, Gao S etal 2016, Li Q etal 2016, Liu Q etal 2017, Song X etal 2017, Sun J etal 2018).  

While addressing the problem of Cas9 toxicity is of scientific interest, from a practical perspective, our finding that Cpf1 behaves very differently from Cas9 suggests that when problems of CRISPR-Cas setup are encountered, assessing different CRISPR-Cas systems, rather than fine-tuning the expression of CEPs and/or the transcription of crRNA, may be an effective solution. 

Figure 2. Roadmap for development of CRISPR-Cas genome editing tool.  

CEP, Cas effector protein; SNHR, single nick-triggered homologous recombination; NHEJ, non-homologous end joining; HR, homologous recombination. 

3) Using efficient genome editing tools, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene and lycopene production in E. coli. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms (Yang C etal 2016). We also engineered an artificial twin-clostridial consortium to convert corn cob to acetone-butanol-ethanol (ABE) more efficient by strengthening its nutrition-detoxify mutualism via pulling carbon flux towards butyrate production Clostridium cellulovorans and butyrate re-assimilation in Clostridium beijerinckii. The titer of ABE approximates to that achieved from a starchy feedstock (Wen Z etal 2017).  

We have constructed a variety of highly efficient biocatalysts cells, including L-arginine and L-proline hyperproducer Corynebacterium glutamicum cells, terpenoids  hyperproducer Yarrowia lipolytica strains (Gao S etal 2017), Saccharomyces cerevisiae cell expressing glucoamylase for starchy ethanol production, and Saccharomyces cerevisiae strains with pentose metabolic pathway for lignocellulosic ethanol industry (Diao L etal 2013, Wang X etal 2018). L-proline and ethanol producer cells have been used in commercial scale. 

c) Contributions 

Using consensus enzyme-producing protocol, we accumulated more than 200 biocatalysts enzymes and built a public enzyme library covering most conventianla enzymatic reactions except the ones catalyzed by bulk or commercial-available enzymes. The supply of biocatalysts enzyme is no longer a bottleneck for biocatalytic process development in China. The plasmids for genome editing tools have been distributed out to hundreds of academic labs and licensed to Novo Nordisk, GenScript and Meihua. 

Till now, over 20 biocatalysts developed by the above mentioned enzyme expression platform or genome editing tools have been applied in commercially production of more than 20 products in recent 5 years (Table 1). Notably, the xylose yeast has been used as an enabler of advanced biofuels in Brazil and USA. These works were honored with Shanghai Science and Technology Progress Award (the first class) and Novozymes Innovation Award in 2015. 

Table 1. Industrial application of biocatalysts developed by our lab in recent 5 years