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Key Laboratory of Synthetic Biology, CAS

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Microbial Metabolic Engineering and Comparative Genomic

Research Direction
The Yang lab aims to understand and manipulate the operation of metabolic networks in microbes. The scientific goal of our research is to provide a quantitative understanding of how genes, proteins, and metabolites interact to generate and regulate functional metabolic networks. To achieve it, we develop experimental techniques for metabolic flux analysis, quantitative metabolomics, and gene expression analysis. Mathematical models are used to interpret the large sets of data within a rigorous metabolic network. We also develop comparative genomic methods for reconstruction of metabolic pathways and transcriptional regulons. Our research focuses on solvent-producing clostridia and cyanobacteria. Research interests include: (i) development of metabolic flux analysis techniques; (ii) regulation and control of metabolic fluxes in microbes; (iii) metabolic engineering of microbes for production of biochemicals.

Principal Investigator: Dr. Chen Yang, Professor. Email: cyang@cibt.ac.cn
Staff: Li-Xia Liu, Fang Gao
Graduate Students: Lei Zhang, Xiang Gao, Jing Xia, Xiao-Qun Nie, Hao Zhang, Bin Yang

Recent Research Progress

1. Technology development for quantitating intracellular fluxes and for reconstructing metabolic pathways and regulons
The quantitative knowledge of in vivo intracellular fluxes is of high importance in deciphering cellular functions and in guiding rational strain engineering for industrial biotechnology. We have developed 13C-based methods for high resolution flux analysis, which enabled us to monitor how microorganisms operate the metabolic network in response to environmental and genetic perturbations. Moreover, we have developed comparative genomic methods for reconstruction of metabolic pathways and transcriptional regulons.
By using the 13C metabolic flux analysis technique, we identified the network of glucose metabolism and quantified intracellular carbon fluxes in Rhodobacter sphaeroides KD131, a nonsulfur purple bacterium that exhibits unusual metabolic versatility and produces hydrogen gas (H2) during photoheterotrophic growth. We investigated how the intracellular fluxes in R. sphaeroides responded to knockout mutations in hydrogenase and poly-β-hydroxybutyrate synthase genes, which led to increased H2 yield. The relative contribution of the Entner-Doudoroff pathway and Calvin-Benson-Bassham cycle to glucose metabolism differed significantly in hydrogenase-deficient mutants and this flux change contributed to the increased formation of the redox equivalent NADH. A remarkable increase in the flux through the tricarboxylic acid cycle, a major NADH producer, was observed for the mutant strains. The in vivo regulation of the tricarboxylic acid cycle flux in photoheterotrophic R. sphaeroides was discussed based on the measurements of in vitro enzyme activities and intracellular concentrations of NADH and NAD+. Our results provide quantitative insights into how photoheterotrophic cells operate the metabolic network and redistribute intracellular fluxes to generate more electrons for increased H2 production. This information will be useful for metabolic engineering of this group of bacteria to improve photobiological production of H2. This work has been published in Journal of Bacteriology (2012, 194: 274).
In a collaborative study with Dr. Guo-Ping Zhao, we investigated the regulatory role of protein acetyltransferase (Pat) and NAD+-dependent deacetylase (CobB) in coordinating carbon source utilization and metabolic flux. We measured in vivo metabolic fluxes in Salmonella enterica using 13C labeled glucose or citrate as the tracer followed by GC-MS analysis. Together with the biochemical studies on key metabolic enzymes, the results indicated that carbon source-associated acetylation affected the relative activities of metabolic enzymes and thus modulated metabolic fluxes. This work has been published in Science (2010, 327: 1004).

2. Pathway and regulon reconstruction and flux quantification of pentose metabolism in clostridia
A number of Clostridium species naturally produce butanol and acetone, which can be used as a solvent for a wide variety of industrial application. Substrate cost is a major factor impacting economics of fermentative solvent production by clostridia. To reduce the substrate cost, abundant and inexpensive lignocellulosic materials could be used, and one of their major components is pentose-rich hemicellulose. Solventogenic clostridia including Clostridium acetobutylicum and Clostridium beijerinckii are capable of utilizing the hemicellulosic pentoses, xylose and arabinose. However, the knowledge about pentose utilization pathways and their regulation in clostridia is rather limited.
We reconstructed the xylose and xyloside utilization pathway and regulons in 24 genomes of the Firmicutes by using a comparative genomic approach. A novel xylose isomerase that is not homologous to previously characterized xylose isomerase, was identified in C. acetobutylicum and other clostridia. The candidate genes for the xylulokinase, xylose transporters, and the transcriptional regulator of xylose metabolism (XylR), were unambiguously assigned in all of the analyzed species. In collaboration with Dr. Weihong Jiang and Dr. Sheng Yang, we confirmed the predicted functions of these genes through a combination of genetic and biochemical techniques. XylR regulons were reconstructed by identification and comparative analysis of XylR-binding sites upstream of xylose and xyloside utilization genes. A novel XylR-binding DNA motif was identified in three Clostridiales species and experimentally validated in C. acetobutylicum by an electrophoretic mobility shift assay. This work has been published in BMC Genomics (2010, 11: 255).
In a recent study we characterized the arabinose utilization pathway and its regulation in clostridia. We combined a comparative genomic reconstruction of AraR regulons in 9 Clostridium species with the detailed experimental characterization of AraR-mediated regulation in C. acetobutylicum. Based on the reconstructed AraR regulons, a novel ribulokinase AraK present in all analyzed Clostridium species was identified, which was a non-orthologous replacement of previously characterized ribulokinases. The predicted function of araK gene in C. acetobutylicum was confirmed by gene inactivation and biochemical assays. In addition to the genes involved in arabinose utilization and arabinoside degradation, extension of the AraR regulon to the pentose phosphate pathway genes in several Clostridium species was revealed. The predicted AraR-binding sites in the C. acetobutylicum genome and the negative effect of L-arabinose on the DNA-regulator complex formation were verified by in vitro binding assays. The predicted AraR-controlled genes in C. acetobutylicum were experimentally validated by comparing gene expression levels between wild-type and araR-inactivated mutant strains. This work has been published in Journal of Bacteriology (2012, 194: 1055). These studies pave the way for improving pentose utilization capability in solventogenic clostridia by genetic engineering.
We also identified the xylose catabolic pathways and quantified their fluxes in Clostridium acetobutylicum based on [1-13C]xylose labeling experiments. The phosphoketolase pathway was found to be active, which contributed up to 40% of the xylose catabolic flux in C. acetobutylicum. The split ratio of the phosphoketolase pathway to the pentose phosphate pathway was markedly increased when the xylose concentration in the culture medium was increased from 10 to 20 g liter-1. To our knowledge, this is the first time that the in vivo activity of the phosphoketolase pathway in clostridia has been revealed. A phosphoketolase from C. acetobutylicum was purified and characterized, and its activity with xylulose-5-P was verified. The phosphoketolase was overexpressed in C. acetobutylicum, which resulted in slightly increased xylose consumption rates during the exponential growth phase and a high level of acetate accumulation. This work has been published in Journal of Bacteriology (2012, 194: 5413). This work makes a contribution towards understanding and maximizing pentose sugar metabolism in C. acetobutylicum.

3. Regulation of central metabolism in cyanobacteria
The cellular metabolism in cyanobacteria is extensively regulated in response to changes of environmental nitrogen availability. Multiple regulators are involved in this process, including a nitrogen-regulated response regulator NrrA. However, the regulatory role of NrrA in most cyanobacteria remains to be elucidated. In this study, we combined a comparative genomic reconstruction of NrrA regulons in 15 diverse cyanobacterial species with detailed experimental characterization of NrrA-mediated regulation in Synechocystis sp. PCC 6803. The reconstructed NrrA regulons in most species included the genes involved in glycogen catabolism, central carbon metabolism, amino acid biosynthesis, and protein degradation. A predicted NrrA-binding motif was verified by in vitro binding assays with purified NrrA protein. The predicted target genes of NrrA in Synechocystis sp. PCC 6803 were experimentally validated by comparing the transcript levels and enzyme activities between the wild-type and nrrA-inactivated mutant strains. The effect of NrrA deficiency on intracellular contents of arginine, cyanophycin, and glycogen was studied. Our results indicate that by directly up-regulating expression of the genes involved in arginine synthesis, glycogen degradation, and glycolysis, NrrA controls cyanophycin accumulation and glycogen catabolism in Synechocystis sp. PCC 6803. This work not only identifies a molecular mechanism coordinately regulating synthesis and degradation of nitrogen and carbon reserves in Synechocystis but also gains an insight into the potential regulatory role of NrrA in diverse cyanobacteria. This work has been published in Journal of Biological Chemistry (2014, 289: 2055).

4. Engineering cyanobacteria for production of biochemicals from CO2
Production of chemicals directly from CO2 is an attractive approach to solve the energy and environmental problems. Isoprene, which is the key building block of a number of elastomers including rubber, is currently produced by chemical processes from petroleum, which is energy intensive, high cost, and low product purity. Our goal is to achieve and optimize photosynthetic production of high-purity isoprene from CO2 using a genetically engineered cyanobacterium Synechocystis sp. PCC 6803. Compared to the previously reported biological isoprene production processes that used sugar as the substrate, direct chemical production from CO2 in photosynthetic organisms recycles the atmospheric CO2 and will not compete with food crops for arable land.
We designed and cloned codon-optimized isoprene synthases from various plants into Synechocystis sp. PCC 6803 and demonstrated photosynthetic production of isoprene from CO2 using the recombinant strains. To increase the precursor supply for isoprene production, we overexpressed the methyl-erythritol-4-phosphate (MEP) pathway genes. The rate-limiting steps of isoprene synthesis were identified based on measurements of intracellular concentrations of MEP pathway intermediates. We showed that the isoprene production rate could be optimized through gene overexpression of rate-limiting steps, colocalization of pathway enzymes, and redirecting electron flow toward product synthesis. Finally, the isoprene production of the engineered strain was further greatly enhanced by using a small scale photobioreactor and optimizing the culture conditions. We have filed a patent application on the key techniques developed in this work.

Major Publications

  1. Deng Liu, Chen Yang* (2014) The nitrogen-regulated response regulator NrrA controls cyanophycin synthesis and glycogen catabolism in the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem. 289(4):2055-71.
  2. Dongfeng Liu, Guomin Ai, Qingxiang Zheng, Chang Liu, Chengying Jiang, Lixia Liu, Bo Zhang, Yiming Liu, Chen Yang*, Shuangjiang Liu* (2014) Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains. Microb Cell Fact. 13: 40.
  3. Yongzhen Tao, Deng Liu, Xing Yang, Zhihua Zhou, Jeong K. Lee, Chen Yang* (2012) Network identification and flux quantification of glucose metabolism in Rhodobacter sphaeroides under photoheterotrophic H2-producing conditions. J Bacteriol. 194(2): 274-283.
  4. Lei Zhang, Semen A. Leyn, Yang Gu, Weihong Jiang, Dmitry A. Rodionov, Chen Yang* (2012) Ribulokinase and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum. J Bacteriol. 194(5): 1055-1064.
  5. Lixia Liu, Lei Zhang, Wei Tang, Yang Gu, Qiang Hua, Sheng Yang, Weihong Jiang, Chen Yang*. (2012) Phosphoketolase pathway for xylose catabolism in Clostridium acetobutylicum revealed by 13C metabolic flux analysis. J Bacteriol. 194(19): 5413-5422.
  6. Semen A. Leyn, Fang Gao, Chen Yang*, Dmitry A. Rodionov* (2012) N-Acetylgalactosamine utilization pathway and regulon in proteobacteria. Genomic reconstruction and experimental characterization in Shewanella. J Biol Chem. 287(33): 28047-56.
  7. Yang Gu, Yi Ding, Cong Ren, Zhe Sun, Dmitry A Rodionov, Weiwen Zhang, Sheng Yang, Chen Yang* and Weihong Jiang* (2010) Reconstruction of xylose utilization pathway and regulons in Firmicutes. BMC Genomics. 11: 255-268.
  8. Qijun Wang, Yakun Zhang, Chen Yang, Hui Xiong, Yan Lin, Jun Yao, Hong Li, Lu Xie, Wei Zhao, Yufeng Yao, Zhibin Ning, Rong Zeng, Yue Xiong, Kunliang Guan, Shimin Zhao*, Guoping Zhao* (2010) Acetylation of metabolic enzymes coordinates carbon source utilization and metabolic flux. Science. 327(5968): 1004-1007.
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