Xiaoming Tan

Photosynthetic production of ethanol from carbon dioxide in genetically engineered cyanobacteria

The rapidly growing demand for energy and the environmental concerns about carbon dioxide emissions make the development of renewable biofuels more and more attractive. Tremendous academic and industrial efforts have been made to produce bioethanol, which is one major type of biofuel. The current production of bioethanol is limited for commercialization because of issues with food competition (from food-based biomass) or cost effectiveness (from lignocellulose-based biomass). In this report we applied a consolidated bioprocessing strategy to integrate photosynthetic biomass production and microbial conversion producing ethanol together into the photosynthetic bacterium, Synechocystis sp. PCC6803, which can directly convert carbon dioxide to ethanol in one single biological system. A Synechocystis sp. PCC6803 mutant strain with significantly higher ethanol-producing efficiency (5.50 g L−1, 212 mg L−1 day−1) compared to previous research was constructed by genetically introducing pyruvate decarboxylase from Zymomonas mobilis and overexpressing endogenous alcohol dehydrogenase through homologous recombination at two different sites of the chromosome, and disrupting the biosynthetic pathway of poly-β-hydroxybutyrate. In total, nine alcohol dehydrogenases from different cyanobacterial strains were cloned and expressed in E. coli to test ethanol-producing efficiency. The effects of different culturing conditions including tap water, metal ions, and anoxic aeration on ethanol production were evaluated.

Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria

The production of high value biochemicals and high energy biofuels from sustainable resources through the use of microbial based, green conversion technologies could reduce the dependence on petrochemical resources. However, a sustainable source of carbon and a clean, cost effective method for its conversion to high quality biofuel products are obstacles that must be overcome. Here we describe the biosynthesis of fatty alcohols in a genetically engineered cyanobacterial system through heterologously expressing fatty acyl-CoA reductase and the effect of environmental stresses on the production of fatty alcohols in the mutant strains. Hydrocarbon production in three representative types of native cyanobacterial model strains and the mutant strain overexpressing acetyl-CoA carboxylase was evaluated. The results of this investigation demonstrate the potential for direct production of high value chemicals and high energy fuels in a single biological system that utilizes solar energy as the energy source and carbon dioxide as the carbon source.

De novo Biosynthesis of Biodiesel by Escherichia coli in Optimized Fed-Batch Cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L−1 FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.

Exploring the photosynthetic production capacity of sucrose by cyanobacteria

Because cyanobacteria are photosynthetic, fast-growing microorganisms that can accumulate sucrose under salt stress, they have a potential application as a sugar source for the biomass-derived production of renewable fuels and chemicals. In the present study, the production of sucrose by the cyanobacteria Synechocystis sp. PCC6803, Synechococcus elongatus PCC7942, and Anabaena sp. PCC7120 was examined. The three species displayed different growth curves and intracellular sucrose accumulation rates in response to NaCl. Synechocystis sp. PCC6803 was used to examine the impact of modifying the metabolic pathway on the levels of sucrose production. The co-overexpression of sps (slr0045), spp (slr0953), and ugp (slr0207) lead to a 2-fold increase in intracellular sucrose accumulation, whereas knockout of ggpS (sll1566) resulted in a 1.5-fold increase in the production of this sugar. When combined, these genetic modifications resulted in a fourfold increase in intracellular sucrose accumulation. To explore methods for optimizing the transport of the intracellular sucrose to the growth medium, the acid-wash technique and the CscB (sucrose permease)-dependent export method were evaluated using Synechocystis sp. PCC6803. Whereas the acid-wash technique proved to be effective, the CscB-dependent export method was not effective. Taken together, these results suggest that using genetic engineering, photosynthetic cyanobacteria can be optimized for efficient sucrose production.

Improved production of fatty alcohols in cyanobacteria by metabolic engineering

BackgroundFatty alcohols are widely used in industrial chemicals. The biosynthetic pathways for fatty alcohols are diverse and widely existing in nature. They display a high capacity to produce fatty alcohols by the metabolic engineering of different microbe hosts. Direct recycling of carbon dioxide to fatty alcohols can be achieved by introducing a fatty alcohol-producing pathway into photosynthetic cyanobacteria. According to our precious reports, a relatively low yield of fatty alcohols was obtained in the genetically engineered cyanobacterium Synechocystis sp. PCC 6803.ResultsThe photosynthetic production of fatty alcohols in Synechocystis sp. PCC 6803 was improved through heterologously expressing fatty acyl-Coenzyme A (acyl-CoA) reductase gene maqu_2220 from the marine bacterium Marinobacter aquaeolei VT8. Maqu_2220 has been proved to catalyze both the four-electron reduction of fatty acyl-CoA or acyl-Acyl Carrier Protein (acyl-ACP) and the two-electron reduction of fatty aldehyde to fatty alcohol. The knockout of the aldehyde-deformylating oxygenase gene (sll0208) efficiently blocked the hydrocarbon accumulation and redirected the carbon flux into the fatty alcohol-producing pathway. By knocking-out both sll0208 and sll0209 (encoding an acyl-ACP reductase), the productivity of fatty alcohols was further increased to 2.87 mg/g dry weight.ConclusionsThe highest yield of fatty alcohols was achieved in cyanobacteria by expressing the prokaryotic fatty acyl-CoA reductase Maqu_2220 and knocking-out the two key genes (sll0208 and sll0209) that are involved in the alka(e)ne biosynthesis pathway. Maqu_2220 was demonstrated as a robust enzyme for producing fatty alcohols in cyanobacteria. The production of fatty alcohols could be significantly increased by blocking the hydrocarbon biosynthesis pathway.

Enhancing photosynthetic production of ethylene in genetically engineered Synechocystis sp. PCC 6803

Ethylene is widely used in the petrochemical industry and has traditionally been produced via the steam cracking of petroleum-based feedstock. The exploration of sustainable and carbon-neutral methods of producing ethylene from the renewable feedstock seems promising. The direct photosynthetic production of ethylene after the recycling of carbon dioxide shows great potential. In this study, continuous and stable ethylene production was achieved in Synechocystis sp. PCC 6803 by introducing a codon-optimized ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. sesami and using 2-oxoglutarate (2-OG) as the substrate. Based on diverse promoter screening, PcpcB was proved to be a highly efficient promoter for ethylene production in cyanobacteria. The genes encoding 2-OG decarboxylase (OGDC) and succinic semialdehyde dehydrogenase (SSADH) in the tricarboxylic acid (TCA) cycle in Synechocystis sp. PCC 6803 were identified, and the TCA cycle was genetically modified by blocking these two enzymes with the simultaneous overexpression of EFE. Meanwhile, a gene encoding 2-OG permease (KgtP) from E. coli was introduced into the phaAB loci to increase the 2-OG supply. A peak volumetric production rate of 9.7 mL L−1 h−1 for ethylene was eventually achieved in the Synechocystis recombinant (XX110), with the genetic modification of the TCA cycle and heterologous expression of 2-oxoglutarate permease by the modified semi-continuous cultivation.

Photosynthetic production of glycerol by a recombinant cyanobacterium

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. Glycerol is an important commodity chemical. Introduction of phosphoglycerol phosphatase 2 from Saccharomyces cerevisiae into the model cyanobacterium Synechocystis sp. PCC6803 resulted in a mutant strain that produced a considerable amount of glycerol from light, water and COPhotosynthetic production . Mild salt stress (200 mM NaCl) on the cells led to an increase of the extracellular glycerol concentration of more than 20%. Under these conditions the mutant accumulated glycerol to an extracellular concentration of 14.3 mM after 17 days of culturing.

Photosynthetic and extracellular production of glucosylglycerol by genetically engineered and gel-encapsulated cyanobacteria.

Glucosylglycerol (GG) has a range of potential applications in health, pharmacy, and cosmetics due to its physiological, protein-stabilizing, and antioxidative properties. In addition to chemical synthesis and enzymatic catalysis, GG can be produced as a protective osmolyte in salt-stressed bacteria, such as the cyanobacterium Synechocystis sp. PCC 6803. Here, we presented an efficient GG production and secretion by genetically modified and encapsulated Synechocystis cells grown in a semicontinuous manner. We improved the production and secretion of GG in Synechocystis by first disrupting both the ggtC and ggtD genes, which encode the subunits of a GG uptake transporter, as well as the ggpR gene, which encodes a repressor for GG synthesis. Then, we confirmed that the rapid GG release from salt-stressed cells of Synechocystis depended on the ion gradient across the cell membrane. Finally, we proved the feasibility of an agar gel encapsulation method in supporting cell growth and the GG production of Synechocystis under semicontinuous culturing conditions.

Role of Rbp1 in the Acquired Chill-Light Tolerance of Cyanobacteria

Synechocystis sp. strain PCC 6803 cultured at 30°C losses viability quickly under chill (5°C)-light stress but becomes highly tolerant to the stress after conditioning at 15°C (Y. Yang, C. Yin, W. Li, and X. Xu, J. Bacteriol. 190:1554-1560, 2008). Hypothetically, certain factors induced during preconditioning are involved in acquisition of chill-light tolerance. In this study, Rbp1 (RNA-binding protein 1) rather than Rbp2 was found to be accumulated during preconditioning, and the accumulation of Rbp1 was correlated with the increase of chill-light tolerance. Inactivation of its encoding gene rbp1 led to a great reduction in the acquired chill-light tolerance, while ectopic expression of rbp1 enabled the cyanobacterium to survive the chill-light stress without preconditioning. Microarray analyses suggested that the Rbp1-dependent chill-light tolerance may not be based on its influence on mRNA abundance of certain genes. Similarly to that in Synechocystis, the Rbp1 homologue(s) can be accumulated in Microcystis cells collected from a subtropic lake in low-temperature seasons. Rbp1 is the first factor shown to be both accumulated early during preconditioning and directly involved in development of chill-light tolerance in Synechocystis. Its accumulation may greatly enhance the overwintering capability in certain groups of cyanobacteria.

Application of the FLP/FRT recombination system in cyanobacteria for construction of markerless mutants.

Due to efficient photosynthetic capability, robust growth, and clear genetic background, cyanobacteria are recently used for production of different biofuel and biochemical molecules by genetic engineering and showed great potentials as the next-generation microbial cell factory. For improving the production of bio-products, a number of genetic modifications are important for cyanobacteria. However, the system-level genetic modification of cyanobacteria is limited by the lack of efficient method for marker recycling. In this investigation, we introduced the self-replicable shutter vectors harboring the flipase (FLP) gene from Saccharomyces cerevisiae into two mutants of Synechocystis sp. PCC6803 and Synechococcus elongatus PCC7942 whose genomes were inserted by a kanamycin resistance gene with flipase recombination target (FRT) flanking, respectively. Transcriptional analysis by reverse transcription polymerase chain reaction showed that FLP gene was transcripted in both the two cyanobacterial strains. Genotyping analysis indicated that FLP performed its function in vivo in both two cyanobacterial strains, and the following DNA sequencing analysis on the targeted loci further confirmed that FLP exactly excised and ligated the two FRT sites between which a kanamycin resistance gene is located. The homozygous mutants free of the kanamycin resistance gene cassette were obtained by conditional expression of FLP and further dilution plating. The shuttle vectors carrying the FLP gene were then lost in these mutants by growing in the absence of antibiotics and the further single colony separation. These results demonstrate that FLP/FRT recombination system is able to be applied to the construction of markerless mutant in both Synechocystis sp. PCC6803 and S. elongatus PCC7942.