Synthetic microbial consortium with specific roles designated by genetic circuits for cooperative chemical production.

Synthetic microbial consortia consisting of microorganisms with different synthetic genetic circuits or divided synthetic metabolic pathway components can exert functions that are beyond the capacities of single microorganisms. However, few consortia of microorganisms with different synthetic genetic circuits have been developed. We designed and constructed a synthetic microbial consortium composed of an enzyme-producing strain and a target chemical-producing strain using Escherichia coli for chemical production with efficient saccharification. The enzyme-producing strain harbored a synthetic genetic circuit to produce beta-glucosidase, which converts cellobiose to glucose, destroys itself via the lytic genes, and release the enzyme when the desired cell density is reached. The target chemical-producing strain was programmed by a synthetic genetic circuit to express enzymes in the synthetic metabolic pathway for isopropanol production when the enzyme-producing strain grows until release of the enzyme. Our results demonstrate the benefits of synthetic microbial consortia with distributed tasks for effective chemical production from biomass.

Recognizability of heterologous co-chaperones with Streptococcus intermedius DnaK and Escherichia coli DnaK.

Streptococcus intermedius DnaK complements the temperature-sensitive phenotype of an Escherichia coli dnaK null mutant only when co-chaperones DnaJ and GrpE are co-expressed. Therefore, whether S. intermedius DnaK and E. coli DnaK can recognize heterologous co-chaperones in vitro was examined. Addition of heterologous GrpE to DnaK and DnaJ partially stimulated adenosine triphosphatase (ATPase) activity, and almost completely stimulated luciferase refolding activity. Addition of heterologous DnaJ to GrpE and DnaK also stimulated ATPase activity; however, significant luciferase refolding activity was not observed. Moreover, E. coli DnaJ had a negative effect on the luciferase refolding activity of the S. intermedius DnaK chaperone system. In E. coli chaperone mutants, with the exception of E. coli DnaJ, stronger expression of the heterologous co-chaperones partially or almost completely complemented the temperature-sensitive-phenotype. These results indicate that all heterologous co-chaperones can at least partially recognize DnaK of a distantly related species. A region of the ATPase domain that is present in the DnaK of gram-negative bacteria is absent from the DnaK of gram-positive bacteria. This region is believed to be important for recognition of co-chaperones from gram-negative bacteria. However, insertion of this segment into S. intermedius DnaK failed to increase its ability to recognize E. coli co-chaperones, implying that this region is unnecessary or insufficient for the recognition of E. coli co-chaperones. Thus, our data suggest that a basic structural similarity is conserved among the components of the S. intermedius and E. coli DnaK chaperone systems, allowing weak associations between heterologous components.

Reconstruction of a metabolic regulatory network in Escherichia coli for purposeful switching from cell growth mode to production mode in direct GABA fermentation from glucose.

γ-aminobutyric acid (GABA) is a drug and functional food additive and is used as a monomer for producing the biodegradable plastic, polyamide 4. Recently, direct GABA fermentation from glucose has been developed as an alternative to glutamate-based whole cell bioconversion. Although total productivity in fermentation is determined by the specific productivity and cell amount responsible for GABA production, the optimal metabolic state for GABA production conflicts with that for bacterial cell growth. Herein, we demonstrated metabolic state switching from the cell growth mode based on the metabolic pathways of the wild type strain to a GABA production mode based on a synthetic metabolic pathway in Escherichia coli through rewriting of the metabolic regulatory network and pathway engineering. The GABA production mode was achieved by multiple strategies such as conditional interruption of the TCA and glyoxylate cycles, engineering of GABA production pathway including a bypass for precursor metabolite supply, and upregulation of GABA transporter. As a result, we achieved 3-fold improvement in total GABA production titer and yield (4.8g/L, 49.2% (mol/mol glucose)) in batch fermentation compared to the case without metabolic state switching (1.6g/L, 16.4% (mol/mol glucose)). This study reports the highest GABA production performance among previous reports on GABA fermentation from glucose using engineered E. coli.

Enhancement of 3-hydroxypropionic acid production from glycerol by using a metabolic toggle switch.

BACKGROUND: 3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli. RESULTS: The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux. CONCLUSION: The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch.

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.

Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light.

Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by adc) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days.

In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli.

In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.

Dual synthetic pathway for 3-hydroxypropionic acid production in engineered Escherichia coli.

3-Hydroxypropionic acid (3-HP) is an important platform C3 chemical; production of 3-HP in recombinant Escherichia coli by synthetic pathways has been the focus of a lot of research. When glycerol is used as a substrate to produce 3-HP in E. coli, only the ALDH pathway (employing aldehyde dehydrogenase (ALDH) for conversion of 3-hydroxypropionaldehyde (3-HPA) into 3-HP) has been utilized as a synthetic pathway. However, several bacteria (including Klebsiella pneumoniae) are known to have the ability to produce 3-HP by the Pdu pathway (employing the PduP, PduL, and PduW enzymes). Here, we report the production of 3-HP in E. coli by using the Pdu pathway from K. pneumoniae as a synthetic pathway. Moreover, a strain harboring a dual synthetic pathways (ALDH and Pdu) exhibited a 70% increase in 3-HP titer compared to one harboring the ALDH pathway alone (56.1 ± 0.736 mM and 33.1 ± 0.920 mM, respectively). To our knowledge, this is the first report of 3-HP production by E. coli harboring the Pdu pathway, with the dual synthetic pathway showing the highest yield ever reported by batch culture [54.1% (mol/mol)].

Role of Streptococcus intermedius DnaK chaperone system in stress tolerance and pathogenicity.

Streptococcus intermedius is a facultatively anaerobic, opportunistic pathogen that causes purulent infections and abscess formation. The DnaK chaperone system has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity. However, the role of DnaK in S. intermedius remains unclear. Therefore, we constructed a dnaK knockout mutant that exhibited slow growth, thermosensitivity, accumulation of GroEL in the cell, and reduced cytotoxicity to HepG2 cells. The level of secretion of a major pathogenic factor, intermedilysin, was not affected by dnaK mutation. We further examined the function and property of the S. intermedius DnaK chaperone system by using Escherichia coli ΔdnaK and ΔrpoH mutant strains. S. intermedius DnaK could not complement the thermosensitivity of E. coli ΔdnaK mutant. However, the intact S. intermedius DnaK chaperone system could complement the thermosensitivity and acid sensitivity of E. coli ΔdnaK mutant. The S. intermedius DnaK chaperone system could regulate the activity and stability of the heat shock transcription factor σ(32) in E. coli, although S. intermedius does not utilize σ(32) for heat shock transcription. The S. intermedius DnaK chaperone system was also able to efficiently eliminate the aggregated proteins from ΔrpoH mutant cells. Overall, our data showed that the S. intermedius DnaK chaperone system has important functions in quality control of cellular proteins but has less participation in the modulation of expression of pathogenic factors.

Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress.

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.