Medium-chain α,ω-dicarboxylic acids (MCDCA) have versatile applications in the chemical industry as chemical products and intermediates, such as polyesters and polyamides, as well as pharmaceuticals. Until recently, much research has been done in the field of biochemical engineering for the production of MCDCA, including monoalkyl esters of ioanedioic acid, showing the potential of bioprocesses with more efficient conversion and higher selectivity than conventional processes. Here, we developed a nonanedioic acid ethyl ester (NDAEE) biosensing system by placing the green fluorescent protein (GFP) gene under the control of the Escherichia coli inaA promoter.
Escherichia coli MG1655 was engineered for the production of NDAEE from the bioconversion of nonanoic acid ethyl ester (NAEE) by introducing a heterologous ω-oxidation pathway. And then a competing pathway was inhibited by deleting fadE, which encodes an acyl-CoA dehydrogenase, thereby blocking the native β-oxidation pathway that degrades NDAEE. Subsequently, by introducing a homologous inaA promoter known to be induced by decanoate upstream of the GFP gene of the pPROBE vector, we successfully developed a biosensor induced by NDAEE with a dynamic range of 2.2-fold when 5mM NDAEE was added externally.
When the biosensor was transformed into the NDAEE-producing strain and produced 6.4 mM NDAEE, the normalized GFP expression level was about 2.0-fold higher than without NDAEE production. This study demonstrated that this biosensor can be used to monitor high-yielding strains, optimize culture conditions, and dynamically control metabolic fluxes.
Introduction
The above studies suggested methods for the sustainable production of MCDCA and MCDCA derivatives, however, their titers are still below those required for industrial applications. Therefore, metabolic engineering approaches are needed for further improvements including the stability of the ω-oxidation system and cell viability during the bioconversion of MCDCA [ 10 , 11 ]. As one of the applications in a metabolic engineering context, biosensors have shown high potential both in high-throughput screening of large mutant libraries and in the implementation of synthetic circuits for dynamic control of cellular metabolism [12].
PinaA has a MarA/Rob/SoxS regulon involved in the membrane stress response to environmental changes by coupling extracellular stimuli [14]. The Rob regulator activates the transcription of the SoxRS/MarA/Rob regulon in the presence of its inducers: dipyridyl, bile salts or decanoate [15, 16]. Decanoate, which is a structural analog of NDAEE with one non-esterified carboxyl group, is also known to directly interact with Rob's C-terminal domain to disperse sequestered Rob [ 17 ].
The expression level of PinaA in the NDAEE biosensor correlated with the concentration of extracellularly added NDAEE. In this work, we developed a biosensor system capable of detecting NDAEE, an ω-oxidized form of NAEE known to have high specific activity with AlkB [ 9 ]. Then, the NDAEE-degrading pathway was removed by deleting the chromosomal fadE, acyl-CoA dehydrogenase-encoding gene.
Methods and materials
- Chemicals and enzymes
- Media and cultivation conditions
- Whole-cell bioconversion
- Product and data analysis
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) or Alfa Aesar (Haverhill, MA) except azelaic acid monoethyl ester (NDAEE, Santa Cruz Biotechnology, Dallas, TX) and dicyclopropyl ketone (DCPK, TCI, Tokyo ). , Japan). When the biosensor system was tested for the interaction of exogenous ligands, chemicals were added at an OD600 of 1.0. In the production of NDAEE with E_BGTHJL containing pPinaA-AT, TB medium was used instead of LB medium, as growth arrest was detected in LB during production.
The produced NDAEE at indicated time was measured by the process of 'Product and data analysis'. The biotransformation was carried out in 250 mL shake flask by directly adding 15 mM NAME or NAEE as a substrate. The biotransformations were performed in a shaking incubator at 30 °C and 200 rpm, and samples were collected for product quantification by gas chromatography.
For quantification of NDA alkyl esters, 500 μl of culture media were supplemented with 50 μL of 12 N HCl and nonadecanoic acid as an internal standard with an initial concentration of 100 mg/l.
Results and discussion
Degradation of NDA alkyl esters by E. coli native β-oxidation pathway
In the case of NDAEE production in BGTJH, almost all of the NDAEE produced was degraded 12 h after the start of bioconversion. We hypothesized that NDAME and NDAEE, synthesized from whole-cell biocatalysts, are degraded by the natural β-oxidation pathway, while other dicarboxylic acids are degraded. NDAME and NDAEE production from BGTJH when NAME and NAEE were added as substrate, respectively.
Blue diamonds represent NDAME production when NAME was added, and green triangles represent NDAEE production when NAEE was added. Bioconversion was performed with resting OD600 16 cells in 10 ml of biotransformation medium. The β-oxidation pathway has been reported to be inactivated by deletion of the fadE gene [ 27 ].
Therefore, fadE was eliminated in BGTJH strain to construct another recombinant strain, E_BGTJH, which not only produces NDA alkyl esters by ω-oxidation pathway, but also accumulates the products instead of degrading them through β-oxidation pathway. In the bioconversion process, unlike BGTJH, the fadE deletion mutant strain E_BGTJH maintained the final ω-oxidation pathway products (Figs. 6 and 7). E_BGTJH accumulated NDAME and NDAEE up to 747 uM and 629 uM, but in BGTJH, NDAME was reduced to 249 uM and NDAEE was not detected after 24 h of bioconversion.
These results indicate that the catabolism of MCDCA with monoalkyl ester can be initiated by fatty acid CoA-ligase and further catabolized by β-oxidation enzymes including FadE, FadA, and FadB. This is because NDAEE has more total carbon atoms than NDAME, therefore, they have different substrate specificity of the fatty acid-CoA ligase FadD. FadD, the rate-limiting step of β-oxidation, has maximal activity on fatty acids in the range of 12 to 18 carbon atoms, and activity decreases as the fatty acid carbon chain becomes shorter [ 28 ].
NDAME production of BGTJH and E_BGTJH when 15 mM NAME was added as a substrate in the bioconversion. NDAEE production of BGTJH and E_BGTJH when 15 mM NAEE was added as a substrate in bioconversion. Green triangles indicate the NDAEE concentration of BGTJH and red circles indicate the same concentration of the compound when the β-oxidation was removed in the strain.
Construction of a biosensor for detection of NDAEE
The mechanism of the PinaA-mediated biosensor detecting exogenously added ligand (herein decanoate) is described. a) At low concentrations of ligand, Rob is sequestered and inactivated, preventing PinaA from being activated. At high concentrations of ligand, Rob is distributed by an unknown mechanism, involving activation of PinaA and expression of GFP. The expression level of GFP was measured in the presence of different concentrations of decanoate, NDAEE, NAEE, IPTG and DCPK.
We then applied pPinaA to NDAEE-producing strains harboring the ω-oxidation pathway to confirm whether pPinaA could also detect NDAEE produced in vivo. This strain produced different amounts of NDAEE depending on the level of IPTG induction and produced the most at induction of 0.01 to 0.02 mM. Although the strain only expressed AlkBGT and the titer was low (up to about 50 µM NDAEE), pPinaA expressed GFP to the titer.
We also introduced the inaA promoter-based biosensor into the fadE mutant strain harboring pBGTHJL (E_BGTHJL pPinaA-AT). E_BGTJHL pPinaA-AT produced 6.4 mM NDAEE during 30 h of bioconversion when NAEE was added as a substrate (Fig. 9b). Overall, these results indicate that the designed PinaA-based biosensor detected not only exogenously added NDAEE but also endogenously produced NDAEE.
The GFP expression level of pPinaA (or pPinaA-AT) was measured during the production of NDAEE. a) E_BGT pPinaA was used for the detection of endogenously produced NDAEE. Green bars represent relative green fluorescence (GFP normalized to OD600 then divided by the normalized GFP value of non-induced E_BGT pPinaA). NAEE- is when E_BGTHJL pPinaA-AT was grown and produced in NAEE-free media while NAEE+ is when the strain was grown and produced in the same media with 15mM NAEE addition.
Green bars represent relative green fluorescence (GFP normalized to OD600 then divided by the normalized GFP value of NAEE-free E_BGTHJL pPinaA-AT).
Conclusions
Design and creation of a synthetic omega oxidation pathway in Saccharomyces Cerevisiae enables the production of medium chain Α, ω-dicarboxylic acids. Production of Long-Chain Α,ω-Dicarboxylic Acids by Engineered Escherichia Coli from Renewable Fatty Acids and Plant Oils. Enhancement of Α,ω-Dicarboxylic acid production by the expression of xylose reductase for refactoring redox cofactor regeneration.
Expansion of the ω-oxidation system AlkBGTL of Pseudomonas Putida GPo1 with AlkJ and AlkH results in the exclusive production of a mono-esterified dicarboxylic acid in E. Kinetic analysis of oxyfunctionalization of the terminal and unactivated C-H bond in fatty acid methyl esters by monooxygenase-based whole-cell biocatalysis. MarA, SoxS and Rob from Escherichia Coli - Global regulators of multidrug resistance, virulence and stress response.
Bile salts and fatty acids induce the expression of Escherichia Coli AcrAB Multidrug Efflux Pump through their interaction with Rob Regulatory Protein. Two Functions of the C-Terminal Domain of Escherichia Coli Rob: Mediating "sequestration-distribution" as a Novel On-Off Switch for Regulating Rob's Activity as a Transcriptional Activator and Preventing Rob Degradation by Lon Protease. Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium-chain ω-functionalized carboxylic acids.
Biochemical analysis of recombinant AlkJ from Pseudomonas Putida reveals a membrane-associated, flavinadenine dinucleotide-dependent dehydrogenase suitable for biosynthetic production of aliphatic aldehydes. Expression, stability and performance of three-component alkane mono-oxygenase of Pseudomonas Oleovorans in Escherichia Coli. Finally, I would like to thank my family, who have endured a difficult time more than anyone else, and thank you for all the hard work.
