Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

Production of polyhydroxyalkanoates by the thermophile Cupriavidus cauae PHS1.

Thermophilic production of polyhydroxyalkanoate is considered a very promising way to overcome the problems that may arise when using mesophilic strains. This study reports the first thermophilic polyhydroxybutyrate-producing Cupriavidus species, which are known as the best polyhydroxybutyrate-producing microorganisms. Cupriavidus cauae PHS1 harbors a phbCABR cluster with high similarity to the corresponding proteins of C. necator H16 (80, 93, 96, and 97 %). This strain can produce polyhydroxybutyrate from a range of substrates, including acetate (5 g/L) and phenol (1 g/L), yielding 7.6 % and 18.9 % polyhydroxybutyrate, respectively. Moreover, the strain produced polyhydroxybutyrate at temperatures ranging from 25 to 50 °C, with the highest polyhydroxybutyrate content (47 °C) observed at 45 °C from gluconate. Additionally, the strain could incorporate 3-hydroxyvalerate (12.5 mol. %) into the polyhydroxybutyrate polymer using levulinic acid as a precursor. Thus, Cupriavidus cauae PHS1 may be a promising polyhydroxybutyrate producer as alternative for mesophilic polyhydroxybutyrate-producing Cupriavidus species.

Characterization of an Entner-Doudoroff pathway-activated Escherichia coli.

BACKGROUND: Escherichia coli have both the Embden-Meyerhof-Parnas pathway (EMPP) and Entner-Doudoroff pathway (EDP) for glucose breakdown, while the EDP primarily remains inactive for glucose metabolism. However, EDP is a more favorable route than EMPP for the production of certain products. RESULTS: EDP was activated by deleting the pfkAB genes in conjunction with subsequent adaptive laboratory evolution (ALE). The evolved strains acquired mutations in transcriptional regulatory genes for glycolytic process (crp, galR, and gntR) and in glycolysis-related genes (gnd, ptsG, and talB). The genotypic, transcriptomic and phenotypic analyses of those mutations deepen our understanding of their beneficial effects on cellulosic biomass bio-conversion. On top of these scientific understandings, we further engineered the strain to produce higher level of lycopene and 3-hydroxypropionic acid. CONCLUSIONS: These results indicate that the E. coli strain has innate capability to use EDP in lieu of EMPP for glucose metabolism, and this versatility can be harnessed to further engineer E. coli for specific biotechnological applications.

Combinatorial Metabolic Engineering Strategies for the Enhanced Production of Free Fatty Acids in Escherichia coli.

In this study, we evaluated the effects of several metabolic engineering strategies in a systematic and combinatorial manner to enhance the free fatty acid (FFA) production in Escherichia coli. The strategies included (i) overexpression of mutant thioesterase I ('TesAR64C) to efficiently release the FFAs from fatty acyl-ACP; (ii) coexpression of global regulatory protein FadR; (iii) heterologous expression of methylmalonyl-CoA carboxyltransferase and phosphoenolpyruvate carboxylase to synthesize fatty acid precursor molecule malonyl-CoA; and (iv) disruption of genes associated with membrane proteins (GusC, MdlA, and EnvR) to improve the cellular state and export the FFAs outside the cell. The synergistic effects of these genetic modifications in strain SBF50 yielded 7.2 ± 0.11 g/L FFAs at the shake flask level. In fed-batch cultivation under nitrogen-limiting conditions, strain SBF50 produced 33.6 ± 0.02 g/L FFAs with a productivity of 0.7 g/L/h from glucose, which is the maximum titer reported in E. coli to date. Combinatorial metabolic engineering approaches can prove to be highly useful for the large-scale production of FA-derived chemicals and fuels.

Bioproduction of propionic acid using levulinic acid by engineered Pseudomonas putida.

The present study elaborates on the propionic acid (PA) production by the well-known microbial cell factory Pseudomonas putida EM42 and its capacity to utilize biomass-derived levulinic acid (LA). Primarily, the P. putida EM42 strain was engineered to produce PA by deleting the methylcitrate synthase (PrpC) and propionyl-CoA synthase (PrpE) genes. Subsequently, a LA-inducible expression system was employed to express yciA (encoding thioesterase) from Haemophilus influenzae and ygfH (encoding propionyl-CoA: succinate CoA transferase) from Escherichia coli to improve the PA production by up to 10-fold under flask scale cultivation. The engineered P. putida EM42:ΔCE:yciA:ygfH was used to optimize the bioprocess to further improve the PA production titer. Moreover, the fed-batch fermentation performed under optimized conditions in a 5 L bioreactor resulted in the titer, productivity, and molar yield for PA production of 26.8 g/L, 0.3 g/L/h, and 83%, respectively. This study, thus, successfully explored the LA catabolic pathway of P. putida as an alternative route for the sustainable and industrial production of PA from LA.

Substrate-inducible and antibiotic-free high-level 4-hydroxyvaleric acid production in engineered Escherichia coli.

In this study, we developed a levulinic acid (LA)-inducible and antibiotic-free plasmid system mediated by HpdR/P hpdH and infA-complementation to produce 4-hydroxyvaleric acid (4-HV) from LA in an engineered Escherichia coli strain. The system was efficiently induced by the addition of the LA substrate and resulted in tight dose-dependent control and fine-tuning of gene expression. By engineering the 5' untranslated region (UTR) of hpdR mRNA, the gene expression of green fluorescent protein (GFP) increased by at least two-fold under the hpdH promoter. Furthermore, by evaluating the robustness and plasmid stability of the proposed system, the engineered strain, IRV750f, expressing the engineered 3-hydroxybutyrate dehydrogenase (3HBDH∗) and formate dehydrogenase (CbFDH), produced 82 g/L of 4-HV from LA, with a productivity of 3.4 g/L/h and molar conversion of 92% in the fed-batch cultivation (5 L fermenter) without the addition of antibiotics or external inducers. Overall, the reported system was highly beneficial for the large-scale and cost-effective microbial production of value-added products and bulk chemicals from the renewable substrate, LA.

Enhanced production of nonanedioic acid from nonanoic acid by engineered Escherichia coli.

In this study, whole-cell biotransformation was conducted to produce nonanedioic acid from nonanoic acid by expressing the alkane hydroxylating system (AlkBGT) from Pseudomonas putida GPo1 in Escherichia coli. Following adaptive laboratory evolution, an efficient E. coli mutant strain, designated as MRE, was successfully obtained, demonstrating the fastest growth (27-fold higher) on nonanoic acid as the sole carbon source compared to the wild-type strain. Additionally, the MRE strain was engineered to block nonanoic acid degradation by deleting fadE. The resulting strain exhibited a 12.8-fold increase in nonanedioic acid production compared to the wild-type strain. Six mutations in acrR, Pcrp , dppA, PfadD , e14, and yeaR were identified in the mutant MRE strain, which was characterized using genomic modifications and RNA-sequencing. The acquired mutations were found to be beneficial for rapid growth and nonanedioic acid production.

Metabolic Engineering of Escherichia coli for Production of Polyhydroxyalkanoates with Hydroxyvaleric Acid Derived from Levulinic Acid.

Polyhydroxyalkanoates (PHAs) are emerging as alternatives to plastics by replacing fossil fuels with renewable raw substrates. Herein, we present the construction of engineered Escherichia coli strains to produce short-chain-length PHAs (scl-PHAs), including the monomers 4-hydroxyvalerate (4HV) and 3-hydroxyvalerate (3HV) produced from levulinic acid (LA). First, an E. coli strain expressing genes (lvaEDABC) from the LA metabolic pathway of Pseudomonas putida KT2440 was constructed to generate 4HV-CoA and 3HV-CoA. Second, both PhaAB enzymes from Cupriavidus necator H16 were expressed to supply 3-hydroxybutyrate (3HB)-CoA from acetyl-CoA. Finally, PHA synthase (PhaCCv) from Chromobacterium violaceum was introduced for the subsequent polymerization of these three monomers. The resulting E. coli strains produced four PHAs (w/w% of dry cell weight): 9.1 wt% P(4HV), 1.7 wt% P(3HV-co-4HV), 24.2 wt% P(3HB-co-4HV), and 35.6 wt% P(3HB-co-3HV-co-4HV).

Inducible and tunable gene expression systems for Pseudomonas putida KT2440.

Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/PhpdH was also efficiently induced by LA. LvaR/PlvaA and XutR/PxutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/Pzwf1 showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.

Levulinic Acid-Inducible and Tunable Gene Expression System for Methylorubrum extorquens.

Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/P hpdH ) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%-100%) to that of the widely used strong promoters such as P mxaF and P L/O4 . The HpdR/P hpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.

Metabolic engineering of Pseudomonas putida for the production of various types of short-chain-length polyhydroxyalkanoates from levulinic acid.

Poly(3-hydroxybutyrate), a short-chain-length polyhydroxyalkanoate (scl-PHA), is considered as a good alternative to conventional synthetic plastics. However, various biopolymers with diverse characteristics are still in demand. In this study, four different types of scl-PHA were successfully produced by engineering levulinic acid (LA) utilization metabolic pathway and expressing heterologous PHA synthase (PhaEC), acetyl-CoA acetyltransferase (PhaA), and acetyl-CoA reductase (PhaB) in Pseudomonas putida EM42. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)], poly(3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HV-co-4HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] were produced by the natural LA pathway, poly(4-hydroxyvalerate) by lvaAB-deleted LA pathway, and P(3HV-co-4HV) and P(3HB-co-3HV-co-4HV) with relatively high 3HV by fadB-deleted LA pathway. PHA with different monomer fractions could be produced using different PHA synthases. Scl-PHA contents reached approximately 40% of cell dry mass under non-optimized flask culture. This demonstrates that the LA catabolic pathway may be a good alternative route to provide monomers for the production of various types of PHA.

Metabolic engineering of Escherichia coli for 2,3-butanediol production from cellulosic biomass by using glucose-inducible gene expression system.

A glucose-inducible gene expression system has been developed using HexR-Pzwf1 of Pseudomonas putida to induce the metabolic pathways. Since the system is controlled by an Entner-Doudoroff pathway (EDP) intermediate, the EDP of Escherichia coli was activated by deleting pfkA and gntR genes. Growth experiment with green fluorescent protein as a reporter indicated that the induction of this system was tightly controlled over a wide range of glucose in E. coli without adding any inducer. 2,3-butanediol (BDO) synthetic pathway genes were expressed by this system in the pfkA-gntR-deleted strain. The resultant engineered strain harbouring this system efficiently produced BDO with a 71% increased titer than the control strain. The strain was also able to produce BDO from a mixture of glucose and xylose which is comparable to glucose alone. Further, the strain produced 11 g/L of BDO at a yield of 0.48 g/g from the hydrolysate of empty palm fruit bunches. This system can also be applied in many other bio-production processes from lignocellulosic biomass.

High-Level Production of 4-Hydroxyvalerate from Levulinic Acid via Whole-Cell Biotransformation Decoupled from Cell Metabolism.

γ-Hydroxyvalerate (4HV) is an important monomer used to produce various valuable polymers and products. In this study, an engineered 3-hydroxybutyrate dehydrogenase that can convert levulinic acid (LA) into 4HV was co-expressed with a cofactor (NADH) regeneration system mediated by an NAD+-dependent formate dehydrogenase (CbFDH) in the Escherichia coli strain, MG1655. The resulting strain produced 23-fold more 4HV in a shake flask. The 4HV production was not dependent on ATP and required low aeration; all of these are considered beneficial characteristics for the production of target compounds, especially at an industrial scale. Under optimized conditions in a 5 L fermenter, the titer, productivity, and molar conversion efficiency for 4HV reached 100 g/L, 4.2 g/L/h, and 92%, respectively. Our system could prove to be a promising method for the large-scale production of 4HV from LA at low-cost and using a renewable biomass source.

Implementation of Combinatorial Genetic and Microenvironmental Engineering to Microbial-Based Field-Deployable Microbead Biosensors for Highly Sensitive and Remote Chemical Detection.

Bioreporters, microbial species genetically engineered to provide measurable signals in response to specific chemicals, have been widely investigated as sensors for biomedical and environmental monitoring. More specifically, the bioreporter encapsulated within a biocompatible material, such as a hydrogel that can provide a suitable microenvironment for its prolonged activity as well as efficient scalable production, has been viewed as a more broadly applicable mode of biosensors. In this study, alginate-based microbeads encapsulated with the bacterial bioreporter capable of expressing green fluorescence protein in response to nitro compounds (e.g., trinitrotoluene and dinitrotoluene) are developed as biosensors. To significantly enhance the sensitivity of the microbial-based microbead biosensors, "multifaceted" modification strategies are simultaneously employed: (1) multiple genetic modifications of the bioreporter, (2) tuning the physicomechanical properties of the encapsulating microbeads, (3) controlling the initial cell density within the microbeads, and (4) enrichment of nitro compounds inside microbeads via functional nanomaterials. These microbial and microenvironmental engineering approaches combine to significantly enhance the sensing capability, even allowing highly sensitive remote detection under a low-vapor phase. Thus, the strategy developed herein is expected to contribute to various cell-based biosensors.

Fluorescence Enhancement from Nitro-Compound-Sensitive Bacteria within Spherical Hydrogel Scaffolds.

For the safety of both production and life, it is a very significant issue to detect explosive nitro compounds in a remote way or over a long distance. Here, we report that nitro compounds were detected by the bacterial sensor based on hydrogel microbeads as a platform. Green fluorescent protein-producing Escherichia coli, which was genetically engineered to be sensitive to nitro compounds, was loaded within poly(2-hydroxyethyl methacrylate) [poly(HEMA)]-based hydrogel beads, in which fluorescent signals from bacteria were concentrated and strong enough to be easily detected. For efficient loading of negatively charged bacteria, the surface charge of poly(HEMA)-based beads was controlled by copolymerization with 2-(methacryloyloxy)ethyltrimethylammonium chloride (MAETC) as a cationic monomer. With the addition of MAETC, the cell affinity was nine times enhanced by the interaction between the positively charged poly(HEMA- co-MAETC) beads and negatively charged bacteria. The increased cell affinity resulted in an enhancement of a sensing signal. After exposure to 2,4,6-trinitrotoluene, a typical explosive nitro compound, the fluorescence intensity of bacterial sensors using poly(HEMA- co-MAETC) beads having 80 wt % MAETC was five times increased compared to those based on poly(HEMA) beads. This amplification of the fluorescent signal enables easier detection of explosives efficiently by a remote detection, even over a long distance.

Engineering the lva operon and Optimization of Culture Conditions for Enhanced Production of 4-Hydroxyvalerate from Levulinic Acid in Pseudomonas putida KT2440.

Monomeric 4-hydroxyvalerate is a versatile chemical used to produce various commodities and fine chemicals. In the present study, the lvaAB gene was deleted from the lva operon in Pseudomonas putida KT2440 and tesB, obtained from Escherichia coli, was overexpressed under the control of the lva operon system, which is induced by the substrate levulinic acid and the product 4-hydroxyvalerate to produce 4-hydroxyvalerate from levulinic acid. The lvaAB-deleted strain showed almost complete conversion of levulinic acid to 4-hydroxyvalerate, compared with 24% conversion in the wild-type strain. In addition, under optimized culture conditions, the final engineered strain produced a maximum of 50 g/L 4-hydroxyvalerate with 97% conversion from levulinic acid. The system presented here could be applied to produce high titers of 4-hydroxyvalerate in a cost-effective manner at a large scale from renewable cellulosic biomass.

High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform.

Screening target microorganisms from a mutated recombinant library plays a crucial role in advancing synthetic biology and metabolic engineering. However, conventional screening tools have several limitations regarding throughput, cost, and labor. Here, we used the fluid array platform to conduct high-throughput screening (HTS) that identified Escherichia coli 'TesA thioesterase mutants producing elevated yields of free fatty acids (FFAs) from a large (106) mutant library. A growth-based screening method using a TetA-RFP fusion sensing mechanism and a reporter-based screening method using high-level FFA producing mutants were employed to identify these mutants via HTS. The platform was able to cover >95% of the mutation library, and it screened target cells from many arrays of the fluid array platform so that a post-analysis could be conducted by gas chromatography. The 'TesA mutation of each isolated mutant showing improved FFA production in E. coli was characterized, and its enhanced FFA production capability was confirmed.

Enhancement of α,ω-Dicarboxylic Acid Production by the Expression of Xylose Reductase for Refactoring Redox Cofactor Regeneration.

The production of α,ω-dicarboxylic acids (DCAs) by whole-cell biocatalysis is often limited by cofactor regeneration. Here, ω-oxidation pathway genes (monooxygenase, alcohol dehydrogenase, and aldehyde dehydrogenase) were coexpressed with a xylose reductase (XR) gene to regenerate cofactors in an engineered Escherichia coli strain that cometabolizes glucose and xylose. The resulting strain exhibited a 180% increase in DCA production compared with the control strain without XR, and produced xylitol in the presence of xylose. Expression of monooxygenase and XR without other ω-oxidation pathway genes resulted in an additional increase in tetradecanedioic acid concentration and a substrate conversion of 95%, which was 198% higher than that associated with the control strain. The expression of XR helped the system to regenerate and balance the cofactors thereby achieving maximum substrate conversion efficiency. It could serve as an efficient platform for the industrial production of α,ω-DCAs.

Increasing Extracellular Free Fatty Acid Production in Escherichia coli by Disrupting Membrane Transport Systems.

Transposon mutagenesis was used to identify three mutants of E. coli that exhibited increased free fatty acid (FFA) production, which resulted from the disruption of genes related to membrane transport. Deletion of envR, gusC, and mdlA individually in a recombinant E. coli strain resulted in 1.4-, 1.8-, and 1.2-fold increases in total FFA production, respectively. In particular, deletion of envR increased the percentage of extracellular FFA to 46%, compared with 29% for the control strain. Multiple deletion of envR, gusC, mdlA, ompF, and fadL had a synergistic effect on FFA production, resulting in high extracellular FFA production, comprising up to 50% of total FFA production. This study has identified new membrane proteins involved in FFA production and showed that genetic engineering targeting these membrane transporters is important to increase both total FFA and extracellular FFA production.

Novosphingobium humi sp. nov., isolated from soil of a military shooting range.

A Gram-stain-negative, strictly aerobic bacterium, designated R1-4T, was isolated from soil from a military shooting range in the Republic of Korea. Cells were non-motile short rods, oxidase-positive and catalase-negative. Growth of R1-4T was observed at 15-45 °C (optimum, 30 °C) and pH 6.0-9.0 (optimum, pH 7.0). R1-4T contained summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c), summed feature 3 (comprising C16 : 1ω7c/C16 : 1ω6c), cyclo-C19 : 0ω8c and C16 : 0 as the major fatty acids and ubiquinone-10 as the sole isoprenoid quinone. Phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, sphingoglycolipid, phosphatidylcholine, an unknown glycolipid and four unknown lipids were detected as polar lipids. The major polyamine was spermidine. The G+C content of the genomic DNA was 64.4 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that R1-4T formed a tight phylogenetic lineage with Novosphingobium sediminicola HU1-AH51T within the genus Novosphingobium. R1-4T was most closely related to N. sediminicola HU1-AH51T with a 98.8 % 16S rRNA gene sequence similarity. The DNA-DNA relatedness between R1-4T and the type strain of N. sediminicola was 37.8±4.2 %. On the basis of phenotypic, chemotaxonomic and molecular properties, it is clear that R1-4T represents a novel species of the genus Novosphingobium, for which the name Novosphingobium humi sp. nov. is proposed. The type strain is R1-4T (=KACC 19094T=JCM 31879T).