Kristine Rose M. Ramos

High yield production of D-xylonic acid from D-xylose using engineered Escherichia coli.

An engineered Escherichia coli was constructed to produce D-xylonic acid, one of the top 30 high-value chemicals identified by US Department of Energy. The native pathway for D-xylose catabolism in E. coli W3110 was blocked by disrupting xylose isomerase (XI) and xylulose kinase (XK) genes. The native pathway for xylonic acid catabolism was also blocked by disrupting two genes both encoding xylonic acid dehydratase (yagE and yjhG). Through the introduction of a D-xylose dehydrogenase from Caulobacter crescentus, a D-xylonic acid producing E. coli was constructed. The recombinant E. coli produced up to 39.2 g L(-1) D-xylonic acid from 40 g L(-1) D-xylose in M9 minimal medium. The average productivity was as high as 1.09 g L(-1) h(-1) and no gluconic acid byproduct was produced. These results suggest that the engineered E. coli has a promising application for the industrial-scale production of D-xylonic acid.

Combination of Entner-Doudoroff pathway with MEP increases isoprene production in engineered Escherichia coli.

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.

Enhanced yield of ethylene glycol production from d-xylose by pathway optimization in Escherichia coli

The microbial production of renewable ethylene glycol (EG) has been gaining attention recently due to its growing importance in chemical and polymer industries. EG has been successfully produced biosynthetically from d-xylose through several novel pathways. The first report on EG biosynthesis employed the Dahms pathway in Escherichia coli wherein 71% of the theoretical yield was achieved. This report further improved the EG yield by implementing metabolic engineering strategies. First, d-xylonic acid accumulation was reduced by employing a weak promoter which provided a tighter control over Xdh expression. Second, EG yield was further improved by expressing the YjgB, which was identified as the most suitable aldehyde reductase endogenous to E. coli. Finally, cellular growth, d-xylose consumption, and EG yield were further increased by blocking a competing reaction. The final strain (WTXB) was able to reach up to 98% of the theoretical yield (25% higher as compared to the first study), the highest reported value for EG production from d-xylose.

Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

Abstract An engineered Escherichia coli strain was developed for enhanced isoprene production using d-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley–Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from d-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct d-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze d-galactose conversion to d-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD–MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD–MEP pathway.

Identification and characterization of a thermostable endolytic β-agarase Aga2 from a newly isolated marine agarolytic bacteria Cellulophaga omnivescoria W5C

Research on the enzymatic breakdown of seaweed-derived agar has recently gained attention due to the progress in green technologies for marine biomass utilization. The enzymes known as agarases catalyze the cleavage of glycosidic bonds within the polysaccharide. In this study, a new β-agarase, Aga2, was identified from Cellulophaga omnivescoria W5C. Aga2 is one of four putative agarases from the W5C genome, and it belongs to the glycoside hydrolase 16 family. It was shown to be exclusive to the Cellulophaga genus. Agarase activity assays showed that Aga2 is an endolytic-type β-agarase that produces tetrameric and hexameric neoagaro-oligosaccharides, with optimum activity at 45°C and pH 8.0. Zinc ions slightly enhanced its activity while manganese ions had inhibitory effects even at very low concentrations. Aga2 has a Km of 2.59mgmL-1 and Vmax of 275.48Umg-1. The Kcat is 1.73×102s-1, while the Kcat/Km is 8.04×106s-1M-1. Aga2 also showed good thermostability at 45°C and above, and retained >90% of its activity after repeated freeze-thaw cycles. Bioinformatic analysis of its amino acid sequence revealed that intrinsic properties of the protein (e.g. presence of certain dipeptides and the relative volume occupied by aliphatic amino acids) and tertiary structural elements (e.g. presence of salt bridges, hydrophobic interactions and H-bonding) contributed to its thermostability.

Overexpression and secretion of AgaA7 from Pseudoalteromonas hodoensis sp. nov in Bacillus subtilis for the depolymerization of agarose.

Interest in agar or agarose-based pharmaceutical products has driven the search for potent agarolytic enzymes. An extracellular β-agarase (AgaA7) recently isolated from Pseudoalteromonas hodoensis sp. nov was expressed in Bacillus subtilis, which was chosen due to its capability to overproduce and secrete functional enzymes. Phenotypic analysis showed that the engineered B. subtilis secreted a functional AgaA7 when fused with the aprE signal peptide (SP) at the amino-terminus. The maximum agarolytic activity was observed during the late logarithmic phase. To further improve the secretion of AgaA7, an expression library of AgaA7 fused to different naturally occurring B. subtilis SPs was created. The amount of AgaA7 secreted by the clones was compared through activity assay, immuno-blot, and purification via affinity chromatography. Although the aprE SP can readily facilitate the secretion of AgaA7, other SPs such as yqgA, pel, and lipA were relatively more efficient. Among these SPs, lipA was the most efficient in improving the secretion of AgaA7.The use of B. subtilis as host for the expression and secretion of agarolytic and other hydrolytic enzymes can be a useful tool in the field of white biotechnology.

l-arabonate and d-galactonate production by expressing a versatile sugar dehydrogenase in metabolically engineered Escherichia coli

The production of L-arabonate and D-galactonate employing a versatile l-arabinose dehydrogenase (AraDH) from Azospirillum brasilense is presented. The promiscuity of AraDH is manifested by its appreciable activity towards L-arabinose and D-galactose as substrates, and NAD(+) and NADP(+) as cofactors. The AraDH was introduced into an engineered Escherichia coli with inactive L-arabinose or D-galactose metabolism, resulting in strains EMA2 and EWG4, respectively. EMA2 produced 43.9 g L(-1)L-arabonate with a productivity of 1.22 g L(-1)h(-1) and 99.1% (mol/mol) yield. After methanol precipitation, 92.6% of L-arabonate potassium salt was recovered with a purity of 88.8%. Meanwhile, EWG4 produced 24.0 g L(-1)D-galactonate, which is 36% higher than that of the strain carrying the specific d-galactose dehydrogenase. Overall results reveal that the versatility of AraDH to efficiently catalyze the formation of L-arabonate and D-galactonate could be a useful tool in advancing industrial viability for sugar acids production.

Performance evaluation of poly-urethane foam packed-bed chemical scrubber for the oxidative absorption of NH3 and H2S gases

ABSTRACT The feasibility of open-pore polyurethane (PU) foam as packing material for wet chemical scrubber was tested for NH3 and H2S removals. The foam is inexpensive, light-weight, highly porous (low pressure drop) and provides large surface area per unit volume, which are desirable properties for enhanced gas/liquid mass transfer. Conventional HCl/HOCl (for NH3) and NaOH/NaOCl (for H2S) scrubbing solutions were used to absorb and oxidize the gases. Assessment of the wet chemical scrubbers reveals that pH and ORP levels are important to maintain the gas removal efficiencies >95%. A higher re-circulation rate of scrubbing solutions also proved to enhance the performance of the NH3 and H2S columns. Accumulation of salts was confirmed by the gradual increase in total dissolved solids and conductivity values of scrubbing solutions. The critical elimination capacities at >95% gas removals were found to be 5.24 g NH3-N/m3-h and 17.2 g H2S-S/m3-h at an empty bed gas residence time of 23.6 s. Negligible pressure drops (< 4 mm H2O) after continuous operation demonstrate the suitability of PU as a practical packing material in wet chemical scrubbers for NH3 and H2S removals from high-volume dilute emissions.

Draft Genome Sequence of Newly Isolated Agarolytic Bacteria Cellulophaga omnivescoria sp. nov. W5C Carrying Several Gene Loci for Marine Polysaccharide Degradation

The continued research in the isolation of novel bacterial strains is inspired by the fact that native microorganisms possess certain desired phenotypes necessary for recombinant microorganisms in the biotech industry. Most studies have focused on the isolation and characterization of strains from marine ecosystems as they present a higher microbial diversity than other sources. In this study, a marine bacterium, W5C, was isolated from red seaweed collected from Yeosu, South Korea. The isolate can utilize several natural polysaccharides such as agar, alginate, carrageenan, and chitin. Genome sequence and comparative genomics analyses suggest that strain W5C belongs to a novel species of the Cellulophaga genus, from which the name Cellulophaga omnivescoria sp. nov. is proposed. Its genome harbors 3,083 coding sequences and 146 carbohydrate-active enzymes (CAZymes). Compared to other reported Cellulophaga species, the genome of W5C contained a higher proportion of CAZymes (4.7%). Polysaccharide utilization loci (PUL) for agar, alginate, and carrageenan were identified in the genome, along with other several putative PULs. These PULs are excellent sources for discovering novel hydrolytic enzymes and pathways with unique characteristics required for biorefinery applications, particularly in the utilization of marine renewable biomass. The type strain is JCM 32108T (= KCTC 13157BPT).

Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms

The d-xylose oxidative pathway (XOP) has recently been employed in several recombinant microorganisms for growth or for the production of several valuable compounds. The XOP is initiated by d-xylose oxidation to d-xylonolactone, which is then hydrolyzed into d-xylonic acid. d-Xylonic acid is then dehydrated to form 2-keto-3-deoxy-d-xylonic acid, which may be further dehydrated then oxidized into α-ketoglutarate or undergo aldol cleavage to form pyruvate and glycolaldehyde. This review introduces a brief discussion about XOP and its discovery in bacteria and archaea, such as Caulobacter crescentus and Haloferax volcanii. Furthermore, the current advances in the metabolic engineering of recombinant strains employing the XOP are discussed. This includes utilization of XOP for the production of diols, triols, and short-chain organic acids in Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Improving the d-xylose uptake, growth yields, and product titer through several metabolic engineering techniques bring some of these recombinant strains close to industrial viability. However, more developments are still needed to optimize the XOP pathway in the host strains, particularly in the minimization of by-product formation.