Development of novel recombinant peroxidase secretion system from Pseudomonas putida for lignin valorisation.

Pseudomonas putida is a promising strain for lignin valorisation. However, there is a dearth of stable and efficient systems for secreting enzymes to enhance the process. Therefore, a novel secretion system for recombinant lignin-depolymerising peroxidase was developed. By adopting a flagellar type III secretion system, P. putida KT-M2, a secretory host strain, was constructed and an optimal secretion signal fusion partner was identified. Application of the dye-decolourising peroxidase of P. putida to this system resulted in efficient oxidation activity of the cell-free supernatant against various chemicals, including lignin model compounds. This peroxidase-secreting strain was examined to confirm its lignin utilisation capability, resulting in the efficient assimilation of various lignin substrates with 2.6-fold higher growth than that of the wild-type strain after 72 h of cultivation. Finally, this novel system will lead efficient bacterial lignin breakdown and utilization through enzyme secretion, paving the way for sustainable lignin-consolidated bioprocessing.

Aqueous two-phase system-derived biofilms for bacterial interaction studies.

We describe patterning of bacterial biofilms using polymer-based aqueous two-phase system (ATPS) microprinting protocols. The fully aqueous but selectively bacteria-partitioning nature of the ATPS allows spatially distinct localization of suspensions of bacteria such as Pseudomonas aeruginosa and Escherichia coli with high precision. The ATPS patterned bacterial suspensions form spatially distinct biofilms over time. Due to the fully aqueous and gentle noncontact printing procedures employed, coculture biofilms composed of multiple types of bacteria could be printed not only adjacent to each other but also directly over another layer of existing biofilm. In addition, the ATPS environment also allows free diffusion of small molecules between spatially distinct and localized bacterial suspensions and biofilms. This enables biofilms to chemically affect or be affected by neighboring biofilms or planktonic cells, even if they consist of different strains or species. We show that a ß-lactamase producing biofilm confers ampicillin resistance to neighboring nonresistant planktonic cells, as seen by a 3,600-fold increase in survival of the ampicillin-sensitive strain. These examples demonstrate the ability of ATPS-based biofilm patterning methods to enable unique studies on commensalistic effects between bacterial species.

Production of polyhydroxyalkanoates containing monomers conferring amorphous and elastomeric properties from renewable resources: Current status and future perspectives.

Petrochemical-based plastics cause environmental pollution and threaten humans and ecosystems. Polyhydroxyalkanoate (PHA) is considered a promising alternative to nondegradable plastics since it is eco-friendly and biodegradable polymer having similar properties to conventional plastics. PHA's material properties are generally determined by composition and type of monomers in PHA. PHA can be designed in tailor-made manner for their suitable application areas. Among many monomers in PHAs, ω-hydroxalkanoates such as 3-hydroxypropionate (3HP), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), and 6-hydroxyhexanoate (6HHx) and medium-chain-length 3-hydroxyalkanoate such as 3-hydroxyhexanoate (3HHx) and 4-hydroxyvalerate (4HV), have been examined as potential monomers able to confer amorphous and elastomer properties when these are incorporated as comonomer in poly(3-hydroxybutyrate) copolymer that has 3HB as main monomer along with comonomers in different monomer fraction. Herein, recent advances in production of PHAs designed to have amorphous and elastomeric properties from renewable sources such as lignocellulose, levulinic acid, crude glycerol, and waste oil are discussed.

Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester.

Lignin valorization depends on microbial upcycling of various aromatic compounds in the form of a complex mixture, including p-coumaric acid and ferulic acid. In this study, an engineered Pseudomonas putida strain utilizing lignin-derived monomeric compounds via biological funneling was developed to produce 2-pyrone-4,6-dicarboxylic acid (PDC), which has been considered a promising building block for bioplastics. The biosynthetic pathway for PDC production was established by introducing the heterologous ligABC genes under the promoter Ptac in a strain lacking pcaGH genes to accumulate a precursor of PDC, i.e., protocatechuic acid. Based on the culture optimization, fed-batch fermentation of the final strain resulted in 22.7 g/L PDC with a molar yield of 1.0 mol/mol and productivity of 0.21 g/L/h. Subsequent purification of PDC at high purity was successfully implemented, which was consequently applied for the novel polyester.

Micropatterning bacterial suspensions using aqueous two phase systems.

Using an aqueous two-phase system comprised of dextran and polyethylene glycol, this article describes the stable spatial patterning of sub-microlitre droplets of bacterial suspensions. Microdroplets of different types of bacterial populations are positioned and maintained adjacent to each other without significant dispersion even though the bacteria are in suspension and not surface bound. Small molecules, in contrast, diffuse relatively freely between the two aqueous phases. The usefulness of these capabilities is demonstrated by generating a small array of suspensions containing different Escherichia coli strains engineered to respond fluorescently or luminescently to different chemical stimuli. When a chemical stimulus is presented, this droplet array produces a pattern of bacterial "illumination" that reflects the type of chemical to which the array was exposed.

Detection of toxic lignin hydrolysate-related compounds using an inaA::luxCDABE fusion strain.

Real-time quantitative PCR analyses of Escherichia coli str. BL21(DE3) exposed to 0.5 g/L ferulic and coumaric acid showed that the inaA gene was significantly induced (7.7- and 3.6-fold higher, respectively). Consequently, a transcriptional fusion of the inaA promoter with the luxCDABE operon was constructed and characterized with several compounds identified within hydrolysates. Tests demonstrated that the phenolics were major inducers, while acetic acid and furfural had only a minor or no effect on the inaA expression respectively. Additional tests with mutant E. coli strains found that a marA partially abolished the response while a marB knock-out led to a 2-3-fold higher basal level expression as evidenced by the bioluminescent levels of the cultures. However, a significant induction was seen even in the marA mutant, suggesting some other control mechanism is involved in regulating inaA expression during an exposure to the hydrolysate compounds. Finally, E. coli str. BL21(DE3)/pSP4 was used to analyze a spruce hydrolysate sample. Real-time quantitative PCR showed a 2.8-fold induction of the inaA expression level while the bioluminescence from the exposed culture was 22-fold higher than the control, demonstrating the possible application of this reporter strain to analyze hydrolysates for the presence of fermentation-inhibiting phenolics.

Biological activities of lignin hydrolysate-related compounds.

Lignin hydrolysates contain many different chemical species, including ferulic acid, coumaric acid, vanillic acid, vanillin, syringaldehyde and furfural. From the perspective of biofuels, these compounds are problematic and can cause downstream loss of product if not removed prior to beginning the fermentative process. In contrast, a search for these compounds within the literature turns up many papers where the same compounds have beneficial properties pertaining to human health, including as antioxidants and in cancer prevention, or are involved in bacterial cell-to-cell signaling. Consequently, this article reviews the dual nature of these and other compounds found in lignin hydrolysates, highlighting both their detrimental and beneficial activities.

Identification of Escherichia coli biomarkers responsive to various lignin-hydrolysate compounds.

Aberrations in the growth and transcriptome of Escherichia coli str. BL21(DE3) were determined when exposed to varying concentrations of ferulic acid (0.25-1 g/L), an aromatic carboxylic acid identified within lignin-cellulose hydrolysate samples. The expression of several individual genes (aaeA, aaeB, inaA and marA) was significantly induced, i.e., more than 4-fold, and thus these genes and the heat shock response gene htpG were selected as biomarkers to monitor E. coli's responses to five additional hydrolysate-related compounds, including vanillic acid, coumaric acid, 4-hydroxybenzoic acid, ferulaldehyde and furfural. While all of the biomarkers showed dose-dependent responses to most of the compounds, expression of aaeA and aaeB showed the greatest induction (5-30-fold) for all compounds tested except furfural. Lastly, the marA, inaA and htpG genes all showed higher expression levels when the culture was exposed to spruce hydrolysate samples, demonstrating the potential use of these genes as biomarkers.