Dietary Hydroxytyrosol Supplementation on Growth Performance, Gut Morphometry, and Oxidative and Inflammatory Status in LPS-Challenged Broilers.

This study assessed the effects of hydroxytyrosol (HT) on 8- to 20-day-old broilers challenged with lipopolysaccharide (LPS); 180 Cobb500™ male chicks were randomly assigned to 3 treatment groups, each comprising 10 replicates with 6 birds per replicate. Treatments included a control diet (CON), CON with LPS administration, and CON + LPS supplemented with 10 mg of HT/kg of feed. LPS was administered intraperitoneally on days 14, 16, 18, and 20. Body weight (BW), body weight gain (BWG), and the feed conversion ratio (FCR) were measured. On day 20, ten birds per treatment were slaughtered for analysis. Bursa, spleen, and liver were collected, and their respective relative weight was determined. The jejunum was destined for morphological analyses of villus height (VH), crypt depth (CD), and their ratio (VH:CD), and for mRNA expression of nuclear factor kappa B (NF-κB), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and interleukins 10 (IL-10), 1 beta (IL-1ß), and 8 (IL-8). HT improved BW, BWG, and FCR, and reduced crypt depth (CD) while increasing the VH:CD ratio in the jejunum. Moreover, HT downregulated mRNA expression of CAT, GPx, IL-10, and IL-1ß. In conclusion, HT enhances broiler growth performance, mitigates jejunal mucosa damage from LPS, and modulates antioxidant and immune responses.

Pseudomonas umsongensis GO16 as a platform for the in vivo synthesis of short and medium chain length polyhydroxyalkanoate blends.

Polyhydroxyalkanoates (PHAs) are biological polyesters, viewed as a replacement for petrochemical plastic. However, they suffer from suboptimal physical and mechanical properties. Here, it was shown that a metabolically versatile Pseudomonas umsongensis GO16 can synthesise a blend of short chain length (scl) and medium chain length (mcl)-PHA. A defined mix of butyric (BA) and octanoic acid (OA) in different ratios was used. The PHA monomer composition varied depending on the feeding strategy. When OA and BA were fed at 80:20 ratio it showed 14, 8, 77 and 1 mol% of (R)-3-hydroxybutyrate, (R)-3-hydroxyhexanoate, (R)-3-hydroxyoctanoate and (R)-3-hydroxydecanoate respectively. The polymer characterisation clearly shows that polyhydroxybutyrate (PHB) and mcl-PHA are produced individually. The two polymers are blended on the PHA granule level, as demonstrated by fluorescence microscopy and yeast two-hybrid assay. The resulting blend has a specific viscoelasticity compared to PHB and PHO. Mcl-PHA acts as a plasticiser and reduces PHB brittleness.

ß-oxidation-polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited.

Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the ß-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids-PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2-14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism. KEY POINTS: • ß-oxidation intermediates are converted to PHA monomers by hydratases PhaJ1, PhaJ4 and MaoC in Pseudomonas putida KT2440. • When these are deleted, the PHA level decreases, but it is not abolished. • PHA non-specific enzyme(s) also contributes to PHA metabolism in KT2440.

Effects of Hydroxytyrosol Supplementation on Performance, Fat and Blood Parameters of Broiler Chickens.

The study aimed to evaluate the effects of dietary supplementation of hydroxytyrosol (HT) on performance, fat, and blood parameters of broilers. In total, 960 male chicks were distributed into four treatments groups with 12 replicates with 20 birds per pen, with varying HT levels (0, 5, 10, and 50 mg/kg of feed) added to the basal diet from 1 to 42 days old. Feed intake, body weight gain, and feed conversion ratio were evaluated. Enzymes related to liver injury were evaluated in blood. Fatty acid profile and malondialdehyde (MDA) concentration were determined in the breast meat. Dietary supplementation of HT did not improve broilers' performance (p > 0.05). Birds fed 50 mg HT/kg had lower AST, ALT, and GGT concentrations (p ≤ 0.05), whereas broilers fed 5, 10, and 50 mg HT/kg, had lower TBIL concentrations (p ≤ 0.05). Breast meat of broilers fed 50 mg HT/kg had lower lipid content, saturated fatty acid, unsaturated fatty acids, MDA concentrations (p ≤ 0.05), and polyunsaturated fatty acids (p < 0.0001). In summary, supplementation of 5, 10, and 50 mg HT/kg does not improve the performance of broilers, but the dose of 50 mg HT/kg helps the liver against inflammation and improves fat parameters.

Identification of genetic variants of the industrial yeast Komagataella phaffii (Pichia pastoris) that contribute to increased yields of secreted heterologous proteins.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, ß-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled ß-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled ß-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies.

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l-1 in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l-1 in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l-1 containing 65% PHA at the end of a 25-h incubation.

CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440.

Genome editing technologies allow us to study the metabolic pathways of cells and the contribution of each associated enzyme to various processes, including polyhydroxyalkanoate (PHA) synthesis. These biodegradable polyesters accumulated by a range of bacteria are thermoplastic, elastomeric, and biodegradable, thus have great applicative potential. However, several challenges are associated with PHA production, mainly the cost and shortcomings in their physical properties. The advances in synthetic biology and metabolic engineering provide us with a tool to improve the production process and allow the synthesis of tailor-made PHAs. CRISPR/Cas9 technology represents a new generation of genome editing tools capable of application in nearly all organisms. However, off-target activity is a crucial issue for CRISPR/Cas9 technology, as it can cause genomic instability and disruption of functions of otherwise normal genes. Here, we provide a detailed protocol for scarless deletion of the genes implicated in PHA metabolism of Pseudomonas putida KT2440 using modified CRISPR/Cas9 systems and methodology.

Robust process for high yield conversion of non-degradable polyethylene to a biodegradable plastic using a chemo-biotechnological approach.

In this study, the optimisation of a process for producing medium-chain-length polyhydroxyalkanoate (mcl-PHA) by Pseudomonas putida KT2440 when fed with a polyethene (PE)-derived fatty acid mixture was investigated. The PE was pyrolysed to produce a hydrocarbon wax that was subsequently oxidised to produce a mixture of fatty acids, purified, and used as a PHA substrate for the growth and selection of microorganisms. Based on the shaken flask screening, a production strain, i.e., Pseudomonas putida KT2440, was selected for conducting bioreactor studies. Feeding PE-derived fatty acids in a 20-L setup resulted in high mcl-PHA yields (83.0 g L-1 CDW with 65% PHA in 25 h). Furthermore, life-cycle assessment (LCA) was conducted to determine the environmental advantages of the proposed process and its impacts compared to those of other technologies for treating PE-derived waste streams. We conclude that processing waste PE into PHA, rather than incineration, produces biodegradable material while also reducing the additional emissions that arise from traditional PE waste treatment processes, such as incineration to gain energy.

Microbiology challenges and opportunities in the circular economy.

Our knowledge and understanding of micro-organisms have led to the development of safe food, clean water, novel foods, antibiotics, vaccines, healthier plants, animals and soils, and more, which feeds into the United Nations Sustainable Development Goals (UN SDGs). The circular economy can contribute to the UN SDGs and micro-organisms are central to circular nutrient cycles. The circular economy as described by the Ellen MacArthur foundation has two halves, i.e. technical and biological. On the technical side, non-biological resources enter manufacturing paths where resource efficiency, renewable energy and design extend the life of materials so that they are more easily reused and recycled. Biological resources exist on the other half of the circular economy. These are used to manufacture products such as bioplastics and paper. The conservation of nature's stocks, resource efficiency and recycling of materials are key facets of the biological half of the circular economy. Microbes play a critical role in both the biological and technical parts of the circular economy. Microbes are key to a functioning circular economy, where natural resources, including biological wastes, are converted by microbes into products of value and use for society, e.g. biogas, bioethanol, bioplastics, building block chemicals and compost for healthy soils. In more recent times, microbes have also been seen as part of the tool kit in the technical side of the circular economy, where microbial enzymes can degrade plastics and microbes can convert those monomers to value-added products.

Genome analysis of the metabolically versatile Pseudomonas umsongensis GO16: the genetic basis for PET monomer upcycling into polyhydroxyalkanoates.

The throwaway culture related to the single-use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET-derived terephthalic acid (TA) into PHA and performed an in-depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.

Polyhydroxyphenylvalerate/polycaprolactone nanofibers improve the life-span and mechanoresponse of human IPSC-derived cortical neuronal cells.

The physico-chemical characteristics of the extracellular matrix (ECM) cause mechanical cues that could elicit responses in the survival rate of cortical neuronal cells. Efficient neurite outgrowth in vitro, is critical for successful cultivation of cortical neuronal cells and the potential for attempts at regeneration of the central nervous system (CNS) in vivo. Relatively soft and hydrophilic, microbially synthesized aromatic polyester, polyhydroxyphenylvalerate (PHPV) was blended 50:50 with the stiff and hydrophobic polycaprolactone (PCL) and electrospun in microfibers for use in a 3D (CellCrown™) configuration and in a 2D coverslip coated configuration. This blend allows a 2.3-fold increase in the life-span of human induced pluripotent stem derived cortical neuronal cells (hiPS) compared to pure PCL fibers. HiPS-derived cortical neuronal cells grown on PHPV/PCL fibers show a 3.8-fold higher cumulative neurite elaboration compared to neurites grown on PCL fibers only. 96% of cortical neuronal cells die after 8 days of growth when plated on PCL fibers alone while >83% and 55% are alive on PHPV/PCL fibers on day 8 and day 17, respectively. An increased migration rate of cortical neuronal cells is also promoted by the blend compared to the PCL fibers alone. The critical survival rate improvement of hiPS derived cortical neuronal cells on PHPV/PCL blend holds promise in using these biocompatible nanofibers as implantable materials for regenerative purposes of an active cortical neuronal population after full maturation in vitro.

Recent Advances in Bioplastics: Application and Biodegradation.

The success of oil-based plastics and the continued growth of production and utilisation can be attributed to their cost, durability, strength to weight ratio, and eight contributions to the ease of everyday life. However, their mainly single use, durability and recalcitrant nature have led to a substantial increase of plastics as a fraction of municipal solid waste. The need to substitute single use products that are not easy to collect has inspired a lot of research towards finding sustainable replacements for oil-based plastics. In addition, specific physicochemical, biological, and degradation properties of biodegradable polymers have made them attractive materials for biomedical applications. This review summarises the advances in drug delivery systems, specifically design of nanoparticles based on the biodegradable polymers. We also discuss the research performed in the area of biophotonics and challenges and opportunities brought by the design and application of biodegradable polymers in tissue engineering. We then discuss state-of-the-art research in the design and application of biodegradable polymers in packaging and emphasise the advances in smart packaging development. Finally, we provide an overview of the biodegradation of these polymers and composites in managed and unmanaged environments.

Novel sodium alkyl-1,3-disulfates, anionic biosurfactants produced from microbial polyesters.

A sodium alkyl disulfate mixture (SADM) synthesised from microbially produced 3-hydroxy fatty acids methyl esters (HFAMEs), showed 13-fold surface tension decrease when compared with the reference surfactant sodium dodecyl sulfate (SDS). Polyhydroxyalkanoates, accumulated by bacteria intracellularly when supplied with a mixture of fatty acids derived from hydrolysed rapeseed oil, were isolated, depolymerised and methylated to produce HFAMEs in very high yield (90%). A sequential chemical reduction and sulfation of the HFAMEs produced the sodium alkyl disulfates in high yields (>65%). SADM performs also 1.3-times better than dodecyl (1,3) disulfate, in surface tension tests. SADM shows also the formation of a specific critical micelle concentration (CMC) at a concentration 21-fold lower than SDS. The wettability of the SADM mixture is similar to SDS but the foaming volume of SADM is 1.5-fold higher. The foam is also more stable with its volume decreasing 3 times slower over time compared to SDS at their respective CMC values. Established sulfation technologies in chemical manufacturing could use the 3-hydroxy fatty acids methyl esters moiety (3-HFAME) given its origin from rapeseed oil and the extra OH residue on 3-position in the molecule, which affords the opportunity to produce disulfate surfactants with a proven superior performance to monosulphated surfactants. Thus, not only addressing environmental issues by avoiding threats of deforestation and monocultivation associated with palm oil use but also achieve a higher performance with lower use of surfactants.

Chemical, physical and biotechnological approaches to the production of the potent antioxidant hydroxytyrosol.

Hydroxytyrosol (HT) is a polyphenol of interest to the food, feed, supplements and pharmaceutical sectors. It is one of the strongest known natural antioxidants and has been shown to confer other benefits such as anti-inflammatory and anti-carcinogenic properties, and it has the potential to act as a cardio- and neuroprotectant. It is known to be one of the compounds responsible for the health benefits of the Mediterranean diet. In nature, HT is found in the olive plant (Olea europaea) as part of the secoiridoid compound oleuropein, in its leaves, fruit, oil and oil production waste products. HT can be extracted from these olive sources, but it can also be produced by chemical synthesis or through the use of microorganisms. This review looks at the production of HT using plant extraction, chemical synthesis and biotechnological approaches.

Unnatural amino acids: production and biotechnological potential.

Unnatural amino acids (UAAs) are valuable building blocks in the manufacture of a wide range of pharmaceuticals. UAAs exhibit biological activity as free acids and they can be incorporated into linear or cyclic peptides with biological activity. However, the scope of biotechnological application of UAAs goes beyond this, as they can be used to investigate the structure and dynamics of proteins, to study protein interactions, or to modulate the activity of proteins in living cells. The means to expand nature's repertoire of amino acids include chemical and biological routes. An UAA can be made through chemical modifications of natural amino acids, or related compounds. These modifications typically rely on utilisation of ligands and palladium catalysts. Employing biocatalysts in the synthesis of UAAs can also afford novel molecules with different physical and chemical properties. A number of transaminases for example have been identified and employed in the production of UAAs. This review will compare the chemical and biological routes for the synthesis of UAAs and provide an overview of their applications.

Plastic waste as a global challenge: are biodegradable plastics the answer to the plastic waste problem?

The strength, flexibility and light weight of traditional oil-derived plastics make them ideal materials for a large number of applications, including packaging, medical devices, building, transportation, etc. However, the majority of produced plastics are single-use plastics, which, coupled with a throw-away culture, leads to the accumulation of plastic waste and pollution, as well as the loss of a valuable resource. In this review we discuss the advances and possibilities in the biotransformation and biodegradation of oil-based plastics. We review bio-based and biodegradable polymers and highlight the importance of end-of-life management of biodegradables. Finally, we discuss the role of a circular economy in reducing plastic waste pollution.

Single point mutations reveal amino acid residues important for Chromobacterium violaceum transaminase activity in the production of unnatural amino acids.

Unnatural amino acids (UAAs) are chiral amines with high application potential in drug discovery and synthesis of other valuable chemicals. Biocatalysis offers the possibility to synthesise novel optically pure UAAs with different physical and chemical properties. While the biocatalytic potential of transaminases in the synthesis of UAAs has been demonstrated, there is still a need to improve the activity with non-native substrates and to understand which amino acids residues are important for activity with these UAAs. Using a rational design approach, six variants of Chromobacterium violaceum DSM30191 transaminase (CV_TA) carrying a single and one variant carrying two substitutions were generated. Among the variants with a single substitution, CV_Y168F showed a 2 to 2.6-fold increased affinity for 2-oxooctanoic acid (2-OOA) and 3-oxobutyric acid (3-OBA) methyl ester used to synthesise an α- and ß-UAA. Analysis of the first half of the transaminase reaction showed no change in the activity with the donor (S)-1-phenylethylamine. The combination of W60C and Y168F substitutions improved the CV_TA affinity for 2-OOA 10-fold compared to the wild type. Other substitutions showed no change, or reduced activity with the tested substrates. Our findings provide structural information on CV_TA and demonstrate the potential of rational design for biosynthesis of UAAs.

Biodegradable Plastic Blends Create New Possibilities for End-of-Life Management of Plastics but They Are Not a Panacea for Plastic Pollution.

Plastic waste pollution is a global environmental problem which could be addressed by biodegradable plastics. The latter are blended together to achieve commercially functional properties, but the environmental fate of these blends is unknown. We have tested neat polymers, polylactic acid (PLA), polyhydroxybutyrate, polyhydroxyoctanoate, poly(butylene succinate), thermoplastic starch, polycaprolactone (PCL), and blends thereof for biodegradation across seven managed and unmanaged environments. PLA is one of the world's best-selling biodegradable plastics, but it is not home compostable. We show here that PLA when blended with PCL becomes home compostable. We also demonstrate that the majority of the tested bioplastics and their blends degrade by thermophilic anaerobic digestion with high biogas output, but degradation times are 3-6 times longer than the retention times in commercial plants. While some polymers and their blends showed good biodegradation in soil and water, the majority of polymers and their blends tested in this study failed to achieve ISO and ASTM biodegradation standards, and some failed to show any biodegradation. Thus, biodegradable plastic blends need careful postconsumer management, and further design to allow more rapid biodegradation in multiple environments is needed as their release into the environment can cause plastic pollution.

Biocatalytic versatility of engineered and wild-type tyrosinase from R. solanacearum for the synthesis of 4-halocatechols.

We evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (kcat/Km) with both reductions in Km and increases in kcat compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in kcat but lowest improvement in catalytic efficiency due to an increase in Km compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of > 90%. Flask experiments with 10 mM 4-iodophenol and 10 µg/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 µg/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme.

Three novel proteins co-localise with polyhydroxybutyrate (PHB) granules in Rhodospirillum rubrum S1.

Polyhydroxybutyrate (PHB), a biodegradable polymer accumulated by bacteria is deposited intracellularly in the form of inclusion bodies often called granules. The granules are supramolecular complexes harbouring a varied number of proteins on their surface, which have specific but incompletely characterised functions. By comparison with other organisms that produce biodegradable polymers, only two phasins have been described to date for Rhodosprillum rubrum, raising the possibility that more await discovery. Using a comparative proteomics strategy to compare the granules of wild-type R. rubrum with a PHB-negative mutant housing artificial PHB granules, we identified four potential PHB granules' associated proteins. These were: Q2RSI4, an uncharacterised protein; Q2RWU9, annotated as an extracellular solute-binding protein; Q2RQL4, annotated as basic membrane lipoprotein; and Q2RQ51, annotated as glucose-6-phosphate isomerase. In silico analysis revealed that Q2RSI4 harbours a Phasin_2 family domain and shares low identity with a single-strand DNA-binding protein from Sphaerochaeta coccoides. Fluorescence microscopy found that three proteins Q2RSI4, Q2EWU9 and Q2RQL4 co-localised with PHB granules. This work adds three potential new granule associated proteins to the repertoire of factors involved in bacterial storage granule formation, and confirms that proteomics screens are an effective strategy for discovery of novel granule associated proteins.