Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

The rhizobacterial Priestia megaterium strain SH-19 mitigates the hazardous effects of heat stress via an endogenous secondary metabolite elucidation network and molecular regulation signalling.

Global warming is a leading environmental stress that reduces plant productivity worldwide. Several beneficial microorganisms reduce stress; however, the mechanism by which plant-microbe interactions occur and reduce stress remains to be fully elucidated. The aim of the present study was to elucidate the mutualistic interaction between the plant growth-promoting rhizobacterial strain SH-19 and soybeans of the Pungsannamul variety. The results showed that SH-19 possessed several plant growth-promoting traits, such as the production of indole-3-acetic acid, siderophore, and exopolysaccharide, and had the capacity for phosphate solubilisation. The heat tolerance assay showed that SH-19 could withstand temperatures up to 45 °C. The strain SH-19 was identified as P. megaterium using the 16S ribosomal DNA gene sequence technique. Inoculation of soybeans with SH-19 improved seedling characteristics under high-temperature stress. This may be due to an increase in the endogenous salicylic acid level and a decrease in the abscisic acid level compared with the negative control group. The strain of SH-19 increased the activity of the endogenous antioxidant defense system, resulting in the upregulation of GSH (44.8%), SOD (23.1%), APX (11%), and CAT (52.6%). Furthermore, this study involved the transcription factors GmHSP, GmbZIP1, and GmNCED3. The findings showed upregulation of the two transcription factors GmbZIP1 (17%), GmNCED3 (15%) involved in ABA biosynthesis and induced stomatal regulation, similarly, a downregulation of the expression pattern of GmHSP by 25% was observed. Overall, the results of this study indicate that the strain SH-19 promotes plant growth, reduces high-temperature stress, and improves physiological parameters by regulating endogenous phytohormones, the antioxidant defense system, and genetic expression. The isolated strain (SH-19) could be commercialized as a biofertilizer.

Pseudochrobactrum asaccharolyticum mitigates arsenic induced oxidative stress of maize plant by enhancing water status and antioxidant defense system.

BACKGROUND: Oxidative stress mediated by reactive oxygen species (ROS) is a common denominator in arsenic toxicity. Arsenic stress in soil affects the water absorption, decrease stomatal conductance, reduction in osmotic, and leaf water potential, which restrict water uptake and osmotic stress in plants. Arsenic-induced osmotic stress triggers the overproduction of ROS, which causes a number of germination, physiological, biochemical, and antioxidant alterations. Antioxidants with potential to reduce ROS levels ameliorate the arsenic-induced lesions. Plant growth promoting rhizobacteria (PGPR) increase the total soluble sugars and proline, which scavenging OH radicals thereby prevent the oxidative damages cause by ROS. The main objective of this study was to evaluate the potential role of Arsenic resistant PGPR in growth of maize by mitigating arsenic stress. METHODOLOGY: Arsenic tolerant PGPR strain MD3 (Pseudochrobactrum asaccharolyticum) was used to dismiss the 'As' induced oxidative stress in maize grown at concentrations of 50 and 100 mg/kg. Previously isolated arsenic tolerant bacterial strain MD3 "Pseudochrobactrum asaccharolyticum was used for this experiment. Further, growth promoting potential of MD3 was done by germination and physio-biochemical analysis of maize seeds. Experimental units were arranged in Completely Randomized Design (CRD). A total of 6 sets of treatments viz., control, arsenic treated (50 & 100 mg/kg), bacterial inoculated (MD3), and arsenic stress plus bacterial inoculated with three replicates were used for Petri plates and pot experiments. After treating with this MD3 strain, seeds of corn were grown in pots filled with or without 50 mg/kg and 100 mg/kg sodium arsenate. RESULTS: The plants under arsenic stress (100 mg/kg) decreased the osmotic potential (0.8 MPa) as compared to control indicated the osmotic stress, which caused the reduction in growth, physiological parameters, proline accumulation, alteration in antioxidant enzymes (Superoxide dismutase-SOD, catalase-CAT, peroxidase-POD), increased MDA content, and H2O2 in maize plants. As-tolerant Pseudochrobactrum asaccharolyticum improved the plant growth by reducing the oxidation stress and antioxidant enzymes by proline accumulation. PCA analysis revealed that all six treatments scattered differently across the PC1 and PC2, having 85.51% and 9.72% data variance, respectively. This indicating the efficiency of As-tolerant strains. The heatmap supported the As-tolerant strains were positively correlated with growth parameters and physiological activities of the maize plants. CONCLUSION: This study concluded that Pseudochrobactrum asaccharolyticum reduced the 'As' toxicity in maize plant through the augmentation of the antioxidant defense system. Thus, MD3 (Pseudochrobactrum asaccharolyticum) strain can be considered as bio-fertilizer.

Degradation of PET microplastic particles to monomers in human serum by PETase.

More than 8 billion tons of plastic waste has been generated, posing severe environmental consequences and health risks. Due to prolonged exposure, microplastic particles are found in human blood and other bodily fluids. Despite a lack of toxicity studies regarding microplastics, harmful effects for humans seem plausible and cannot be excluded. As small plastic particles readily translocate from the gut to body fluids, enzyme-based treatment of serum could constitute a promising future avenue to clear synthetic polymers and their corresponding oligomers via their degradation into monomers of lower toxicity than the material they originate from. Still, whereas it is known that the enzymatic depolymerization rate of synthetic polymers varies by orders of magnitude depending on the buffer and media composition, the activity of plastic-degrading enzymes in serum was unknown. Here, we report how an engineered PETase, which we show to be generally trans-selective via induced fit docking, can depolymerize two different microplastic-like substrates of the commodity polymer polyethylene terephthalate (PET) into its non-toxic monomer terephthalic acid (TPA) alongside mono(2-hydroxyethyl)terephthalate (MHET) in human serum at 37 °C. We show that the application of PETase does not influence cell viability in vitro. Our work highlights the potential of applying biocatalysis in biomedicine and represents a first step towards finding a future solution to the problem that microplastics in the bloodstream may pose.

Characterizing the "fungal shunt": Parasitic fungi on diatoms affect carbon flow and bacterial communities in aquatic microbial food webs.

Microbial interactions in aquatic environments profoundly affect global biogeochemical cycles, but the role of microparasites has been largely overlooked. Using a model pathosystem, we studied hitherto cryptic interactions between microparasitic fungi (chytrid Rhizophydiales), their diatom host Asterionella, and cell-associated and free-living bacteria. We analyzed the effect of fungal infections on microbial abundances, bacterial taxonomy, cell-to-cell carbon transfer, and cell-specific nitrate-based growth using microscopy (e.g., fluorescence in situ hybridization), 16S rRNA gene amplicon sequencing, and secondary ion mass spectrometry. Bacterial abundances were 2 to 4 times higher on individual fungal-infected diatoms compared to healthy diatoms, particularly involving Burkholderiales. Furthermore, taxonomic compositions of both diatom-associated and free-living bacteria were significantly different between noninfected and fungal-infected cocultures. The fungal microparasite, including diatom-associated sporangia and free-swimming zoospores, derived ∼100% of their carbon content from the diatom. By comparison, transfer efficiencies of photosynthetic carbon were lower to diatom-associated bacteria (67 to 98%), with a high cell-to-cell variability, and even lower to free-living bacteria (32%). Likewise, nitrate-based growth for the diatom and fungi was synchronized and faster than for diatom-associated and free-living bacteria. In a natural lacustrine system, where infection prevalence reached 54%, we calculated that 20% of the total diatom-derived photosynthetic carbon was shunted to the parasitic fungi, which can be grazed by zooplankton, thereby accelerating carbon transfer to higher trophic levels and bypassing the microbial loop. The herein termed "fungal shunt" can thus significantly modify the fate of photosynthetic carbon and the nature of phytoplankton-bacteria interactions, with implications for diverse pelagic food webs and global biogeochemical cycles.

Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity.

The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.

Biodegradation of different PET variants from food containers by Ideonella sakaiensis.

The accumulation of macro-, micro- and nano-plastic wastes in the environment is a major global concern, as these materials are resilient to degradation processes. However, microorganisms have evolved their own biological means to metabolize these petroleum-derived polymers, e.g., Ideonella sakaiensis has recently been found to be capable of utilizing polyethylene terephthalate (PET) as its sole carbon source. This study aims to prove its potential capacity to biodegrade two commercial PET materials, obtained from food packaging containers. Plastic pieces of different crystallinity were simultaneously introduced to Ideonella sakaiensis during a seven-week lasting investigation. Loss in weight, appearance of plastics, as well as growth of Ideonella sakaiensis-through quantitative real-time PCR-were determined. Both plastics were found enzymatically attacked in a two-stage degradation process, reaching biodegradation capacities of up to 96%. Interestingly, the transparent, high crystallinity PET was almost fully degraded first, followed by the colored low-crystallinity PET. Results of quantitative real-time PCR-based gene copy numbers were found in line with experimental results, thus underlining its potential of this method to be applied in future studies with Ideonella sakaiensis.

Overexpression and kinetic analysis of Ideonella sakaiensis PETase for polyethylene terephthalate (PET) degradation.

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETaseMut, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETaseMut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L-1 purified soluble IsPETaseMut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L-1 purified soluble NusA-IsPETaseMut, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETaseMut (GroEL/ES) displayed the same catalytic behavior as IsPETaseMut, while the fusion of NusA conferred new enzymatic properties to IsPETaseMut. Although NusA-IsPETaseMut displayed a lower initial hydrolysis capacity than IsPETaseMut, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETaseMut. Taken together, the latter two catalytic properties of NusA-IsPETaseMut are more likely to contribute to the enhanced product release by NusA-IsPETaseMut PET degradation for two weeks.

Assessment of the PETase conformational changes induced by poly(ethylene terephthalate) binding.

Recently, a bacterium strain of Ideonella sakaiensis was identified with the uncommon ability to degrade the poly(ethylene terephthalate) (PET). The PETase from I. sakaiensis strain 201-F6 (IsPETase) catalyzes the hydrolysis of PET converting it to mono(2-hydroxyethyl) terephthalic acid (MHET), bis(2-hydroxyethyl)-TPA (BHET), and terephthalic acid (TPA). Despite the potential of this enzyme for mitigation or elimination of environmental contaminants, one of the limitations of the use of IsPETase for PET degradation is the fact that it acts only at moderate temperature due to its low thermal stability. Besides, molecular details of the main interactions of PET in the active site of IsPETase remain unclear. Herein, molecular docking and molecular dynamics (MD) simulations were applied to analyze structural changes of IsPETase induced by PET binding. Results from the essential dynamics revealed that the ß1-ß2 connecting loop is very flexible. This loop is located far from the active site of IsPETase and we suggest that it can be considered for mutagenesis to increase the thermal stability of IsPETase. The free energy landscape (FEL) demonstrates that the main change in the transition between the unbound to the bound state is associated with the ß7-α5 connecting loop, where the catalytic residue Asp206 is located. Overall, the present study provides insights into the molecular binding mechanism of PET into the IsPETase structure and a computational strategy for mapping flexible regions of this enzyme, which can be useful for the engineering of more efficient enzymes for recycling plastic polymers using biological systems.

Comparative Biochemistry of Four Polyester (PET) Hydrolases*.

The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insoluble substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The analysis provided values of kcat and KM , as well as an apparent specificity constant in the conventional units of M-1 s-1 . These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high kcat rather than a low KM . Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, whereas the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.

Development of a Targeted Gene Disruption System in the Poly(Ethylene Terephthalate)-Degrading Bacterium Ideonella sakaiensis and Its Applications to PETase and MHETase Genes.

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo, we have developed a gene disruption system in I. sakaiensis. The pT18mobsacB-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (pyrF) from the genome of the wild-type strain, producing the ΔpyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the ΔpyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δpetase and Δmhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δpetase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis, which was then applied for constructing Δpetase and Δmhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis, while PETase and MHETase play essential roles in its PET assimilation.

Inhella proteolytica sp. nov. and Inhella gelatinilytica sp. nov., two novel species of the genus Inhella isolated from aquaculture water.

The two novel bacterial strains designated 1Y17T and 4Y10T from aquaculture water were characterized using a polyphasic taxonomic approach. Phylogenetic analysis of 16S rRNA gene sequences revealed that strains 1Y17T and 4Y10T belonged to the genus Inhella and were close to Inhella crocodyli CCP-18T, Inhella inkyongensis IMCC1713T and Inhella fonticola TNR-25T. Strains 1Y17T and 4Y10T shared 98.6% identity with each other and less than 99.0% identity with their relatives above. The phylogenomic analysis indicated that the two strains formed two independent branches distinct from their relatives. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the two strains were 21.3 and 80.9% below the two thresholds of 70% dDDH and 95-96% ANI for species definition; those between the two novel strains and their relatives were far below thresholds for species definition, implying that they represent two novel genospecies. The predominant fatty acids of the two strains were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and C16:0; the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol; the major quinone and polyamine were Q-8 and putrescine. Their genomic DNA G + C contents were 69.3 and 65.0%. The two novel strains can produce poly-ß-hydroxybutyrate, matching with the presence of the three synthetic related genes of the phaC-phaA-phaB in their genomes. Based on the genotypic and phenotypic characteristics such as aesculin and gelatin hydrolysis, strains 1Y17T and 4Y10T are concluded to represent two novel species of the genus Inhella, for which the names Inhella proteolytica sp. nov. (type strain 1Y17T = GDMCC 1.1830T = KACC 21948T) and Inhella gelatinilytica sp. (type strain 4Y10T = GDMCC 1.1829T = KCTC 82338T) are proposed.

Genome analysis provides insights into the biocontrol ability of Mitsuaria sp. strain TWR114.

Mitsuaria sp. TWR114 is a biocontrol agent against tomato bacterial wilt (TBW). We aimed to gain genomic insights relevant to the biocontrol mechanisms and colonization ability of this strain. The draft genome size was found to be 5,632,523 bp, with a GC content of 69.5%, assembled into 1144 scaffolds. Genome annotation predicted a total of 4675 protein coding sequences (CDSs), 914 pseudogenes, 49 transfer RNAs, 3 noncoding RNAs, and 2 ribosomal RNAs. Genome analysis identified multiple CDSs associated with various pathways for the metabolism and transport of amino acids and carbohydrates, motility and chemotactic capacities, protection against stresses (oxidative, antibiotic, and phage), production of secondary metabolites, peptidases, quorum-quenching enzymes, and indole-3-acetic acid, as well as protein secretion systems and their related appendages. The genome resource will extend our understanding of the genomic features related to TWR114's biocontrol and colonization abilities and facilitate its development as a new biopesticide against TBW.

Mixotrophic Iron-Oxidizing Thiomonas Isolates from an Acid Mine Drainage-Affected Creek.

Natural attenuation of heavy metals occurs via coupled microbial iron cycling and metal precipitation in creeks impacted by acid mine drainage (AMD). Here, we describe the isolation, characterization, and genomic sequencing of two iron-oxidizing bacteria (FeOB) species: Thiomonas ferrovorans FB-6 and Thiomonas metallidurans FB-Cd, isolated from slightly acidic (pH 6.3), Fe-rich, AMD-impacted creek sediments. These strains precipitated amorphous iron oxides, lepidocrocite, goethite, and magnetite or maghemite and grew at a pH optimum of 5.5. While Thiomonas spp. are known as mixotrophic sulfur oxidizers and As oxidizers, the FB strains oxidized Fe, which suggests they can efficiently remove Fe and other metals via coprecipitation. Previous evidence for Thiomonas sp. Fe oxidation is largely ambiguous, possibly because of difficulty demonstrating Fe oxidation in heterotrophic/mixotrophic organisms. Therefore, we also conducted a genomic analysis to identify genetic mechanisms of Fe oxidation, other metal transformations, and additional adaptations, comparing the two FB strain genomes with 12 other Thiomonas genomes. The FB strains fall within a relatively novel group of Thiomonas strains that includes another strain (b6) with solid evidence of Fe oxidation. Most Thiomonas isolates, including the FB strains, have the putative iron oxidation gene cyc2, but only the two FB strains possess the putative Fe oxidase genes mtoAB The two FB strain genomes contain the highest numbers of strain-specific gene clusters, greatly increasing the known Thiomonas genetic potential. Our results revealed that the FB strains are two distinct novel species of Thiomonas with the genetic potential for bioremediation of AMD via iron oxidation.IMPORTANCE As AMD moves through the environment, it impacts aquatic ecosystems, but at the same time, these ecosystems can naturally attenuate contaminated waters via acid neutralization and catalyzing metal precipitation. This is the case in the former Ronneburg uranium-mining district, where AMD impacts creek sediments. We isolated and characterized two iron-oxidizing Thiomonas species that are mildly acidophilic to neutrophilic and that have two genetic pathways for iron oxidation. These Thiomonas species are well positioned to naturally attenuate AMD as it discharges across the landscape.

Biosynthesis of polyketides by trans-AT polyketide synthases in Burkholderiales.

Widely used as drugs and agrochemicals, polyketides are a family of bioactive natural products, with diverse structures and functions. Polyketides are produced by megaenzymes termed as polyketide synthases (PKSs). PKS biosynthetic pathways are divided into the cis-AT PKSs and trans-AT PKSs; a division based mainly on the absence of an acyltransferase (AT) domain in the trans-AT PKS modules. In trans-AT biosynthesis, the AT activity is contributed via one or several independent proteins, and there are few other characteristics that distinguish trans-AT PKSs from cis-AT PKSs, especially in the formation of the ß-branch. The trans-AT PKSs constitute a major PKS pathway, and many are found in Burkholderia species, which are prevalent in the environment and prolific sources of polyketides. This review summarizes studies from 1973 to 2017 on the biosynthesis of natural products by trans-AT PKSs from Burkholderia species.

Using a marine microalga as a chassis for polyethylene terephthalate (PET) degradation.

BACKGROUND: The biological degradation of plastics is a promising method to counter the increasing pollution of our planet with artificial polymers and to develop eco-friendly recycling strategies. Polyethylene terephthalate (PET) is a thermoplast industrially produced from fossil feedstocks since the 1940s, nowadays prevalently used in bottle packaging and textiles. Although established industrial processes for PET recycling exist, large amounts of PET still end up in the environment-a significant portion thereof in the world's oceans. In 2016, Ideonella sakaiensis, a bacterium possessing the ability to degrade PET and use the degradation products as a sole carbon source for growth, was isolated. I. sakaiensis expresses a key enzyme responsible for the breakdown of PET into monomers: PETase. This hydrolase might possess huge potential for the development of biological PET degradation and recycling processes as well as bioremediation approaches of environmental plastic waste. RESULTS: Using the photosynthetic microalga Phaeodactylum tricornutum as a chassis we generated a microbial cell factory capable of producing and secreting an engineered version of PETase into the surrounding culture medium. Initial degradation experiments using culture supernatant at 30 °C showed that PETase possessed activity against PET and the copolymer polyethylene terephthalate glycol (PETG) with an approximately 80-fold higher turnover of low crystallinity PETG compared to bottle PET. Moreover, we show that diatom produced PETase was active against industrially shredded PET in a saltwater-based environment even at mesophilic temperatures (21 °C). The products resulting from the degradation of the PET substrate were mainly terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) estimated to be formed in the micromolar range under the selected reaction conditions. CONCLUSION: We provide a promising and eco-friendly solution for biological decomposition of PET waste in a saltwater-based environment by using a eukaryotic microalga instead of a bacterium as a model system. Our results show that via synthetic biology the diatom P. tricornutum indeed could be converted into a valuable chassis for biological PET degradation. Overall, this proof of principle study demonstrates the potential of the diatom system for future biotechnological applications in biological PET degradation especially for bioremediation approaches of PET polluted seawater.

RETRACTED: Effluent containing Rubrivivax gelatinosus promoting the yield, digestion system, disease resistance, mTOR and NF-kB signaling pathway, intestinal microbiota and aquaculture water quality of crucian carp.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of Editors-in-Chief and first Author. The article duplicates significant parts of a paper that had already appeared in Fish & Shellfish Immunology, Volume 93 (2019) 726-731, https://doi.org/10.1016/j.fsi.2019.06.052. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process. The first author informed the journal that the article was published without the knowledge of the co-authors.

Precursor-feeding and altered-growth conditions reveal novel blue pigment production by Rubrivivax benzoatilyticus JA2.

OBJECTIVE: To explore the secondary metabolite biosynthetic potential of Rubrivivax benzoatilyticus JA2 using a new metabolite mining strategy. RESULTS: Combination of precursor-feeding and altered growth conditions were used to mine new biomolecules. Strain JA2 utilised L-phenylalanine as sole source of nitrogen and showed pigments production only under phenylalanine-amended aerobic cultures. Stable isotope based precursor feeding studies indicated the blue pigment consists of 4-phenyl rings derived from L-phenylalanine. The purified blue pigment displayed characteristic visible-absorption and pH-dependent color variations. Precursor-feeding under altered growth conditions activated the plausible novel aromatic pigment production in strain JA2. CONCLUSION: Our approach unraveled the previously unknown pigment synthesis in strain JA2 and demonstrated the potential of mining strategy in discovering the hidden secondary metabolite repertoire in microorganisms.

Efficient circular gene knockout system for Burkholderiales strain DSM 7029 and Mycobacterium smegmatis mc2 155.

Multiple gene knockouts are often employed in studies of microbial physiology and genetics. However, the selective markers that confer antibiotic resistance are generally limited, so it is necessary to remove these resistance genes before the next round of using, which is time consuming and labor intensive. Here, we created a universal circular gene knockout system for both the gram-negative bacterial Burkholderiales strain DSM 7029 and the gram-positive bacterial Mycobacterium smegmatis mc2 155, by combining the homologous recombination with multiple serine integrase-meditated site-specific recombination systems. In this system, a resistance gene and an integrase gene were constructed within the two attachment sites corresponding to a second, different integrase to form a cassette for gene disruption, which could be easily removed by the second integrase during the subsequent round of gene knockout. The sacB gene was also employed for negative selection. As the integrase-mediated deletion of the resistance/integrase gene cassette was highly efficient and concurrent with the following knockout round, the cyclic use of three cassettes could achieve multiple gene knockout in a sequential manner. Following the modularity concept in synthetic biology, common components of the knockout plasmids were retained as BioBricks, accelerating the knockout plasmids construction process. The circular gene knockout system can also be used for multiple gene insertions and applied to other microorganisms.