Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation

Polyhydroxyalkanoates (PHAs) are highly reduced bacterial storage compounds that increase fitness in changing environments. We have previously shown that phaRBAC genes from the Antarctic bacterium Pseudomonas sp. 14-3 are located in a genomic island containing other genes probably related with its adaptability to cold environments. In this paper, Pseudomonas sp. 14-3 and its PHA synthase-minus mutant (phaC) were used to asses the effect of PHA accumulation on the adaptability to cold conditions. The phaC mutant was unable to grow at 10°C and was more susceptible to freezing than its parent strain. PHA was necessary for the development of the oxidative stress response induced by cold treatment. Addition of reduced compounds cystine and gluthathione suppressed the cold sensitive phenotype of the phaC mutant. Cold shock produced very rapid degradation of PHA in the wild type strain. The NADH/NAD ratio and NADPH content, estimated by diamide sensitivity, decreased strongly in the mutant after cold shock while only minor changes were observed in the wild type. Accordingly, the level of lipid peroxidation in the mutant strain was 25-fold higher after temperature downshift. We propose that PHA metabolism modulates the availability of reducing equivalents, contributing to alleviate the oxidative stress produced by low temperature.     

Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria.

The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus, purple non-sulfur bacteria, such as Rhodospirillum rubrum, purple sulfur bacteria, such as Chromatium vinosum, pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens, and for the Gram-positive bacterium Rhodococcus ruber. Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.     

Polyhydroxyalkanoates production from carbohydrates by a genetic recombinant Aeromonas sp.

Aims: To develop an Aeromonas strain able to utilize inexpensive carbon sources such as starch for the synthesis of polyhydroxyalkanoates (PHA). Methods and Results: A recombinant Aeromonas sp. (strain KC007‐1) was constructed by introducing the PHB synthesis genes (phaCAB) into the bacterium. Strain KC001‐R1 can not only use carbohydrate (including starch) for growth but also accumulate significant amounts of polyhydroxybutyrate (PHB) in the cells. Conclusions: One of the present focuses on PHA production has been on lowering the production costs. Starch is an example of an inexpensive carbohydrate for use in industrial production of PHA. We have demonstrated that by introducing the phaCAB operon into Aeromonas sp. allowed the bacterium able to accumulated PHB using this substrate. Significance and Impact of the Study: Aeromonas spp. are able to synthesize PHA using fatty acids as carbon source. Although good robust growth results with use of starch as sole carbon source for Aeromonas, PHA synthesis does not occur. Strain KC007‐R1 showed the ability to accumulate PHA in relative high amount with both carbohydrates and fatty acids as carbon source, and can be cultivated to a significant amount of cell mass and hence is a potential strain for further development for industrial applications.     

Molecular basis for biosynthesis and accumulation of polyhydroxyalkanoic acids in bacteria

Abstract The current knowledge on the structure and on the organization of polyhydroxyalkanoic acid (PHA)-biosynthetic genes from a wide range of different bacteria, which rely on different pathways for biosynthesis of this storage polyesters, is provided. Molecular data will be shown for genes of Alcaligenes eutrophus , purple non-sulfur bacteria, such as Rhodospirillum rubrum , purple sulfur bacteria, such as Chromatium vinosum , pseudomonads belonging to rRNA homology group I, such as Pseudomonas aeruginosa, Methylobacterium extorquens , and for the Gram-positive bacterium Rhodococcus ruber . Three different types of PHA synthases can be distinguished with respect to their substrate specificity and structure. Strategies for the cloning of PHA synthase structural genes will be outlined which are based on the knowledge of conserved regions of PHA synthase structural genes and of the PHA-biosynthetic routes in bacteria as well as on the heterologous expression of these genes and on the availability of mutants impaired in the accumulation of PHA. In addition, a terminology for the designation of PHAs and of proteins and genes relevant for the metabolism of PHA is suggested.     

A novel mcl PHA-producing bacterium, Pseudomonas guezennei sp. nov., isolated from a 'kopara' mat located in Rangiroa, an atoll of French Polynesia

Aims: The aim of the present study was to describe an aerobic, mesophilic and heterotrophic bacterium, designated RA26, able to produce a medium‐chain‐length polyhydroxyalkanoate (PHA). It was isolated from a French Polynesian bacterial mat located in the atoll of Rangiroa. Methods and Results: This micro‐organism, on the basis of the phenotypical features and genotypic investigations can be clearly assigned to the Pseudomonas genus and the name of Pseudomonas guezennei is proposed. Optimal growth occurs between 33 and 37°C, at a pH between 6·4 and 7·1 and at ionic strength of 15 g l^?1 of sea salts. The G+C content of DNA is 63·2%. Under laboratory conditions, this bacterium produced a novel, medium‐chain‐length PHA, mainly composed of 3‐hydroxydecanaote (64 mol.%) and 3‐hydroxyoctanoate (24 mol.%) (GC‐MS, NMR) from a single nonrelated carbon substrate, i.e. glucose. Conclusions: The bacterium P. guezennei produces a novel PHA mcl with elastomeric properties. Significance and Impact of the Study: PHAs share physical and material properties that recommend them for application in various areas, and are considered as an alternative to nonbiodegradable plastics produced from fossil oils. In this study, we describe a new bacteria with the capability to synthesize a novel PHA with promising biotechnological applications.     

A marine bacterium accumulates polyhydroxyalkanoate consisting of mainly 3-hydroxydodecanoate and 3-hydroxydecanoate

A deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913, isolated from abyssalbenthic sediments in Bohai Sea, was found to be able to synthesize polyhydroxyalkanoate (PHA) polymer consisting of mainly 3-hydroxydecanoate and 3-hydroxydodecanoate. PHA accumulation in Pseudoaltermonas sp. SM9913 was confirmed by transmission electron microscope, Nile red dye and gas chromatography/mass spectral. The PHA content was determined as high as 2–3% of cell dry weight when the strain was grown in a sea water-based liquid medium. The relationship between PHA and exopolysaccharide accumulation in this strain was discussed.     

Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli

Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, β-ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a pH-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of 194.1 and 141.6 g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness.     

Biotechnological Coproduction of Compatible Solutes and Polyhydroxyalkanoates using the Genus Halomonas

An increased usage of poly‐β‐hydroxyalkanoates (PHA), for instance as bulk biodegradable and biocompatible plastics, will require a cheaper production and downstream processing. If the synthesis of this intracellular biopolyester could be combined with the production of another valuable intracellular product, the economic balance of the process could be improved. It was found that the moderately halophilic bacterium Halomonas elongata simultaneously synthesizes PHA and a protector molecule, called ectoine. Whereas the synthesis of PHA is a response to the shortage of nutrients, the production of ectoine counteracts osmotic imbalances. This behavior is in so far surprising as the conditions of a bi‐factorial stress initiate the fast simultaneous synthesis of ectoine and PHA. In the presence of 100 g/L NaCl, Halomonas elongata accumulated up to 50 % w/w PHA and up to 14 % ectoine within 2–3 days under so far non‐optimized conditions. Furthermore, it was found that other Halomonas species (e.g. Halomonas halodenitrificans and own isolates of Halomonas halodeneurihalina and Halomonas salina) were able to produce both ectoine and PHA.     

Biosynthesis of Poly(3-Hydroxyalkanoic Acid) Copolymer from CO(inf2) in Pseudomonas acidophila through Introduction of the DNA Fragment Responsible for Chemolithoautotrophic Growth of Alcaligenes hydrogenophilus

Pseudomonas acidophila is a bacterial strain producing a poly(3-hydroxyalkanoic acid) (PHA) copolymer from low-molecular-weight organic compounds such as formate and acetate. The genes responsible for PHA production were cloned in cosmid pIK7 containing a 14.8-kb HindIII fragment of P. acidophila DNA. With the aim of developing a means of producing a PHA copolymer from CO(inf2), cosmid pIK7 was introduced into a polymer-negative mutant of the chemolithoautotrophic bacterium Alcaligenes eutrophus PHB(sup-)4. However, the recombinant strain produced a homopolymer of 3-hydroxybutyric acid (polyhydroxybutyric acid) from CO(inf2). Since it was thought that the composition of the accumulated polymer might depend not on the PHA biosynthetic genes but on the metabolism of the host strain, a recombinant plasmid, pFUS, containing the genes for chemolithoautotrophic growth of the hydrogen-oxidizing bacterium A. hydrogenophilus was introduced into P. acidophila by conjugation. The recombinant plasmid pFUS was stably maintained in P. acidophila in the absence of chemolithoautotrophic or antibiotic selection. This pFUS-harboring strain possessed the ability to grow under a gas mixture of H(inf2), O(inf2), and CO(inf2) in a mineral salts medium, and PHA copolymer accumulation was confirmed by nuclear magnetic resonance spectral analysis. A gas chromatogram obtained by gas chromatography-mass spectrometry showed the composition of the polymer to be 52.8% 3-hydroxybutyrate, 41.1% 3-hydroxyoctanoate, and 6.1% 3-hydroxydecanoate. This is the first report of the production of a PHA copolymer from CO(inf2) as sole carbon source.     

Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.     

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

Expression of PHA polymerase genes of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular approach to elucidate the genes involved in the formation of two homopolymers consisting of short-chain-length 3-hydroxyalkanoic acids

Burkholderia sp. accumulates polyhydroxyalkanoates (PHAs) containing 3-hydroxybutyrate and 3-hydroxy-4-pentenoic acid when grown on mineral media under limited phosphate or nitrogen, and using sucrose or gluconate as a carbon and energy source. Solvent fractionation and NMR spectroscopic characterization of these polyesters revealed the simultaneous accumulation of two homopolyesters rather than a co-polyester with random sequence distribution of the monomers [Valentin HE, Berger PA, Gruys KJ, Rodrigues MFA, Steinbüchel A, Tran M, Asrar J (1999) Macromolecules 32: 7389–7395]. To understand the genetic requirements for such unusual polyester accumulation, we probed total genomic DNA from Burkholderia sp. by Southern hybridization experiments using phaC-specific probes. These experiments indicated the presence of more than one PHA synthase gene within the genome of Burkholderia sp. However, when total genomic DNA from Burkholderia sp. was used to complement a PHA-negative mutant of Ralstonia eutropha for PHA accumulation, only one PHA synthase gene was obtained resembling the R. eutropha type of PHA synthases, based on amino acid sequence similarity. In addition to the PHA synthase gene, based on high sequence homology, genes encoding a β-ketothiolase and acetoacetyl-CoA reductase were identified in a gene cluster with the PHA synthase gene. The arrangement of the three genes is quite similar to the R. eutropha poly-β-hydroxybutyrate biosynthesis operon. Received: 3 September 1999 / Received revision: 29 October 1999 / Accepted: 5 November 1999     

红树林土壤中产聚羟基脂肪酸酯细菌的分离及其评估

[背景] 细菌能通过合成聚羟基脂肪酸酯（Polyhydroxyalknoates,PHA）在细胞内储存物质和能量,提高对环境的适应能力。在红树林中,由于土壤周期性受海水浸没,形成营养物质种类丰富和含量波动大的特殊生境,为细菌进化出特殊的PHA合成途径提供了条件。[目标] 为了增加对红树林产PHA细菌资源的了解,获得产PHA细菌,使用纯培养方法分离和鉴定细菌,并评估菌株的产PHA能力。[方法] 采集红树植物海桑根系和红树滩涂土壤样品,连续5周培养、分离纯化获得细菌菌株;通过16S rRNA基因相似性及系统进化分析鉴定细菌分类地位,利用PHA合成酶基因（phaC）鉴定细菌合成PHA的能力;通过基因组草图测序,分析细菌的phaC基因种类、代谢通路及系统进化关系;通过气相色谱分析细菌产PHA的累积量及组成。[结果] 从红树林土壤样品中分离得到97株细菌,其中13株带有phaC基因,包括坚强芽孢杆菌（Cytobacillus firmus）、弯曲芽孢杆菌（Bacillus flexus）、除烃海杆菌（Marinobacter hydrocarbonoclasticus）和酯香微杆菌（Microbacterium esteraromaticum）。B. flexus MN15-19以丙酮酸盐为碳源,可累积细胞干重11%的PHA,同时具有固碳功能的还原性三羧酸循环通路,有开发成为固碳产PHA工程菌株的潜力。酯香微杆菌可产PHA,但是其phaC基因结构特殊,基因组注释未能识别出任何已知phaC基因。[结论] 研究发现红树林土壤可培养细菌中存在未知的PHA合成途径,说明红树林生态系统中的细菌具有资源挖掘的重要价值。     

Mutants of Pseudomonas putida affected in poly-3-hydroxyalkanoate synthesis

The generation and characterization of Pseudomonas putida KT2442 mutants affected in poly-3-hydroxyalkanoate (PHA) synthesis are reported. The mutants from P. putida KT2442 carrying several copies of the PHA-polymerase-encoding gene (phaC) were isolated via N-methyl-N′-nitro-N-nitrosoguanidine chemical mutagenesis and contained mutation(s) on genes that are involved in PHA accumulation other than the phaC genes. No PHA-free mutants were obtained, suggesting that there must be various routes for the synthesis of PHA polymerase precursors. One of the isolated mutants (GPp120) accumulated more PHA than the parental strain, and there was virtually no down-regulation of PHA formation by growth in non-limiting amounts of nitrogen, which normally block or reduce formation of PHA. Compared to the parental strain, GPp120 exhibited significant changes in physiology and morphology when grown in minimal medium: the growth rate was reduced more than twofold and cells formed filaments. The other four groups of isolated mutants, with P. putida strains GPp121 to GPp124 as characteristic type strains, exhibited morphological characteristics similar to those of the parental strain. However, they showed reduced PHA production compared to the parental PHA⁺ strain, and especially GPp121 and GPp122 showed PHA formation tightly controlled by nutrient conditions. All of these mutants provide starting points for genetically dissecting the biosynthesis and regulation of PHA precursors. Received: 10 November 1997 / Received revision: 6 February 1998 / Accepted: 6 February 1998     

Altered composition of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> poly(hydroxyalkanoate) through expression of PHA synthase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 35206

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved α/β-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB⁻4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4–5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.     

Expression of polyhydroxyalkanoic-acid-biosynthesis genes in methylotrophic bacteria relying on the ribulose monophosphate pathway

Four representatives of methylotrophic bacteria relying on the ribulose monophosphate (RMP) pathway were investigated for their capability to synthesize polyhydroxyalkanoic acids (PHA). In Methylophilus methylotrophus B115, Methylobacillus glycogenes strains B121 and B53 and Acetobacter methanolicus B58 no \-ketothiolase, acetoacetyl-coenzyme A (CoA) reductase or PHA synthase could be detected, and hybridization experiments using heterologous DNA probes derived from PHA-biosynthesis genes of Methylobacterium extorquens or Alcaligenes eutrophus gave no evidence for the presence of the corresponding genes in these PHA-negative methylotrophic bacteria. Fragments harbouring a cluster of PHA-biosynthesis genes of A. eutrophus or Chromatium vinosum or isolated PHA synthase structural genes of M. extorquens, Rhodospirillum rubrum or Rhodobacter sphaeroides were mobilized into the RMP pathway bacteria mentioned above. Only transconjugants, which harboured the PHA-biosynthesis genes of A. eutrophus or C. vinosum, expressed active \-ketothiolase, acetoacetyl-CoA reductase and PHA synthase and accumulated poly(3-hydroxybutyric acid) (PHB). Highest amounts of PHB (up to 15% of the cellular dry weight) were accumulated in transconjugants of Methylophilus methylotrophus B115 or of Methylobacillus glycogenes strains B121 and B53 harbouring the PHA-biosynthesis genes of C. vinosum. Correspondence to: A. Steinbüchel     

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.