Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

/pdf/characterization-and-engineering-of-a-plastic-degrading-2rf8vuokpw.pdf

Characterization and engineering of a plastic-degrading aromatic polyesterase

With the sharp increase in population and modernization of society, environmental pollution resulting from petroleum hydrocarbons has increased, resulting in an urgent need for remediation. Petroleum hydrocarbon-degrading bacteria are ubiquitous in nature and can utilize these compounds as sources of carbon and energy. Bacteria displaying such capabilities are often exploited for the bioremediation of petroleum oil-contaminated environments. Recently, microbial remediation technology has developed rapidly and achieved major gains. However, this technology is not omnipotent. It is affected by many environmental factors that hinder its practical application, limiting the large-scale application of the technology. This paper provides an overview of the recent literature referring to the usage of bacteria as biodegraders, discusses barriers regarding the implementation of this microbial technology, and provides suggestions for further developments.

/pdf/petroleum-hydrocarbon-degrading-bacteria-for-the-remediation-zmaxlkbj56.pdf

Petroleum Hydrocarbon-Degrading Bacteria for the Remediation of Oil Pollution Under Aerobic Conditions: A Perspective Analysis.

The previous part discusses the GI/BSI/DFKI Protection Profile which constitutes after the implementation of the identified improvements as the proposed evaluation methodology for remote electronic voting systems. The result can now be applied to available systems. Currently, there is no system that has been evaluated against the GI/BSI/DFKI Protection Profile or even against the improved version.

Proof of Concept

https://backend.orbit.dtu.dk/ws/files/160753786/1_s2.0_S1096717618301502_main.pdf

Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism.

Most petroleum-derived plastics, as exemplified by poly(ethylene terephthalate) (PET), are chemically inactive and highly resistant to microbial attack. The accumulation of plastic waste results in environmental pollution and threatens ecosystems, referred to as the “microplastic issue”. Recently, PET hydrolytic enzymes (PHEs) have been identified and we reported PET degradation by a microbial consortium and its bacterial resident, Ideonella sakaiensis. Bioremediation may thus provide an alternative solution to recycling plastic waste. The mechanism of PET degradation into benign monomers by PET hydrolase and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase from I. sakaiensis has been elucidated; nevertheless, biodegradation may require additional development for commercialization owing to the low catalytic activity of these enzymes. Here, we introduce PET degrading microorganisms and the enzymes involved, along with the evolution of PHEs to address the issues that hamper microbial and enzymatic PE...

Biodegradation of PET: Current Status and Application Aspects

https://digital.csic.es/bitstream/10261/161580/1/Bioremediation%203.0%20Engineering%20pollutant-removing....pdf

Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology.

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose.

Given its capacity to tolerate stress, NAD(P)H/NAD(P) balance, and increased ATP levels, the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. The knocked-in beta-glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage of the disaccharide to D-glucose, which was then channeled to the default central metabolism. Xylose oxidation to the dead end product D-xylonate was prevented by by deleting the gcd gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. These results not only show the broadening of the metabolic capacity of a soil bacterium towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new biochemical pathways for utilization and valorization of carbohydrate mixtures from lignocellulose processing.

https://www.biorxiv.org/content/biorxiv/early/2018/03/18/284182.full.pdf

Pavel Dvořák

Papers

Bioremediation 3.0: Engineering pollutant-removing bacteria in the times of systemic biology.

Refactoring the upper sugar metabolism of Pseudomonas putida for co-utilization of cellobiose, xylose, and glucose.

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Biology and Bioinformatics