Recent developments in non-biodegradable biopolymers: Precursors, production processes, and future perspectives.

During the last decades, biopolymers experienced a renaissance. The increasing limitation of fossil resources in combination with a public demand for environmental-friendly and sustainable processes has led to the formation of a market for biobased plastics. Especially non-biodegradable bioplastics are very interesting materials, as they combine the benefits of reduced carbon footprint during production and increased resource efficiency with the persistence to microbial degradation. Consequently, persistent biomass-derived plastic materials are highly promising to substitute conventional fossil-based plastics in applications, which require durability and longevity. Non-biodegradable bioplastics derived from renewable resources represent 57% of all bioplastics with partially biobased polyethylene terephthalate currently leading the market, followed by biobased polyamides and fully biomass-derived polyethylene. An exceptional biopolymer with thermoplastic properties was discovered only two decades ago, when-for the first time-polythioesters were synthesized by microbial fermentation. Though synthesized by bacteria, it turned out that polythioesters are non-biodegradable by microorganisms in contrast to all other biopolymers and thus, represent a novel non-biodegradable bioplastic material. This review gives an overview about the recent development and progress regarding bioplastics with special focus on persistent bioplastics. We describe the generation of the respective monomers from biomass-derived substrates and summarize the current status of production, which range from the laboratory-scale up to large-scale industrial processes.

In vitro biosynthesis of 3-mercaptolactate by lactate dehydrogenases.

3-Mercaptolactate (3ML) is an interesting mercapto compound with special regard to the biosynthesis of new polythioesters (PTEs). Unfortunately, this thioester analog of lactic acid is currently not commercially available. For this reason, we developed an in vitro biosynthesis pathway to convert cysteine to 3-mercaptopyruvate (3MPy), which is then rapidly and efficiently converted to 3ML by suitable lactate dehydrogenases (LDHs). As liver LDH from Rattus norvegicus (LDHRn) was previously described to Exhibit 3MPy reduction activity, in silico studies based on homology to LDHRn were performed and led to the identification of four potentially suitable bacterial LDH candidates from Escherichia coli (LDHEc), Corynebacterium glutamicum (LDHCg), Bacillus cereus (LDHBc) and Gloeobacter violaceus (LDHGv). After heterologous expression in E. coli followed by purification, the enzymes were assessed for their potential to reduce 3MPy to 3ML in comparison to LDHRn. With 3MPy, LDHs from E. coli, C. glutamicum and B. cereus showed no or only very low specific activities of 0.23±0.1U/mg (LDHCg) and 0.08±0.2U/mg (LDHBc), respectively. In contrast, LDHGv exhibited a remarkable specific activity of 63.6±8.1U/mg, being even twice as active as the R. norvegicus LDH. To verify LDH-catalyzed biosynthesis of 3ML we developed and optimized a detection method allowing qualitative analysis and quantification of 3MPy and 3ML by derivatization with Ellman's reagent and liquid chromatography-mass spectrometry. This study shows once more the impressive versatility of LDHs and presents a rapid and efficient biosynthesis process for 3ML, a biotechnologically interesting, yet hard-to-obtain, compound.

Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.