The limited number of well-characterised model bacteria cannot address all the challenges in a circular bioeconomy. Therefore, there is a growing demand for new production strains with enhanced resistance to extreme conditions, versatile metabolic capabilities and the ability to utilise cost-effective renewable resources while efficiently generating attractive biobased products. Particular thermophilic microorganisms fulfil these requirements. Non-virulent Gram-negative Caldimonas thermodepolymerans DSM15344 is one such attractive thermophile that efficiently converts a spectrum of plant biomass sugars into high quantities of polyhydroxyalkanoates (PHA)-a fully biodegradable substitutes for synthetic plastics. However, to enhance its biotechnological potential, the bacterium needs to be 'domesticated'. In this study, we established effective homologous recombination and transposon-based genome editing systems for C. thermodepolymerans. By optimising the electroporation protocol and refining counterselection methods, we achieved significant improvements in genetic manipulation and constructed the AI01 chassis strain with improved transformation efficiency and a ΔphaC mutant that will be used to study the importance of PHA synthesis in Caldimonas. The advances described herein highlight the need for tailored approaches when working with thermophilic bacteria and provide a springboard for further genetic and metabolic engineering of C. thermodepolymerans, which can be considered the first model of thermophilic PHA producer.

• Keywords
• Caldimonas thermodepolymerans, gene deletion, genetic engineering, polyhydroxyalkanoates, thermophiles,
• MeSH
• Gene Editing * methods   MeSH
• Electroporation MeSH
• Genome, Bacterial MeSH
• Homologous Recombination MeSH
• Metabolic Engineering methods   MeSH
• Polyhydroxyalkanoates * biosynthesis   metabolism   MeSH
• DNA Transposable Elements MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polyhydroxyalkanoates * MeSH
• DNA Transposable Elements MeSH

Purple photosynthetic bacteria (PPB) are versatile microorganisms capable of producing various value-added chemicals, e.g., biopolymers and biofuels. They employ diverse metabolic pathways, allowing them to adapt to various growth conditions and even extreme environments. Thus, they are ideal organisms for the Next Generation Industrial Biotechnology concept of reducing the risk of contamination by using naturally robust extremophiles. Unfortunately, the potential of PPB for use in biotechnology is hampered by missing knowledge on regulations of their metabolism. Although Rhodospirillum rubrum represents a model purple bacterium studied for polyhydroxyalkanoate and hydrogen production, light/chemical energy conversion, and nitrogen fixation, little is known regarding the regulation of its metabolism at the transcriptomic level. Using RNA sequencing, we compared gene expression during the cultivation utilizing fructose and acetate as substrates in case of the wild-type strain R. rubrum DSM 467T and its knock-out mutant strain that is missing two polyhydroxyalkanoate synthases PhaC1 and PhaC2. During this first genome-wide expression study of R. rubrum, we were able to characterize cultivation-driven transcriptomic changes and to annotate non-coding elements as small RNAs.

• Keywords
• Acetate, Depolymerase knock-out, Fructose, Gene ontology, Genome, Metabolism, Polyhydroxyalkanoates, RNA-Seq, Rhodospirillum rubrum, Transcriptome,
• Publication type
• Journal Article MeSH

An ever-growing body of literature evidences the protective role of polyhydroxyalkanoates (PHAs) against a plethora of mostly physical stressors in prokaryotic cells. To date, most of the research done involved bacterial strains isolated from habitats not considered to be life-challenging or extremely impacted by abiotic environmental factors. Polar region microorganisms experience a multitude of damaging factors in combinations rarely seen in other of Earth's environments. Therefore, the main objective of this investigation was to examine the role of PHAs in the adaptation of psychrophilic, Arctic-derived bacteria to stress conditions. Arctic PHA producers: Acidovorax sp. A1169 and Collimonas sp. A2191, were chosen and their genes involved in PHB metabolism were deactivated making them unable to accumulate PHAs (ΔphaC) or to utilize them (Δi-phaZ) as a carbon source. Varying stressors were applied to the wild-type and the prepared mutant strains and their survival rates were assessed based on CFU count. Wild-type strains with a functional PHA metabolism were best suited to survive the freeze-thaw cycle - a common feature of polar region habitats. However, the majority of stresses were best survived by the ΔphaC mutants, suggesting that the biochemical imbalance caused by the lack of PHAs induced a permanent cell-wide stress response thus causing them to better withstand the stressor application. Δi-phaZ mutants were superior in surviving UV irradiation, hinting that PHA granule presence in bacterial cells is beneficial despite it being biologically inaccessible. Obtained data suggests that the ability to metabolize PHA although important for survival, probably is not the most crucial mechanism in the stress-resistance strategies arsenal of cold-loving bacteria. KEY POINTS: • PHA metabolism helps psychrophiles survive freezing • PHA-lacking psychrophile mutants cope better with oxidative and heat stresses • PHA granule presence enhances the UV resistance of psychrophiles.

• Keywords
• Arctic bacteria, Environmental stressors, Polyhydroxyalkanoates, Survival,
• MeSH
• Bacteria metabolism   MeSH
• Polyhydroxyalkanoates * metabolism   MeSH
• Carbon metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polyhydroxyalkanoates * MeSH
• Carbon MeSH

In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.

• Keywords
• Arctic, Bioplastics, Extremophile, Low-temperature biotechnology, Oligotrophy,
• MeSH
• Comamonadaceae * genetics   MeSH
• Genetic Engineering MeSH
• Polyhydroxyalkanoates * MeSH
• Temperature MeSH
• Carbon MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Polyhydroxyalkanoates * MeSH
• Carbon MeSH

Polyhydroxyalkanoates (PHAs) are hydrolyzable bio-polyesters. The possibility of utilizing lignocellulosic waste by-products and grape pomace as carbon sources for PHA biosynthesis was investigated. PHAs were biosynthesized by employing Cupriavidus necator grown on fructose (PHBV-1) or grape sugar extract (PHBV-2). Fifty grams of lyophilized grape sugar extract contained 19.2 g of glucose, 19.1 g of fructose, 2.7 g of pectin, 0.52 g of polyphenols, 0.51 g of flavonoids and 7.97 g of non-identified rest compounds. The grape sugar extract supported the higher production of biomass and modified the composition of PHBV-2. The biosynthesized PHAs served as matrices for the preparation of the scaffolds. The PHBV-2 scaffolds had about 44.2% lower crystallinity compared to the PHBV-1 scaffolds. The degree of crystallinity markedly influenced the mechanical behavior and enzymatic hydrolysis of the PHA scaffolds in the synthetic gastric juice and phosphate buffer saline solution with the lipase for 81 days. The higher proportion of amorphous moieties in PHBV-2 accelerated enzymatic hydrolysis. After 81-days of lasting enzymatic hydrolysis, the morphological changes of the PHBV-1 scaffolds were negligible compared to the visible destruction of the PHBV-2 scaffolds. These results indicated that the presence of pectin and phenolic moieties in PHBV may markedly change the semi-crystalline character of PHBV, as well as its mechanical properties and the course of abiotic or enzymatic hydrolysis.

• Keywords
• degradation kinetics, grape pomace, hydrolysis, mechanical properties, morphology, polyhydroxyalkanoates, scaffolds, thermal properties,
• Publication type
• Journal Article MeSH