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Organohalides are recalcitrant, toxic environmental pollutants. Reductive dehalogenase enzymes (RDases) found in organohalide respiring bacteria (OHRB) utilise organohalides as electron acceptors for cellular energy and growth, producing lesser-halogenated compounds. Consequently, microbial reductive dehalogenation via organohalide respiration represents a promising solution for clean-up of organohalide pollutants. Dehalobacter sp. UNSWDHB is an OHRB capable of respiring highly toxic chloroform (CF) and converting it to dichloromethane (DCM). TmrA has been identified as an RDase responsible for this conversion and different strategies for generation of functional recombinant TmrA is the focus of this article. In this study, TmrA was recovered from inclusion bodies expressed in E. coli and refolded in the presence of FeCl 3 , Na 2 S and cobalamin to yield functional enzyme. TmrA has been previously expressed in a soluble and functional form in the corrinoid-producing Bacillus megaterium. Using a fractional experimental design for cultivation and induction combined with purification under anaerobic conditions resulted in substantially higher activity of recombinant and native TmrA than previously reported. TmrA was then expressed in a soluble and active form in Shimwellia blattae. Co-expression with two different putative chaperone proteins from the original host did not increase the level of soluble expression in S. blattae , however activity assays showed that removing the TAT signal from TmrA increases the dechlorination activity compared to when the TAT signal is present. Finally, TmrA was successfully expressed in a soluble and active form in the H 2 -oxidizing C. necator H16, a novel host for the expression of RDases. • Active recombinant reductive dehalogenase (TmrA) in four different microbial hosts • Improved activity was achieved in Bacillus megaterium by process optimisation • Removal of the Tat signal improved activity when expressed in Shimwella blattae • Functional RDase expression in Cupravidius necator for the first time [ABSTRACT FROM AUTHOR]

Protein self-assembly is a process that often occurs via liquid–liquid phase separation (LLPS), a sequence-specific and environmentally influenced phenomenon. Heuristic analysis of these self-assembling proteins has highlighted the conservation and importance of tyrosine residues and their ability to initiate LLPS and subsequent liquid–solid phase transitions. Generating tyrosine-templated constructs such as those found in nature serves as a promising method to generate responsive, self-assembling systems for a diverse range of applications. We discuss the importance of tyrosine in mediating the self-assembly of proteins, the interactions that enable this, and how it can be exploited for beneficial purposes. Protein self-assembly, guided by the interplay of sequence- and environment-dependent liquid–liquid phase separation (LLPS), constitutes a fundamental process in the assembly of numerous intrinsically disordered proteins. Heuristic examination of these proteins has underscored the role of tyrosine residues, evident in their conservation and pivotal involvement in initiating LLPS and subsequent liquid–solid phase transitions (LSPT). The development of tyrosine-templated constructs, designed to mimic their natural counterparts, emerges as a promising strategy for creating adaptive, self-assembling systems with diverse applications. This review explores the central role of tyrosine in orchestrating protein self-assembly, delving into key interactions and examining its potential in innovative applications, including responsive biomaterials and bioengineering. [ABSTRACT FROM AUTHOR]