Towards using bacterial microcompartments as a platform for spatial metabolic engineering in the industrially important and metabolically versatile Zymomonas mobilis.

Advances in synthetic biology have enabled the incorporation of novel biochemical pathways for the production of high-value products into industrially important bacterial hosts. However, attempts to redirect metabolic fluxes towards desired products often lead to the buildup of toxic or undesirable intermediates or, more generally, unwanted metabolic cross-talk. The use of shells derived from self-assembling protein-based prokaryotic organelles, referred to as bacterial microcompartments (BMCs), as a scaffold for metabolic enzymes represents a sophisticated approach that can both insulate and integrate the incorporation of challenging metabolic pathways into industrially important bacterial hosts. Here we took a synthetic biology approach and introduced the model shell system derived from the myxobacterium Haliangium ochraceum (HO shell) into the industrially relevant organism Zymomonas mobilis with the aim of constructing a BMC-based spatial scaffolding platform. SDS-PAGE, transmission electron microscopy, and dynamic light scattering analyses collectively demonstrated the ability to express and purify empty capped and uncapped HO shells from Z. mobilis. As a proof of concept to internally load or externally decorate the shell surface with enzyme cargo, we have successfully targeted fluorophores to the surfaces of the BMC shells. Overall, our results provide the foundation for incorporating enzymes and constructing BMCs with synthetic biochemical pathways for the future production of high-value products in Z. mobilis.

Characterization of a novel aromatic substrate-processing microcompartment in Actinobacteria.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta-cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro, in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta-cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs.

ZnJ2 Is a Member of a Large Chaperone Family in the Chloroplast of Photosynthetic Organisms that Features a DnaJ-Like Zn-Finger Domain.

Photosynthesis is performed by large complexes, composed of subunits encoded by the nuclear and chloroplast genomes. Assembly is assisted by general and target-specific chaperones, but their mode of action is yet unclear. We formerly showed that ZnJ2 is an algal chaperone resembling BSD2 from land plants. In algae, it co-migrates with the rbcL transcript on chloroplast polysomes, suggesting it contributes to the de-novo synthesis of RbcL (Doron et al., 2014). ZnJ2 contains four CXXCXGXG motifs, comprising a canonical domain typical also of DnaJ-type I (DNAJA). It contributes to the binding of protein substrates to DnaK and promotes an independent oxidoreductase activity (Mattoo et al., 2014). To examine whether ZnJ2 has oxidoreductase activity, we used the RNaseA assay, which measures the oxidation-dependent reactivation of reduced-denatured RNaseA. Although ZnJ2 assisted the native refolding of reduced-denatured RNaseA, its activity was restricted to an oxidizing environment. Thus, ZnJ2 did not carry the exclusive responsibility for the formation of disulfide bridges, but contributed to the stabilization of its target polypeptides, until they reached their native state. A ZnJ2 cysteine deficient mutant maintained a similar holding chaperone activity as the wild-type and did not induce the formation of disulfide bonds. ZnJ2 is devoid of a J-domain. It thus does not belong to the J-domain co-chaperones that target protein substrates to DnaK. As expected, in vitro, its aggregation-prevention activity was not synergic to the ATP-fueled action of DnaK/DnaJ/GrpE in assisting the native refolding of denatured malate dehydrogenase, nor did it show an independent refolding activity. A phylogenetic analysis showed that ZnJ2 and BSD2 from land plants, are two different proteins belonging to a larger group containing a cysteine-rich domain, that also includes the DNAJAs. Members of this family are apparently involved in specific assembly of photosynthetic complexes in the chloroplast.

Transgene Expression in Microalgae-From Tools to Applications.

Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while C. reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.

Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF) with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

The BSD2 ortholog in Chlamydomonas reinhardtii is a polysome-associated chaperone that co-migrates on sucrose gradients with the rbcL transcript encoding the Rubisco large subunit.

The expression of the CO2 -fixation enzyme ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which is affected by light, involves the cysteine-rich protein bundle-sheath defective-2 (BSD2) that was originally identified in maize bundle-sheath cells. We identified the BSD2 ortholog in Chlamydomonas reinhardtii as a small protein (17 kDa) localized to the chloroplast. The algal BSD2-ortholog contains four CXXCXGXG DnaJ-like elements, but lacks the other conserved domains of DnaJ. BSD2 co-migrated with the rbcL transcript on heavy polysomes, and both BSD2 and rbcL mRNA shifted to the lighter fractions under oxidizing conditions that repress the translation of the Rubisco large subunit (RbcL). This profile of co-migration supports the possibility that BSD2 is required for the de novo synthesis of RbcL. Furthermore, BSD2 co-migrated with the rbcL transcript in a C. reinhardtii premature-termination mutant that encodes the first 60 amino acids of RbcL. In both strains, BSD2 shared its migration profile with the rbcL transcript but not with psbA mRNA. The chaperone activity of BSD2 was exemplified by its ability to prevent the aggregation of both citrate synthase (CS) and RbcL in vitro following their chemical denaturation. This activity did not depend on the presence of the thiol groups on BSD2. In contrast, the activity of BSD2 in preventing the precipitation of reduced ß-chains in vitro in the insulin turbidity assay was thiol-dependent. We conclude that BSD2 combines a chaperone 'holdase' function with the ability to interact with free thiols, with both activities being required to protect newly synthesized RbcL chains.