Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

RIKEN Center for Sustainable Resource Science

Polyhydroxyalkanoate (PHA) is a potential substitute for some petrochemical-based plastics. This biodegradable plastic is derived from microbial fermentation using various carbon substrates. Since carbon source has been identified as one of the major cost-absorbing factors in PHA production, cheap and renewable substrates are currently being investigated as substitutes for existing sugar-based feedstock. Plant oils have been found to result in high-yield PHA production. Malaysia, being the world's second largest producer of palm oil, is able to ensure continuous supply of palm oil products for sustainable PHA production. The biosynthesis and characterization of various types of PHA using palm oil products have been described in detail in this review. Besides, by-products and waste stream from palm oil industry have also demonstrated promising results as carbon sources for PHA biosynthesis. Some new applications in cosmetic and wastewater treatment show the diversity of PHA usage. With proper management practices and efficient milling processes, it may be possible to supply enough palm oil-based raw materials for human consumption and other biotechnological applications such as production of PHA in a sustainable manner.

Synthesis of polyhydroxyalkanoate from palm oil and some new applications.

The synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolated Chromobacterium sp. USM2 (PhaC(Cs)). PhaC(Cs) showed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. An in vitro assay of recombinant PhaC(Cs) expressed in Escherichia coli showed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strain C. necator (307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaC(Cs) was 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC from C. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation in Escherichia coli expressing PhaC(Cs) of up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaC(Cs) is a naturally occurring, highly active PHA synthase with superior polymerizing ability.

Characterization of the highly active polyhydroxyalkanoate synthase of Chromobacterium sp. strain USM2.

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA(Co) gene, 1182 bp), acetoacetyl-CoA reductase (phaB(Co) gene, 738 bp) and PHA synthase, class I (phaC(Co) gene, 1694 bp) were identified. Sequence analysis of the phaA(Co), phaB(Co) and phaC(Co) genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaC(Co) and phaAB(Co)) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM'-phaC(Co)AB(Re) and pGEM'-phaC(Re)AB(Co) were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCAB(Co)) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCAB(Co) could accumulate P(3HB) at 2g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively.

Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109.

Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaC(Cs)) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaC(Cs) for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C(4) and C(5)) or MCL (C(6)) substrates substantiates the fundamental classification of PHA synthases.

Characterization of site-specific mutations in a short-chain-length/medium-chain-length polyhydroxyalkanoate synthase: in vivo and in vitro studies of enzymatic activity and substrate specificity.

A new method is developed to prepare silk hydrogels and silk-pectin hydrogels via dialysis against methanol to obtain hydrogels with high concentrations of silk fibroin. The relationship between the mechanical and biological properties and the structure of the silk-pectin hydrogels is subsequently evaluated. The present results suggest that pectin associates with silk molecules when the silk concentration exceeds 15 wt%, suggesting that a silk concentration of over 15 wt% is critical to construct interacting silk-pectin networks. The silk-pectin hydrogel reported here is composed of a heterogeneous network, which is different from fiber-reinforced, interpenetrated networks and double-network hydrogels, as well as high-stiffness hydrogels (elastic modulus of 4.7 ± 0.9 MPa, elastic stress limit of 3.9 ± 0.1 MPa, and elastic strain limit of 48.4 ± 0.5%) with regard to biocompatibility and biodegradability.

Silk-pectin hydrogel with superior mechanical properties, biodegradability, and biocompatibility.

Available methods in plant genetic transformation are nuclear and plastid transformations because similar procedures have not yet been established for the mitochondria. The double membrane and small size of the organelle, in addition to its large population in cells, are major obstacles in mitochondrial transfection. Here we report the intracellular delivery of exogenous DNA localized to the mitochondria of Arabidopsis thaliana using a combination of mitochondria-targeting peptide and cell-penetrating peptide. Low concentrations of peptides were sufficient to deliver DNA into the mitochondria and expression of imported DNA reached detectable levels within a short incubation period (12 h). We found that electrostatic interaction with the cell membrane is not a critical factor for complex internalization, instead, improved intracellular penetration of mitochondria-targeted complexes significantly enhanced gene transfer efficiency. Our results delineate a simple and effective peptide-based method, as a starting point for the development of more sophisticated plant mitochondrial transfection strategies.

Gene introduction into the mitochondria of Arabidopsis thaliana via peptide-based carriers.

Cationic peptides such as poly(l-lysine) and poly(l-arginine) are important tools for gene delivery since they can efficiently condense DNA. It is difficult to produce cationic peptides by recombinant bacterial expression, and its chemical synthesis requires several steps of protection/deprotection and toxic agents. Chemo-enzymatic synthesis of peptides is a clean chemistry technique that allows fast production under mild conditions. With the aim to simplify the production of cationic peptides, the present work develops an enzymatic reaction which enables the synthesis of linear cationic peptides and, through terminal functionalization with tris(2-aminoethyl)amine, of branched cationic peptide conjugates, which show improved DNA complex formation. Cytotoxicity and transfection efficiency of all the chemo-enzymatically synthesized cationic peptides are evaluated for their novel use as gene delivery agents. Synthesized peptides exhibit transfection efficiencies comparable to previously reported monodisperse peptides. Chemo-enzymatic synthesis opens the door for efficient production of cationic peptides for their use as gene delivery carriers.

Chemo-Enzymatic Synthesis of Linear and Branched Cationic Peptides: Evaluation as Gene Carriers.

Amyloid fibers are classified as a new generation of tunable bionanomaterials that exhibit new functions related to their distinctive characteristics, such as their universality, tunability, and stiffness. Here, we introduce the catalytic residues of serine protease into a peptide catalyst (PC) via an enzyme-mimic approach. The rational design of a repeating pattern of polar and nonpolar amino acids favors the conversion of the peptides into amyloid-like fibrils via self-assembly. Distinct fibrous morphologies have been observed at different pH values and temperatures, which indicates that different fibril packing schemes can be designed; hence, fibrillar peptides can be used to generate efficient artificial catalysts for amidolytic activities at mild pH values. The results of atomic force microscopy, Raman spectroscopy, and wide-angle X-ray scattering analyses are used to discuss and compare the fibril structure of a fibrillar PC with its amidolytic activity. The pH of the fibrillation reaction crucially affects the pKa of the side chains of the catalytic triads and is important for stable fibril formation. Temperature is another important parameter that controls the self-assembly of peptides into highly stacked and laminated morphologies. The morphology and stability of fibrils are crucial and represent important factors for demonstrating the capability of the peptides to exert amidolytic activity. The observed amidolytic activity of PC4, one of the PCs, was validated using an inhibition assay, which revealed that PC4 can perform enzyme-like amidolytic catalysis. These results provide insights into the potential use of designed peptides in the generation of efficient artificial enzymes.

Enzyme-Mimic Peptide Assembly To Achieve Amidolytic Activity.

Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection.

Jo-Ann Chuah

Enzyme Research Team

RIKEN Center for Sustainable Resource Science

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Jo-Ann Chuah

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Enzyme Research Team

RIKEN Center for Sustainable Resource Science

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Enzyme Research Team