Jo-Ann Chuah

Synthesis of polyhydroxyalkanoate from palm oil and some new applications

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.

Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

ABSTRACT 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 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

ABSTRACT Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) 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 PhaCCs 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 (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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

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.

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

Chapter 1:Design Considerations for Properties of Nanocarriers on Disposition and Efficiency of Drug and Gene Delivery

The delivery of drugs or genetic material into cells is one of the emerging areas of biotechnology. Nanoparticle (NP)-based drug carriers are especially interesting due to their ability to deliver drugs inside target cells, thereby reducing the side-effects of non-specific treatments. Among the various cargoes that are transportable by NPs, genetic material allows the reprogramming of cells both temporarily as well as permanently. Several approaches are available for gene delivery, such as the use of natural vectors, like modified viruses, or artificial ones, in the form of liposomes or peptidic complexes. Among the key consideration for designing NPs to effectively overcome biological barriers are their physicochemical properties and effects they produce in living organism. This chapter discusses how the properties of nanocarriers can affect the biological response as well as the functionality of drug and gene delivery systems.

Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants

For efficient gene delivery in plant systems, nonviral vector and DNA complexes require extracellular stability, cell wall/membrane translocation capability, and the ability to mediate both endosomal escape and intracellular DNA release. Peptides make appealing gene delivery vectors due to their DNA-binding, cell-penetrating, and endosome escape properties. However, DNA release within cells has so far been inefficient, which results in poor and delayed gene expression, while the lack of understanding of both internalization and trafficking mechanisms is a further obstacle to the design of efficient peptide gene delivery vectors. Here, we report successful gene delivery into plants using a cellular environment-responsive vector, BPCH7, which is an efficient cell-penetrating peptide with a cyclic DNA-binding domain that is formed by a disulfide bond between two cysteines. The cyclic structure of BPCH7 confers high avidity attachment to DNA in vitro. Following endocytosis into cells, disulfide bond cleavage facilitated by intracellular glutathione induces structural changes within BPCH7 that enable the release of the associated DNA cargo. Comparative studies with BPKH, a cell-penetrating peptide with a linear DNA-binding domain, show that BPCH7 maximized and expedited gene transfer in cells and unveil for the first time the crucial role of plant stomata in the internalization of peptide-DNA complexes.

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 well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide–DNA Complexes

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

Interplay between Silk Fibroin’s Structure and Its Adhesive Properties

Bombyx mori-derived silk fibroin (SF) is a well-characterized protein employed in numerous biomedical applications. Structurally, SF consists of a heavy chain (HC) and a light chain (LC), connected via a single disulfide bond. The HC sequence is organized into 12 crystalline domains interspersed with amorphous regions that can transition between random coil/alpha helix and beta-sheet configurations, giving silk its hallmark properties. SF has been reported to have adhesive properties and shows promise for development of medical adhesives; however, the mechanism of these interactions and the interplay between SFs structure and adhesion is not understood. In this context, the effects of physical parameters (i.e., concentration, temperature, pH, ionic strength) and protein structural changes on adhesion were investigated in this study. Our results suggest that amino acid side chains that have functionalities capable of coordinate (dative) bond or hydrogen bond formation (such as those of serine and tyrosine), might be important determinants in SFs adhesion to a given substrate. Additionally, the data suggest that fibroin amino acids involved in beta-sheet formation are also important in the proteins adhesion to substrates.