Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.
A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.
We have developed an integrative transformation system for metabolic engineering of the tetraacetyl phytosphingosine (TAPS)-secreting yeast Pichia ciferrii. The system uses (i) a mutagenized ribosomal protein L41 gene of P. ciferrii as a dominant selection marker that confer resistance to the antibiotic cycloheximide and (ii) a ribosomal DNA (rDNA) fragment of P. ciferrii as a target for multicopy gene integration into the chromosome. A locus within the nontranscribed region located between 5S and 26S rDNAs was selected as the integration site. A maximum frequency of integrative transformation of approximately 1,350 transformants/g of DNA was observed. To improve the de novo synthesis of sphingolipid, the LCB2 gene, encoding a subunit of serine palmitoyltransferase, which catalyzes the first committed step of sphingolipid synthesis, was cloned from P. ciferrii and overexpressed under the control of the P. ciferrii glyceraldehyde-3-phosphate dehydrogenase promoter. After transformation of an LCB2 gene expression cassette, several transformants that contained approximately five to seven copies of transforming DNA in the chromosome and exhibited about 50-fold increase in LCB2 mRNA relative to the wild type were identified. These transformants were observed to produce approximately two times more TAPS than the wild type.Sphingolipids are integral membrane components found in mammalian cells. They are also distributed widely in plants and microbes (14). Sphingolipids constitute a diverse group of complex membrane lipids, all containing a long-chain amino acid alcohol, usually of 18 carbons, connected by an amide linkage to a fatty acid to form ceramide. Sphingolipids have structural functions in maintaining cell membrane integrity, and they act as anchors for proteins. Metabolites of sphingolipids, such as ceramide, sphingosine, and sphingosine 1-phosphate, are involved as bioeffector molecules and second messengers in cell growth, differentiation, cell senescence, apoptosis, and stress responses (for reviews, see references 9, 29, and 20). Ceramide is a major end product of differentiation through the keratinization process in the mammalian epidermis and is secreted into the extracellular space to form a mantle surrounding individual keratinized cells (19). Extracellular ceramide is a major part of the permeability barrier and the water reservoir of the skin (11). Therefore, ceramide has attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. Extraction of ceramides from natural sources, however, is difficult, and in some cases the resulting product is not acceptable for cosmetic use. Chemically synthesized ceramides are also available as a mixture of stereoisomers that must be separated prior to use. Biological production of sphingoid base, such as sphinganine and phytosphingosine, can provide the base materials for conversion to various ceramides. This sphingoid base can be obtained from several yeasts and easily converted to ceramides by N a...
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