Browsing by Author "Liaw, Li-Ling"

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Biomass and lipid production of a novel microalga, Chlorellaceae sp P5, through heterotrophic and swine wastewater cultivation
(2018-05-01)
Chiang, Yin-Ling
;
Chen, Yu-Pei
;
Yu, Ming-Chen
;
Hsieh, Sung-Yuan
;
Hwang, Ing-Er
A potential novel genus of microalgae was isolated from swine wastewater. According to a phylogenetic analysis of 18S rRNA and internal transcribed spacer (ITS) sequences, as well as the ITS2 secondary structure, this strain was designated as Chlorellaceae sp. P5. Acclimatization to various temperatures, pH conditions, and salinities demonstrated that this strain can grow at temperatures of 20 degrees C-40 degrees C, in an acidity range of pH 4-7, and with salinity as high as 3% NaCl. To optimize biomass production, response surface methodology was used which included variables associated with carbon and nitrogen sources. In this study, flask cultivation yielded 1.66 g l(-1) d(-1) of biomass under 15.19 g l(-1) glucose and 8.26 mM ammonium bicarbonate. The fatty acid composition suggested that the lipid from Chlorellaceae sp. P5 can be typically used for biodiesel production, with a low degree of unsaturated fatty acids. Moreover, a high content of the first-limiting amino acid (lysine) and 9,12-Octadecadienoic acid (linoleic acid, C18:2) for nutritional supplements was also observed. With respect to swine wastewater treatment, the isolated strain showed an excellent removal rate of ammonia and phosphate over 97.3 mg l(-1) of NH3-N and 20.3 mg of l(-1) PO43- in the sterilized and none-sterilized wastewater. The biomass and lipid derived from Chlorellaceae sp. P5 can be attained at 0.49 g l(-1) d(-1) and 0.13 g l(-1) d(-1) by swine wastewater cultivation with 5% CO2 supply, respectively. According to the various characteristic analyses, Chlorellaceae sp. P5 has great potential for different industrial developments such as biodiesel and wastewater treatment. Published by AIP Publishing.
Characterization of MRT, a new non-LTR retrotransposon in Monascus spp
(2007-10-01)
Chen, Yi-Pei
;
Tseng, Ching-Ping
;
Liaw, Li-Ling
;
Wang, Chun-Lin
;
Yuan, Gwo-Fang
A new non-LTR retrotransposon, named MRT, was discovered in the filamentous fungus Monascus pilosus BCRC38072. The entire nucleotide sequence of the MRT element was 5.5-kb long, including two open reading frames. These two ORFs showed homologies to gag-like and pol-like gene products, and an A-rich sequence at the 3' end of pol-like gene. ORF1 encoded a protein of 5 17 amino acids and contained a cysteine-rich zinc finger motif ORF2 encoded a protein of 1181 amino acids and contained apurinic/apyrimidinic endonuclease (APE), reverse transcriptase (RT), RNaseH domains, and a CCHC motif. The phylogenetic analyses demonstrated that the MRT element should be classified into the Tad1 clade. The results of Southern hybridizations showed that MRT elements were distributed within M pilosus, M ruber, M sanguineus, and M barkeri. In addition, the species of Monascus can be grouped by the presence or absence of MRT elements in the hybridization pattern according to phylogenctic subgroups established with the partial beta-tubulin gene.
Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus
(2008-07-23)
Chen, Yi-Pei
;
Tseng, Ching-Ping
;
Liaw, Li-Ling
;
Wang, Chun-Lin
;
Chen, I-Ching
Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus, and it has the same structure as lovastatin, which is mainly produced by Aspergillus terreus. In the present study, a bacterial artificial chromosome (BAC) clone, mps01, was screened from the BAC library constructed from Monascus pilosus BCRC38072 genomic DNA. The putative monacolin K biosynthetic gene cluster was found within a 42 kb region in the mps01 clone. The deduced amino acid sequences encoded by the nine genes designated as mokA-mokl, which share over 54% similarity with the lovastatin biosynthetic gene cluster in A. terreus, were assumed to be involved in monacolin K biosynthesis. A gene disruption construct designed to replace the central part of mokA, a polyketide synthase gene, in wild-type M. pilosus BCRC38072 with a hygromycin B resistance gene through homologous recombination, resulted in a mokA-disrupted strain. The disruptant did not produce monacolin K, indicating that mokA encoded the PKS responsible for monacolin K biosynthesis in M. pilosus BCRC38072.
Exploring the Distribution of Citrinin Biosynthesis Related Genes among Monascus Species
(2008-12-24)
Chen, Yi-Pei
;
Tseng, Ching-Ping
;
Chien, I-Ling
;
Wang, Wei-Yi
;
Liaw, Li-Ling
Citrinin, a hepato-nephrotoxic compound to humans, can be produced by the food fermentation microorganisms Monascus spp. In this study, we investigated the distribution of mycotoxin citrinin biosynthesis genes in 18 Monascus strains. The results show that the acyl-transferase and keto-synthase domains of the pksCT gene encoding citrinin polyketide synthase were found in Monascus purpureus, Monascus kaoliang, and Monascus sanguineus. Furthermore, the ctnA gene, a major activator for citrinin biosynthesis, was found in M. purpureus and U kaoliang, but was absent in M. sanguineus. The orf3 gene encoding oxygenase, located between pksCT and ctnA, was also present in M. purpureus and M. kaoliang. The pksCT gene was highly conserved in M. purpureus, U kaoliang, and M. sanguineus, while the ctnA and orf3 genes were shown to be highly homologous in M. purpureus and M. kaoliang. In contrast, the PCR and Southern blot analyses suggest that pksCT, ctnA, and orf3 were absent or significantly different in Monascus pilosus, Monascus ruber, Monascus barkeri, Monascus floridanus, Monascus lunisporas, and Monascus pallens. A citrinin-producing phenotype was detected only in M. purpureus and M., kaoliang using high performance liquid chromatography (HPLC). These results clearly indicate that the highly conserved citrinin gene cluster in M. purpureus and U kaoliang carry out citrinin biosynthesis. In addition, according to the phylogenetic subgroups established with the beta-tubulin gene, the citrinin gene cluster can group the species of Monascus.
Identification of the mokH Gene Encoding Transcription Factor for the Upregulation of Monacolin K Biosynthesis in Monascus pilosus
(2010-01-13)
Chen, Yi-Pei
;
Yuan, Gwo-Fang
;
Hsieh, Sung-Yuan
;
Lin, Yu-Shan
;
Wang, Wei-Yi
Monacolin K is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus. The monacolin K biosynthetic gene cluster, mokA-mokI, has been characterized in Monascus pilosus. The mokH gene encoding Zn(II)2Cys6 binuclear DNA binding protein is assumed to be an activator for monacolin K production. In this study, the mokH gene was cloned and driven by the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter for overexpression in M. pilosus. The transformants containing an extra copy of the mokH gene were obtained and verified by PCR and Southern hybridization. The transcripts of mokH in the transformants were expressed significantly higher than those of the wild-type strain. The transformants were stably inherited through the next generation, as determined by observation of the enhanced green fluorescent protein (EGFP). The transformant T-mokH1 also showed a 1.7-fold higher production of monacolin K than the wild-type strain in a time course analysis. Analysis of the RT-PCR products demonstrated that the monacolin K biosynthetic genes in the transformant were expressed to a greater extent than those in the wildtype strain. These results indicated that mokH upregulated the transcription of monacolin K biosynthetic genes and increased monacolin K production.
Selection of an Effective Red-Pigment Producing Monascus pilosus by Efficient Transformation with Aurintricarboxylic Acid
(2008-11-01)
Chen, Yi-Pei
;
Chen, I-Ching
;
Hwang, Ing-Er
;
Yuan, Gwo-Fang
;
Liaw, Li-Ling
The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.