1-Aminocyclopropane-1-Carboxylate Oxidase 2 Reduction Effects on Physical and Physiological Responses of Eksotika Papaya

Sekeli, Rogayah; Abdullah, Janna Ong; Muda, Pauziah; Namasivayam, Parameswari; Bakar, Umi Kalsom Abu; Shin, Sew Yun

doi:10.1080/15427528.2013.795205

Cited by 5 publications

(3 citation statements)

References 21 publications

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“…Genomic DNA of the selected transformants was isolated from L. starkeyi cell pellets using the procedure described previously [ 16 ]. Transgene copy number was estimated using a real-time qPCR method described by Weng et al [ 32 ], which had been used in numerous studies [ 33 – 35 ] and is described in our recent publication [ 16 ]. Briefly, the target gene (X) copy number was calculated versus the reference gene (R) by X o /R o .…”

Section: Methodsmentioning

confidence: 99%

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Alahuhta

Wei

et al. 2018

Biotechnol Biofuels

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The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1301-y) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Alahuhta

Wei

et al. 2018

Biotechnol Biofuels

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“…Real-time quantitative PCR (qPCR) has been widely used to determine gene copy number in engineered organisms [ 65 , 66 ]. In this study, the copy number of expressed chimeric cbh1 and eg2 genes in the transformants were estimated according to a method described by Weng et al [ 59 ], which has been used in numerous studies [ 66 – 68 ]. For comparison, the endogenous eukaryotic initiation factor 5 (eif5) gene was used as a single-copy reference gene [ 69 ].…”

Section: Methodsmentioning

confidence: 99%

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

Knoshaug

Wang

et al. 2017

Microb Cell Fact

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Background Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates.ResultsTo evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method.ConclusionsOur results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0742-5) contains supplementary material, which is available to authorized users.

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“…In 1990, MARDI started the research on delayed ripening papaya with the successful development of a tissue culture regeneration and transformation system, and identification of 1-aminocyclopropane-1-carboxylic acid oxidase 2 ( ACO 2) gene associated with ripening. Following which, transgenic and silencing technologies were used to develop new Eksotika papaya variety with delayed fruit ripening trait by manipulating the ACO 2 gene in the pathway ( Sekeli et al, 2013 ). The research findings showed ethylene production was greatly reduced by manipulating ACO 2 gene using antisense technique.…”

Section: Long-term Strategymentioning

confidence: 99%

Malaysian Carica papaya L. var. Eksotika: Current Research Strategies Fronting Challenges

et al. 2018

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Carica papaya L. or commonly known as papaya, is a major tropical crop consumed worldwide either as a vegetable or fresh fruit or processed products. In Malaysia, papaya was initially planted as a smallholder crop throughout the country. Eventually after 15 years of breeding and selection, a new variety, named C. papaya L. var. Eksotika, was released by the Malaysian Agricultural Research and Development Institute (MARDI) in 1987. This event changed the outlook of papaya planting from a smallholder crop to a plantation crop. Despite the blooming papaya business, the industry faced various disease issues that jeopardize its future. The most devastating was the papaya dieback disease, which affected approximately 800 hectares of plantation, destroyed approximately 1 million trees nationwide with total losses estimated at US$ 58 million. Even though Eksotika is a favored commercial variety with good eating and aesthetic quality fruit, its potential for more lucrative distant markets is tarnished with its short-shelf life fruits. Several strategies had been reported to address the challenges faced by Eksotika specifically against the dieback disease and the fruit’s short shelf-life. This review focuses on C. papaya L. var. Eksotika particularly on the strategies to address the challenges faced in order to sustain the economic value of this crop plant, which had contributed significantly to the Malaysian economy.

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1-Aminocyclopropane-1-Carboxylate Oxidase 2 Reduction Effects on Physical and Physiological Responses of Eksotika Papaya

Cited by 5 publications

References 21 publications

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

Malaysian Carica papaya L. var. Eksotika: Current Research Strategies Fronting Challenges

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