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Friday, 20 October 2017
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Biotechnology and Bioengineering
Wiley Online Library : Biotechnology and Bioengineering

  • Enhanced pyruvate production in Candida glabrata by carrier engineering
    Pyruvate is an important organic acid that plays a key role in the central metabolic pathway. Manipulating transporters is an efficient strategy to enhance production of target organic acids and a means to understand the effects of altered intracellular pyruvate content on global metabolic networks. Efforts have been made to manipulate mitochondrial pyruvate carrier (MPC) to transport pyruvate into different subcellular compartments in Candida glabrata to demonstrate the effects of the subcellular distribution of pyruvate on central carbon metabolism. By increasing the mitochondrial pyruvate content through enhancing the rate of pyruvate transport into mitochondria, a high central carbon metabolism rate, specific growth rate and specific pyruvate production rate were obtained. Comparing the intracellular pyruvate content of engineered and control strains showed that higher intracellular pyruvate levels were not conducive to improving pyruvate productivity or central carbon metabolism. Plasma membrane expression of MPCs significantly increased the expression levels of key rate?limiting glycolytic enzymes. Moreover, pyruvate production of CG?ura3?Sp?MPC1, CG?ura3?Sp?MPC2 and CG?ura3?Sp?MPC1?Sp?MPC2 increased 134.4%, 120.3% and 30.0%, respectively. In conclusion, lower intracellular pyruvate content enhanced central carbon metabolism and provided useful clues for improving the production of other organic acids in microorganisms. This article is protected by copyright. All rights reserved

  • Overproduction of MCL?PHA with high 3?Hydroxydecanoate Content
    Methods of producing medium?chain?length poly?3?hydroxyalkanoate (mcl?PHA) with high content of the dominant subunit, 3?hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a ??oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA?F1) or by inhibiting ??oxidation with addition of acrylic acid to wild type P. putida KT2440 in carbon?limited, fed?batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4?g/g, P. putida DBA?F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5?g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18?g/L biomass containing 59% mcl?PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone addition allowed the mutant strain to accumulate 74% mcl?PHA by weight with 97 mol% HD at a production rate of 0.41?g/L/h, at least twice that of published data for any ??oxidation knock?out mutant. Higher biomass concentration was achieved with acrylic acid inhibition of ??oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity and rate of crystallization. This article is protected by copyright. All rights reserved

  • Metabolic reduction of resazurin; location within the cell for cytotoxicity assays
    Resazurin is widely used as a metabolic indicator for living cells, however, there has been considerable debate in the literature with regards to the specific location in the cell where the non?fluorescent resazurin is reduced to the strongly fluorescent resorufin. This lack of clarity about the reduction site makes the use of resazurin reduction data in cytotoxicity studies difficult to interpret. In this study, E. faecalis, a Gram?positive and facultative anaerobic bacterial strain, and the most toxic chlorophenol, pentachlorophenol (PCP), were chosen as models for an anaerobe and toxicant, respectively. By studying the kinetics of resazurin reduction by E. faecalis after different treatments (cell disruption, bacterial filtration and pre?exposure to toxicant), we confirmed that resazurin reduction to resorufin by live Gram?positive and facultative anaerobic bacterial cells can only happen intracellularly under anaerobic conditions, while resorufin reduction to dihydroresorufin can happen both intracellularly and extracellularly. Based on the understanding of these fundamental mechanisms, we suggest that resazurin reduction can be used as a quick bioassay for measuring cytotoxicity. This article is protected by copyright. All rights reserved

  • Types of Cell Death and Apoptotic Stages in Chinese Hamster Ovary Cells Distinguished by Raman Spectroscopy
    Cell death is the ultimate cause of productivity loss in bioreactors that are used to produce therapeutic proteins. We investigated the ability of Raman spectroscopy to detect the onset and types of cell death for Chinese Hamster Ovary (CHO) cells ? the most widely used cell type for therapeutic protein production. Raman spectroscopy was used to compare apoptotic, necrotic, autophagic and control CHO cells. Several specific nucleic acid?, protein? and lipid?associated marker bands within the 650?850 cm?1 spectral region were identified that distinguished among cells undergoing different modes of cell death; supporting evidence was provided by principal component analysis of the full spectral data. In addition to comparing the different modes of cell death, normal cells were compared to cells sorted at several stages of apoptosis, in order to explore the potential for early detection of apoptosis. Different stages of apoptosis could be distinguished via Raman spectroscopy, with multiple changes observed in nucleic acid peaks at early stages whereas an increase in lipid signals was a feature of late apoptosis/secondary necrosis. This article is protected by copyright. All rights reserved

  • A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of ??carotene
    The increasing market demands of ??carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. Yarrowia lipolytica, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl?CoA. We first found that a lipid overproducer strain was capable of producing more ??carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter?gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5?g/L and a maximum yield of 0.048?g/g of glucose in flask. ??carotene production was further increased in controlled conditions using a fed?batch fermentation. A total production of ??carotene of 6.5?g/L and 90?mg/g DCW with a concomitant production of 42.6?g/L of lipids was achieved. Such high titers suggest that engineered Y. lipolytica is a competitive producer organism of ??carotene. This article is protected by copyright. All rights reserved

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