Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Calado, Cecı́lia R.C.; Mannesse, Maurice; Egmond, Maarten R.; Cabral, J. M. S.; Fonseca, Luís

doi:10.1002/bit.10252.abs

Cited by 5 publications

(5 citation statements)

References 10 publications

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“…Cutinases are natural extracellular enzymes in their native organisms. Previously, secretory expression of cutinases in different hosts was mediated by a secretion signal peptide (9)(10)(11)(12)(13)(14). In the present study, cutinase activity in the culture medium from a 3-liter fermentor of T. fusca cutinase, heterologously expressed in E. coli without a signal peptide, reached 1,063.5 U/ml (2,380.8 mg/ liter) after 24 h. This represents a 4.4-fold increase over the highest previously reported yield (546 mg/liter) (12).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11,12), and Pichia pastoris (13). The highest yield of extracellular protein, 546 mg/liter, was obtained in a 5-liter bioreactor utilizing an engineered S. cerevisiae cellular system (12).…”

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confidence: 99%

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Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Woodard

Chen

et al. 2013

Appl Environ Microbiol

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Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase NS ) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase NS showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase NS S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase NS S130A, the cells expressing cutinase NS showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase NS " was proposed to explain the underlying mechanism for the extracellular release of cutinase NS . Cutinase not only catalyzes the cleavage of the ester bonds of cutin, but is also capable of hydrolyzing soluble esters, insoluble triglycerides, and a variety of polyesters (1). In addition to its hydrolytic capability, cutinase is also used in ester synthesis (2, 3) and transesterification (4). Therefore, as a multifunctional enzyme, cutinase has potential uses in the food, chemical, textile, and other industries (5).Enzymes with cutinase activity have been found in both fungi and bacteria, such as Fusarium solani pisi and Thermobifida fusca, respectively (5-7). It has been demonstrated that cutinases from both groups belong to the ␣/␤-hydrolase superfamily, share similar spatial structures, and display similar catalytic properties, as well as substrate specificities. In addition, consistent with their ability to accommodate large substrates like cutin, they contain an open active site, and the lid structure normally seen in most lipases is absent in cutinase. Despite these similarities, there does not appear to be significant sequence homology between fungal and bacterial cutinases; thus, it has been suggested that they be classified into prokaryotic and eukaryotic cutinase subfamilies, respectively (6).To date, all the studied cutinases from microorganisms have been found to be secretory enzymes (6). Therefore, for heterologous expression, signal peptides are usually utilized to mediate the secretion of recombinant cutinase. Using this approach, F. solani cutinase has been expressed in a variety of host cells, such as Escherichia coli (8), Aspergillus awamori (9), Fusarium venenatum (10), Saccharomyces cerevisiae (11, 12), and Pichia pastoris (13). The highest yield of extracellu...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Woodard

Chen

et al. 2013

Appl Environ Microbiol

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“…A constant specific growth rate of 0.14 h À1 was considered during the exponential feed to maintain very low glucose concentration in the culture medium. 9 With the first fed-batch fermentation (FB1), higher cutinase activity, specific cell activity, specific cutinase activity and productivity were obtained in relation to the previously performed BB batch fermentation (Table 2). In spite of a lower biomass yield, higher cutinase activity yields were obtained, except for the cutinase yield on glucose of the SF1 fermentation.…”

Section: Effect Of Cultivation Strategymentioning

confidence: 99%

Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase

Calado

Fonseca

2002

J of Chemical Tech & Biotech

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The effect of culture media composition, and fermentation conditions and strategy on the growth and cutinase production of recombinant Saccharomyces cerevisiae and subsequent cutinase purification by expanded bed adsorption (EBA) was studied. The reduction in the amount of yeast extract used as nitrogen source from 20 g dm À3 to 10 g dm À3 in batch cultures led to a 29% decrease in the heterologous cutinase production, while the 5% cutinase dynamic adsorption capacity (q5%) on the cation Streamline SP XL was increased 6.7-fold. By dilution of the whole fermentation broth, performed with the lowest yeast extract content, which reduces conductivity, the q5% was additionally increased by 1.9-fold. After implementation of a fed-batch strategy the cutinase concentration, cutinase yield on carbon source, cutinase yield on nitrogen source and productivity were increased by 10.8-, 2.9-, 5.3-and 6.4-fold, respectively, in relation to the previously-mentioned batch fermentation. However, the increased cutinase production was compromised by heterologous protein loss during the EBA recovery operation and the cutinase dynamic adsorption capacity and purification productivity decreased by 90% and 75%, respectively. Thus, target protein production by S cerevisiae fermentation and a downstream process with EBA cannot be considered as separate entities, where the understanding of the factors that affect the interactions among them are crucial towards optimization of the overall production process of heterologous proteins.

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“…An exponential feeding phase was started on cultures B and C with a feeding of 0.3 L of medium containing 22.5 g of yeast extract, 22.5 g of Bactotryptone, and 45 g of glucose, considering a maximum specific growth rate of 0.18 h −1 and a constant yield of biomass per glucose of 0.6 g/g as described previously. 46 Samples were taken from the bioreactor along the culture and subsequently used for off-line reference analysis of biomass, glucose, glycerol, acetate, and plasmid.…”

Section: Methodsmentioning

confidence: 99%

In Situ Near-Infrared (NIR) versus High-Throughput Mid-Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

et al. 2015

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The development of biopharmaceutical manufacturing processes presents critical constraints, with the major constraint being that living cells synthesize these molecules, presenting inherent behavior variability due to their high sensitivity to small fluctuations in the cultivation environment. To speed up the development process and to control this critical manufacturing step, it is relevant to develop high-throughput and in situ monitoring techniques, respectively. Here, high-throughput mid-infrared (MIR) spectral analysis of dehydrated cell pellets and in situ near-infrared (NIR) spectral analysis of the whole culture broth were compared to monitor plasmid production in recombinant Escherichia coli cultures. Good partial least squares (PLS) regression models were built, either based on MIR or NIR spectral data, yielding high coefficients of determination (R(2)) and low predictive errors (root mean square error, or RMSE) to estimate host cell growth, plasmid production, carbon source consumption (glucose and glycerol), and by-product acetate production and consumption. The predictive errors for biomass, plasmid, glucose, glycerol, and acetate based on MIR data were 0.7 g/L, 9 mg/L, 0.3 g/L, 0.4 g/L, and 0.4 g/L, respectively, whereas for NIR data the predictive errors obtained were 0.4 g/L, 8 mg/L, 0.3 g/L, 0.2 g/L, and 0.4 g/L, respectively. The models obtained are robust as they are valid for cultivations conducted with different media compositions and with different cultivation strategies (batch and fed-batch). Besides being conducted in situ with a sterilized fiber optic probe, NIR spectroscopy allows building PLS models for estimating plasmid, glucose, and acetate that are as accurate as those obtained from the high-throughput MIR setup, and better models for estimating biomass and glycerol, yielding a decrease in 57 and 50% of the RMSE, respectively, compared to the MIR setup. However, MIR spectroscopy could be a valid alternative in the case of optimization protocols, due to possible space constraints or high costs associated with the use of multi-fiber optic probes for multi-bioreactors. In this case, MIR could be conducted in a high-throughput manner, analyzing hundreds of culture samples in a rapid and automatic mode.

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Production of wild‐type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

Cited by 5 publications

References 10 publications

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Extracellular Location of Thermobifida fusca Cutinase Expressed in Escherichia coli BL21(DE3) without Mediation of a Signal Peptide

Effect of Saccharomyces cerevisiae fermentation conditions on expanded bed adsorption of heterologous cutinase

In Situ Near-Infrared (NIR) versus High-Throughput Mid-Infrared (MIR) Spectroscopy to Monitor Biopharmaceutical Production

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