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An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

ABSTRACT: Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified Escherichia coli strains that had previously undergone fed-batch testing. Firstly, we compared the induction temperatures in small-scale whole-cell biocatalysis studies and then examined the optimal temperature in a large volume bioreactor. By lowering the induction temperature, we were able to double the yield of HT produced thereby, reaching 82% when utilizing tyrosine or L-DOPA as substrates. Hence, without the need to further modify our original strains, we were able to increase the HT yield.

SUBMITTER: Mermigka G

PROVIDER: S-EPMC10672836 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

Mermigka Glykeria G Vavouraki Aikaterini I AI Nikolaou Chrysoula C Cheiladaki Ioanna I Vourexakis Michail M Goumas Dimitrios D Ververidis Filippos F Trantas Emmanouil E

Metabolites 20231103 11

Hydroxytyrosol (HT) is a phenolic substance primarily present in olive leaves and olive oil. Numerous studies have shown its advantages for human health, making HT a potentially active natural component with significant added value. Determining strategies for its low-cost manufacturing by metabolic engineering in microbial factories is hence still of interest. The objective of our study was to assess and improve HT production in a one-liter bioreactor utilizing genetically modified <i>Escherichi ...[more]

PMID: 37999222

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Project description:Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7 mN/mm2) and conduction velocity (52.5 cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2 mN/mm2 and conduction velocity of 25.8 cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function in vitro.

Project description:BACKGROUND: Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. RESULTS: In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5? had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process. CONCLUSIONS: High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD+ transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis.

Dataset Information

An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

Publications

An Engineered Plant Metabolic Pathway Results in High Yields of Hydroxytyrosol Due to a Modified Whole-Cell Biocatalysis in Bioreactor.

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