| 1. |
- Cao, Xuan, et al.
(författare)
-
Engineering yeast for high-level production of diterpenoid sclareol
- 2023
-
Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 75, s. 19-28
-
Tidskriftsartikel (refereegranskat)abstract
- The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.
|
|
| 2. |
- Zhou, Yongjin, 1984, et al.
(författare)
-
Engineering NAD + availability for Escherichia coli whole-cell biocatalysis: A case study for dihydroxyacetone production
- 2013
-
Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- 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 DH5α had the highest DHA specific titer of 0.81 g/g DCW 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/g DCW . 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/g DCW /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. © 2013 Zhou et al.; licensee BioMed Central Ltd.
|
|
