| 1. |
- Zhou, Yongjin, 1984, et al.
(författare)
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Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 7, s. 11709-11709
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Tidskriftsartikel (refereegranskat)abstract
- Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g/L of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg /L) and fatty alcohols (1.5 g/L), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.
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| 2. |
- Zhiwei, Zhu, 1985, et al.
(författare)
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Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast
- 2017
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Ingår i: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 44, s. 81-88
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Tidskriftsartikel (refereegranskat)abstract
- Microbial synthesis of medium chain aliphatic hydrocarbons, attractive drop-in molecules to gasoline and jet fuels, is a promising way to reduce our reliance on petroleum-based fuels. In this study, we enabled the synthesis of straight chain hydrocarbons (C7–C13) by yeast Saccharomyces cerevisiae through engineering fatty acid synthases to control the chain length of fatty acids and introducing heterologous pathways for alkane or 1-alkene synthesis. We carried out enzyme engineering/screening of the fatty aldehyde deformylating oxygenase (ADO), and compartmentalization of the alkane biosynthesis pathway into peroxisomes to improve alkane production. The two-step synthesis of alkanes was found to be inefficient due to the formation of alcohols derived from aldehyde intermediates. Alternatively, the drain of aldehyde intermediates could be circumvented by introducing a one-step decarboxylation of fatty acids to 1-alkenes, which could be synthesized at a level of 3 mg/L, 25-fold higher than that of alkanes produced via aldehydes.
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| 3. |
- Zhou, Yongjin, 1984, et al.
(författare)
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Harnessing yeast peroxisomes for biosynthesis of fatty-acid-derived biofuels and chemicals with relieved side-pathway competition
- 2016
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 138:47, s. 15368-15377
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Tidskriftsartikel (refereegranskat)abstract
- Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and/or insufficient precursor supply. Compartmentalization in cellular organelles can isolate synthetic pathways from competing pathways, and provide a compact and suitable environment for biosynthesis. Peroxisomes are cellular organelles where fatty acids are degraded, a process that is inhibited under typical fermentation conditions making them an interesting workhouse for production of fatty-acid-derived molecules. Here, we show that targeting synthetic pathways to peroxisomes can increase the production of fatty-acid-derived fatty alcohols, alkanes and olefins up to 700%. In addition, we demonstrate that biosynthesis of these chemicals in the peroxisomes results in significantly decreased accumulation of byproducts formed by competing enzymes. We further demonstrate that production can be enhanced up to 3-fold by increasing the peroxisome population. The strategies described here could be used for production of other chemicals, especially acyl-CoA-derived molecules.
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| 4. |
- Hu, Yating, 1991, et al.
(författare)
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Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:37, s. 22974-22983
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Tidskriftsartikel (refereegranskat)abstract
- Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme’s substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.
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| 5. |
- Hu, Yating, 1991, et al.
(författare)
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Engineering Saccharomyces cerevisiae cells for production of fatty acid-derived biofuels and chemicals
- 2019
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Ingår i: Open Biology. - : The Royal Society. - 2046-2441. ; 9:5
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, in particular ethanol, a biofuel produced in large quantities. With a need for high-energy-density fuels for jets and heavy trucks, there is, however, much interest in the biobased production of hydrocarbons that can be derived from fatty acids. Fatty acids also serve as precursors to a number of oleochemicals and hence provide interesting platform chemicals. Here, we review the recent strategies applied to metabolic engineering of S. cerevisiae for the production of fatty acid-derived biofuels and for improvement of the titre, rate and yield (TRY). This includes, for instance, redirection of the flux towards fatty acids through engineering of the central carbon metabolism, balancing the redox power and varying the chain length of fatty acids by enzyme engineering. We also discuss the challenges that currently hinder further TRY improvements and the potential solutions in order to meet the requirements for commercial application.
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| 6. |
- Hu, Yating, 1991, et al.
(författare)
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Heterologous transporter expression for improved fatty alcohol secretion in yeast
- 2018
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Ingår i: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 45, s. 51-58
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.
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| 7. |
- Zhiwei, Zhu, 1985, et al.
(författare)
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Expanding the product portfolio of fungal type I fatty acid synthases
- 2017
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 13:4, s. 360-362
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Tidskriftsartikel (refereegranskat)abstract
- Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/ medium-chain fatty acids and methyl ketones.
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| 8. |
- Zhiwei, Zhu, 1985, et al.
(författare)
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Multidimensional engineering of Saccharomyces cerevisiae for efficient synthesis of medium-chain fatty acids
- 2020
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Ingår i: Nature Catalysis. - : Springer Science and Business Media LLC. - 2520-1158. ; 3:1, s. 64-74
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Tidskriftsartikel (refereegranskat)abstract
- Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain.
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| 9. |
- Zhou, Yongjin, et al.
(författare)
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Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast.
- 2018
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 2:7, s. 584-590
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Tidskriftsartikel (refereegranskat)abstract
- Microbial production of fatty acid-derived hydrocarbons offers a great opportunity to sustainably supply biofuels and oleochemicals. One challenge is to achieve a high production rate. Besides, low efficiency in secretion will cause high separation costs and it is therefore desirable to have product secretion. Here, we engineered the budding yeast Saccharomyces cerevisiae, to produce and secrete 1-alkenes by manipulation of the fatty acid metabolism, enzyme selection, engineering the electron transfer system and expressing a transporter. Furthermore, we implemented a dynamic regulation strategy to control the expression of membrane enzyme and transporter, which improved 1-alkene production and cell growth by relieving the possible toxicity of overexpressed membrane proteins. With these efforts, the engineered yeast cell factory produced 35.3 mg/L 1-alkenes with more than 80% being secreted. This represents a 10-fold improvement compared with earlier reported hydrocarbon production by S. cerevisiae.
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| 10. |
- Zhou, Yongjin, 1984, et al.
(författare)
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Engineering NAD + availability for Escherichia coli whole-cell biocatalysis: A case study for dihydroxyacetone production
- 2013
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 12:1
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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.
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