| 1. |
- Buijs, Nicolaas, 1985, et al.
(författare)
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Long-chain Alkane Production by the Yeast Saccharomyces cerevisiae
- 2015
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 112:6, s. 1275-1279
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Tidskriftsartikel (refereegranskat)abstract
- In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfdl and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFDI together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol.
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| 2. |
- Cao, Xuan, et al.
(författare)
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Engineering yeast for high-level production of diterpenoid sclareol
- 2023
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Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 75, s. 19-28
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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.
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| 3. |
- Chen, Yun, 1978, et al.
(författare)
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Enabling Technologies to Advance Microbial Isoprenoid Production
- 2015
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Ingår i: Advances in Biochemical Engineering/Biotechnology. - Cham : Springer International Publishing. - 0724-6145 .- 1616-8542. ; 148, s. 143-160
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Tidskriftsartikel (refereegranskat)abstract
- Microbial production of isoprenoids provides an attractive alternative to biomass extraction and chemical synthesis. Although widespread research aims for isoprenoid biosynthesis, it is still in its infancy in terms of delivering commercial products. Large barriers remain in realizing a cost-competitive process, for example, developing an optimal microbial cell factory. Here, we summarize the many tools and methods that have been developed in the metabolic engineering of isoprenoid production, with the advent of systems biology and synthetic biology, and discuss how these technologies advance to accelerate the design–build–test engineering cycle to obtain optimum microbial systems. It is anticipated that innovative combinations of new and existing technologies will continue to emerge, which will enable further development of microbial cell factories for commercial isoprenoid production.
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| 4. |
- Gong, Z. W., et al.
(författare)
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Engineering Robustness of Microbial Cell Factories
- 2017
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Ingår i: Biotechnology journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 12:10
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Forskningsöversikt (refereegranskat)abstract
- Metabolic engineering and synthetic biology offer great prospects in developing microbial cell factories capable of converting renewable feedstocks into fuels, chemicals, food ingredients, and pharmaceuticals. However, prohibitively low production rate and mass concentration remain the major hurdles in industrial processes even though the biosynthetic pathways are comprehensively optimized. These limitations are caused by a variety of factors unamenable for host cell survival, such as harsh industrial conditions, fermentation inhibitors from biomass hydrolysates, and toxic compounds including metabolic intermediates and valuable target products. Therefore, engineered microbes with robust phenotypes is essential for achieving higher yield and productivity. In this review, the recent advances in engineering robustness and tolerance of cell factories is described to cope with these issues and briefly introduce novel strategies with great potential to enhance the robustness of cell factories, including metabolic pathway balancing, transporter engineering, and adaptive laboratory evolution. This review also highlights the integration of advanced systems and synthetic biology principles toward engineering the harmony of overall cell function, more than the specific pathways or enzymes.
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| 5. |
- Hu, Yating, 1991, et al.
(författare)
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Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene
- 2017
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Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 44:7, s. 1065-1072
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Tidskriftsartikel (refereegranskat)abstract
- Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.
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| 6. |
- Kang, Min-Kyoung, 1981, et al.
(författare)
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Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae
- 2017
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Ingår i: Microbial Cell Factories. - : BioMed Central. - 1475-2859. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Low catalytic activities of pathway enzymes are often a limitation when using microbial based chemical production. Recent studies indicated that the enzyme activity of aldehyde decarbonylase (AD) is a critical bottleneck for alkane biosynthesis in Saccharomyces cerevisiae. We therefore performed functional screening to identify efficient ADs that can improve alkane production by S. cerevisiae. Results: A comparative study of ADs originated from a plant, insects, and cyanobacteria were conducted in S. cerevisiae. As a result, expression of aldehyde deformylating oxygenases (ADOs), which are cyanobacterial ADs, from Synechococcus elongatus and Crocosphaera watsonii converted fatty aldehydes to corresponding Cn-1 alkanes and alkenes. The CwADO showed the highest alkane titer (0.13 mg/L/OD600) and the lowest fatty alcohol production (0.55 mg/L/OD600). However, no measurable alkanes and alkenes were detected in other AD expressed yeast strains. Dynamic expression of SeADO and CwADO under GAL promoters increased alkane production to 0.20 mg/L/OD600 and no fatty alcohols, with even number chain lengths from C8 to C14, were detected in the cells. Conclusions: We demonstrated in vivo enzyme activities of ADs by displaying profiles of alkanes and fatty alcohols in S. cerevisiae. Among the AD enzymes evaluated, cyanobacteria ADOs were found to be suitable for alkane biosynthesis in S. cerevisiae. This work will be helpful to decide an AD candidate for alkane biosynthesis in S. cerevisiae and it will provide useful information for further investigation of AD enzymes with improved activities.
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| 7. |
- Qin, J., et al.
(författare)
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Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine
- 2015
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 6:Sept., s. Art. no. 8224-
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Tidskriftsartikel (refereegranskat)abstract
- Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l-1 with a yield of 67 mg (g glucose)-1 in shake-flask cultures and a titre of 5.1 g l-1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.
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| 8. |
- Teixeira, Paulo, 1990, et al.
(författare)
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Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae
- 2017
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 16:1, s. 45-
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Tidskriftsartikel (refereegranskat)abstract
- Background: In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields. Results: We analyzed the metabolite dynamics of a faa1 Delta faa4 Delta strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain. Conclusions: Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.
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| 9. |
- Yu, Tao, 1986, et al.
(författare)
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Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723 .- 2041-1723. ; 8, s. Article number: 15587-
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Tidskriftsartikel (refereegranskat)abstract
- Production of chemicals and biofuels through microbial fermentation is an economical and sustainable alternative for traditional chemical synthesis. Here we present the construction of a Saccharomyces cerevisiae platform strain for high-level production of very-long-chain fatty acid (VLCFA)-derived chemicals. Through rewiring the native fatty acid elongation system and implementing a heterologous Mycobacteria FAS I system, we establish an increased biosynthesis of VLCFAs in S. cerevisiae . VLCFAs can be selectively modified towards the fatty alcohol docosanol (C22H46O) by expressing a specific fatty acid reductase. Expression of this enzyme is shown to impair cell growth due to consumption of VLCFA-CoAs. We therefore implement a dynamic control strategy for separating cell growth from docosanol production. We successfully establish high-level and selective docosanol production of 83.5 mg l(-1) in yeast. This approach will provide a universal strategy towards the production of similar high value chemicals in a more scalable, stable and sustainable manner.
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| 10. |
- Yu, Tao, 1986, et al.
(författare)
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Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis
- 2018
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 174:6, s. 1549-1572
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Tidskriftsartikel (refereegranskat)abstract
- Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.
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