| 1. |
- Gong, Guiping, et al.
(författare)
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GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae
- 2021
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:6, s. 1328-1337
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Tidskriftsartikel (refereegranskat)abstract
- Targeted genome disruptions and single-nucleotide conversions with the CRISPR/Cas system have greatly facilitated the development of gene therapy, basic biological research, and synthetic biology. With vast progress in this field, there are still aspects to be optimized, including the target range, the ability to multiplex, the mutation efficiency and specificity, as well as the requirement of adjusting protospacer adjacent motifs (PAMs). Here, we report the development of a highly efficient genome disruption and single-nucleotide conversion tool with a gRNA-tRNA array and SpCas9-NG (GTR 2.0). We performed gene disruptions in yeast cells covering all 16 possible NGN PAMs and all 12 possible single-nucleotide conversions (N to N) with near 100% efficiencies. Moreover, we applied GTR 2.0 for multiplexed single-nucleotide conversions, resulting in 66.67% mutation efficiency in simultaneous generation of 4 single-nucleotide conversions in one gene, as well as 100% mutation efficiency for simultaneously generating 2 single-nucleotide conversions in two different genes. GTR 2.0 will substantially expand the scope, efficiency, and capabilities of yeast genome editing, and will be a versatile and invaluable addition to the toolbox of synthetic biology and metabolic engineering.
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| 2. |
- Lin, Zhenquan, et al.
(författare)
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Characterization of cross-species transcription and splicing from Penicillium to Saccharomyces cerevisiae
- 2021
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Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 48:9-10
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Tidskriftsartikel (refereegranskat)abstract
- Heterologous expression of eukaryotic gene clusters in yeast has been widely used for producing high-value chemicals and bioactive secondary metabolites. However, eukaryotic transcription cis-elements are still undercharacterized, and the cross-species expression mechanism remains poorly understood. Here we used the whole expression unit (including original promoter, terminator, and open reading frame with introns) of orotidine 5'-monophosphate decarboxylases from 14 Penicillium species as a showcase, and analyzed their cross-species expression in Saccharomyces cerevisiae. We found that pyrG promoters from the Penicillium species could drive URA3 expression in yeast, and that inefficient cross-species splicing of Penicillium introns might result in weak cross-species expression. Thus, this study demonstrates cross-species expression from Penicillium to yeast, and sheds light on the opportunities and challenges of cross-species expression of fungi expression units and gene clusters in yeast without refactoring for novel natural product discovery.
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| 3. |
- Qin, Ning, et al.
(författare)
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Increased CO 2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast
- 2024
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Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- CO2 fixation plays a key role to make biobased production cost competitive. Here, we use 3-hydroxypropionic acid (3-HP) to showcase how CO2 fixation enables approaching theoretical-yield production. Using genome-scale metabolic models to calculate the production envelope, we demonstrate that the provision of bicarbonate, formed from CO2, restricts previous attempts for high yield production of 3-HP. We thus develop multiple strategies for bicarbonate uptake, including the identification of Sul1 as a potential bicarbonate transporter, domain swapping of malonyl-CoA reductase, identification of Esbp6 as a potential 3-HP exporter, and deletion of Uga1 to prevent 3-HP degradation. The combined rational engineering increases 3-HP production from 0.14 g/L to 11.25 g/L in shake flask using 20 g/L glucose, approaching the maximum theoretical yield with concurrent biomass formation. The engineered yeast forms the basis for commercialization of bio-acrylic acid, while our CO2 fixation strategies pave the way for CO2 being used as the sole carbon source.
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| 4. |
- Wang, Kai, et al.
(författare)
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A one-carbon chemicals conversion strategy to produce precursor of biofuels with Saccharomyces cerevisiae
- 2023
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Ingår i: Renewable Energy. - : Elsevier BV. - 0960-1481 .- 1879-0682. ; 208, s. 331-340
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Tidskriftsartikel (refereegranskat)abstract
- Utilization of one-carbon chemicals such as CO2, formate, and methanol by microorganisms can enable the sustainable production of fuels and chemicals. However, the low conversion efficiency of these chemicals by microorganisms is a major challenge. To address this, we designed a one-carbon strategy that can utilize CO2 and its derivative formate. Here, a platform yeast strain with improved formate utilization and NAD(P)H production was constructed and evaluated for its ability to produce free fatty acids (FFAs). Based on 13C-marked analysis, the one-carbon assimilation efficiency of the platform strain reached 11.24%. Through continuous optimization, under conditions of glucose feeding the formate utilization rate of the final strain reached 0.48 g/L/h, with the final titer of FFAs reached 10.1 g/L, which represented improvements of 21.8 times and 33.7 times, respectively. As such, the produced FFAs can be easily transformed into biodiesel by combining them with downstream technologies in future research.
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| 5. |
- Wang, Kai, et al.
(författare)
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The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals
- 2023
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Ingår i: Green Energy and Environment. - 2468-0257 .- 2096-2797. ; In Press
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Tidskriftsartikel (refereegranskat)abstract
- For decades micoorganisms have been engineered for the utilization of lignocellulose-based second-generation (2G) feedstocks, but with the concerns of increased levels of atmospheric CO2 causing global warming there is an emergent need to transition from the utilization of 2G feedstocks to third-generation (3G) feedstocks such as CO2 and its derivatives. Here, we established a yeast platform that is capable of simultaneously converting 2G and 3G feedstocks into bulk and value-added chemicals. We demonstrated that by adopting 3G substrates such as CO2 and formate, the conversion of 2G feedstocks could be substantially improved. Specifically, formate could provide reducing power and energy for xylose conversion into valuable chemicals. Simultaneously, it can form a concentrated CO2 pool inside the cell, providing thermodynamically and kinetically favoured amounts of precursors for CO2 fixation pathways, e.g. the Calvin–Benson–Bassham (CBB) cycle. Furthermore, we demonstrated that formate could directly be utilized as a carbon source by yeast to synthesize endogenous amino acids. The engineered strain achieved a one-carbon (C1) assimilation efficiency of 9.2 %, which was the highest efficiency observed in the co-utilization of 2G and 3G feedstocks. We applied this strategy for productions of both bulk and value-added chemicals, including ethanol, free fatty acids (FFAs), and longifolene, resulting in yield enhancements of 18.4 %, 49.0 %, and ∼100 %, respectively. The strategy demonstrated here for co-utilization of 2G and 3G feedstocks sheds lights on both basic and applied research for the up-coming establishment of 3G biorefineries.
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| 6. |
- Lin, Zhenquan, et al.
(författare)
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Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters
- 2020
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 8
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Forskningsöversikt (refereegranskat)abstract
- Since the discovery of penicillin, natural products and their derivatives have been a valuable resource for drug discovery. With recent development of genome mining approaches in the post-genome era, a great number of natural product biosynthetic gene clusters (BGCs) have been identified and these can potentially be exploited for the discovery of novel natural products that can find application as pharmaceuticals. Since many BGCs are silent or do not express in native hosts under laboratory conditions, heterologous expression of BGCs in genetically tractable hosts becomes an attractive route to activate these BGCs to discover the corresponding products. Here, we highlight recent achievements in cloning and discovery of natural product biosynthetic pathways via intact BGC capturing, and discuss the prospects of high-throughput and multiplexed cloning of rational-designed gene clusters in the future.
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| 7. |
- Liu, Zihe, 1984, et al.
(författare)
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Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery
- 2021
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Ingår i: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 6:1, s. 20-22
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Tidskriftsartikel (refereegranskat)abstract
- Fungi are well known for production of antibiotics and other bioactive secondary metabolites, that can be served as pharmaceuticals, therapeutic agents and industrially useful compounds. However, compared with the characterization of prokaryotic biosynthetic gene clusters (BGCs), less attention has been paid to evaluate fungal BGCs. This is partially because heterologous expression of eukaryotic gene constructs often requires replacement of original promoters and terminators, as well as removal of intron sequences, and this substantially slow down the workflow in natural product discovery. It is therefore of interest to investigate the possibility and effectiveness of heterologous expression and library screening of intact BGCs without refactoring in industrial friendly microbial cell factories, such as the yeast Saccharomyces cerevisiae. Here, we discuss the importance of developing new research directions on library screening of fungal BGCs in yeast without refactoring, followed by outlooking prominent opportunities and challenges for future advancement.
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| 8. |
- Qin, Ning, 1990, et al.
(författare)
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Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast
- 2023
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 186:4, s. 748-763.e15
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Tidskriftsartikel (refereegranskat)abstract
- Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.
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| 9. |
- Qin, Ning, et al.
(författare)
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Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production
- 2020
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:12, s. 3236-3244
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Tidskriftsartikel (refereegranskat)abstract
- The central carbon metabolite acetyl-CoA and the cofactor NADPH are important for the synthesis of a wide array of biobased products. Here, we constructed a platform yeast strain for improved provision of acetyl-CoA and NADPH, and used the production of 3-hydroxypropionic acid (3-HP) as a case study. We first demonstrated that the integration of phosphoketolase and phosphotransacetylase improved 3-HP production by 41.9% and decreased glycerol production by 48.1% compared with that of the control strain. Then, to direct more carbon flux toward the pentose phosphate pathway, we reduced the expression of phosphoglucose isomerase by replacing its native promoter with a weaker promoter, and increased the expression of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase by replacing their native promoters with stronger promoters. This further improved 3-HP production by 26.4%. Furthermore, to increase the NADPH supply we overexpressed cytosolic aldehyde dehydrogenase, and improved 3-HP production by another 10.5%. Together with optimizing enzyme expression of acetyl-CoA carboxylase and malonyl-CoA reductase, the final strain is able to produce 3-HP with a final titer of 864.5 mg/L, which is a more than 24-fold improvement compared with that of the starting strain. Our strategy combines the PK pathway with the oxidative pentose phosphate pathway for the efficient provision of acetyl-CoA and NADPH, which provides both a higher theoretical yield and overall yield than the reported yeast-based 3-HP production strategies via the malonyl-CoA reductase-dependent pathway and sheds light on the construction of efficient platform cell factories for other products.
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| 10. |
- Wang, Junyang, et al.
(författare)
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Synthetic biology advanced natural product discovery
- 2021
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Ingår i: Metabolites. - : MDPI AG. - 2218-1989 .- 2218-1989. ; 11:11
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Forskningsöversikt (refereegranskat)abstract
- A wide variety of bacteria, fungi and plants can produce bioactive secondary metabolites, which are often referred to as natural products. With the rapid development of DNA sequencing technology and bioinformatics, a large number of putative biosynthetic gene clusters have been reported. However, only a limited number of natural products have been discovered, as most biosynthetic gene clusters are not expressed or are expressed at extremely low levels under conventional laboratory conditions. With the rapid development of synthetic biology, advanced genome mining and engineering strategies have been reported and they provide new opportunities for discovery of natural products. This review discusses advances in recent years that can accelerate the design, build, test, and learn (DBTL) cycle of natural product discovery, and prospects trends and key challenges for future research directions.
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