| 11. |
- Sjöström, Staffan L., et al.
(author)
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High-throughput screening for industrial enzyme production hosts by droplet microfluidics
- 2014
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In: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:4, s. 806-813
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Journal article (peer-reviewed)abstract
- A high-throughput method for single cell screening by microfluidic droplet sorting is applied to a whole-genome mutated yeast cell library yielding improved production hosts of secreted industrial enzymes. The sorting method is validated by enriching a yeast strain 14 times based on its a-amylase production, close to the theoretical maximum enrichment. Furthermore, a 105 member yeast cell library is screened yielding a clone with a more than 2-fold increase in a-amylase production. The increase in enzyme production results from an improvement of the cellular functions of the production host in contrast to previous droplet-based directed evolution that has focused on improving enzyme protein structure. In the workflow presented, enzyme producing single cells are encapsulated in 20 pL droplets with a fluorogenic reporter substrate. The coupling of a desired phenotype (secreted enzyme concentration) with the genotype (contained in the cell) inside a droplet enables selection of single cells with improved enzyme production capacity by droplet sorting. The platform has a throughput over 300 times higher than that of the current industry standard, an automated microtiter plate screening system. At the same time, reagent consumption for a screening experiment is decreased a million fold, greatly reducing the costs of evolutionary engineering of production strains.
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| 12. |
- Wang, Kai, et al.
(author)
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A one-carbon chemicals conversion strategy to produce precursor of biofuels with Saccharomyces cerevisiae
- 2023
-
In: Renewable Energy. - : Elsevier BV. - 0960-1481 .- 1879-0682. ; 208, s. 331-340
-
Journal article (peer-reviewed)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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| 13. |
- Wang, Kai, et al.
(author)
-
The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals
- 2023
-
In: Green Energy and Environment. - 2468-0257 .- 2096-2797. ; In Press
-
Journal article (peer-reviewed)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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| 14. |
- Hou, Jin, 1982, et al.
(author)
-
Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae
- 2012
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 14:2, s. 120-127
-
Journal article (peer-reviewed)abstract
- The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Secl/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and alpha-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases alpha-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.
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| 15. |
- Hou, Jin, 1982, et al.
(author)
-
Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae
- 2012
-
In: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 12:5, s. 491-510
-
Journal article (peer-reviewed)abstract
- The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals, and it is also provides a platform for the production of many heterologous proteins of medical or industrial interest. Therefore, many studies have focused on metabolic engineering S similar to cerevisiae to improve the recombinant protein production, and with the development of systems biology, it is interesting to see how this approach can be applied both to gain further insight into protein production and secretion and to further engineer the cell for improved production of valuable proteins. In this review, the protein post-translational modification such as folding, trafficking, and secretion, steps that are traditionally studied in isolation will here be described in the context of the whole system of protein secretion. Furthermore, examples of engineering secretion pathways, high-throughput screening and systems biology applications of studying protein production and secretion are also given to show how the protein production can be improved by different approaches. The objective of the review is to describe individual biological processes in the context of the larger, complex protein synthesis network.
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| 16. |
- Lin, Zhenquan, et al.
(author)
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Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters
- 2020
-
In: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 8
-
Research review (peer-reviewed)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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| 17. |
- Liu, Zihe, 1984, et al.
(author)
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Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae
- 2013
-
In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:20, s. 8955-8962
-
Journal article (peer-reviewed)abstract
- With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas alpha-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h(-1) that influences protein production. Thus, for lower specific growth rates, the alpha-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate.
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| 18. |
- Liu, Zihe, 1984, et al.
(author)
-
Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae
- 2012
-
In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 109:5, s. 1259-1268
-
Journal article (peer-reviewed)abstract
- Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and a-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and a-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (mu mol/L) of IP was found to be higher than that of a-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, that is, the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, (Tyo et al. submitted) in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter). For the IP there is more than 10-fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.
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| 19. |
- Liu, Zihe, 1984, et al.
(author)
-
Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery
- 2021
-
In: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 6:1, s. 20-22
-
Journal article (peer-reviewed)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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| 20. |
- Liu, Zihe, 1984
(author)
-
Metabolic Engineering of Recombinant Protein Production by Saccharomyces cerevisiae
- 2012
-
Doctoral thesis (other academic/artistic)abstract
- The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and it also provides a platform for the production of many heterologous proteins of medical or industrial interest. In this thesis, random and rational approaches, such as vector design, host engineering, fermentation analysis, UV Mutation, coupled with high-throughput systems biology techniques (including whole genomic sequencing, microarray analysis and flux analysis) and integrated analysis (Reporter feature technique), were employed to engineer cellular properties more effectively and purposefully to construct cell factories for protein production. We reported that insulin production mainly depends on the expression level of the gene, whereas amylase tends to achieve higher secretion at lower growth conditions in order to reduce ER stress. Moreover, based on large data generated and systems biology tools, we proposed several models to address unknown questions regarding recombinant protein production: i) the futile cycle of protein folding in the ER and the thermodynamic model of non-stoichiometric production of reactive oxygen species explains the oxidative stress that occurred during recombinant protein production, and ii) the final electron acceptor for protein folding and the electron transferring model at anaerobic condition proposed potential electron consuming pathway for protein folding in the ER. Our research provided a deep understanding of the processing of protein secretory pathway, potential targets for future engineering, as well as shed lights for basic cellular metabolisms.
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