| 1. |
- Liu, Zihe, 1984, et al.
(författare)
-
Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering
- 2014
-
Ingår i: Applied and Environmental Microbiology. - : American Society for Microbiology. - 1098-5336 .- 0099-2240. ; 80:17, s. 5542-5550
-
Tidskriftsartikel (refereegranskat)abstract
- The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is widely used as a cell factory for recombinant protein production. However, many recombinant proteins are produced at only 1% (or less) of the theoretical capacity due to the complexity of the secretory pathway, which has not been fully exploited. In this study, we applied the concept of inverse metabolic engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be associated with higher amylase secretion were identified through whole-genome sequencing. Several single-point mutations, including an S196I point mutation in the VTA1 gene coding for a protein involved in vacuolar sorting, were evaluated by introducing these to the starting strain. By applying this modification alone, the amylase secretion could be improved by 35%. As a complement to the identification of genomic variants, transcriptome analysis was also performed in order to understand on a global level the transcriptional changes associated with the improved amylase production caused by UV mutagenesis.
|
|
| 2. |
- Gong, Guiping, et al.
(författare)
-
GTR 2.0: GRNA-tRNA Array and Cas9-NG Based Genome Disruption and Single-Nucleotide Conversion in Saccharomyces cerevisiae
- 2021
-
Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 10:6, s. 1328-1337
-
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.
|
|
| 3. |
- Hou, Jin, 1982, et al.
(författare)
-
Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae
- 2013
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:8, s. 3559-3568
-
Tidskriftsartikel (refereegranskat)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 low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous alpha-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously.
|
|
| 4. |
- Hou, Jin, 1982, et al.
(författare)
-
Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast
- 2014
-
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 14:3, s. 481-494
-
Tidskriftsartikel (refereegranskat)abstract
- In yeast Saccharomyces cerevisiae, accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates the unfolded protein response (UPR), which is mediated by Hac1p. The heat shock response (HSR) mediated by Hsf1p, mainly regulates cytosolic processes and protects the cell from stresses. Here, we find that a constitutive activation of the HSR could increase ER stress resistance in both wild-type and UPR-deficient cells. Activation of HSR decreased UPR activation in the WT (as shown by the decreased HAC1 mRNA splicing). We analyzed the genome-wide transcriptional response in order to propose regulatory mechanisms that govern the interplay between UPR and HSR and followed up for the hypotheses by experiments in vivo and in vitro. Interestingly, we found that the regulation of ER stress response via HSR is (1) only partially dependent on over-expression of Kar2p (ER resident chaperone induced by ER stress); (2) does not involve the increase in protein turnover via the proteasome activity; (3) is related to the oxidative stress response. From the transcription data, we also propose that HSR enhances ER stress resistance mainly through facilitation of protein folding and secretion. We also find that HSR coordinates multiple stress-response pathways, including the repression of the overall transcription and translation.
|
|
| 5. |
- Huang, Mingtao, 1984, et al.
(författare)
-
Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
- 2015
-
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. ; 112:34, s. E4689-E4696
-
Tidskriftsartikel (refereegranskat)abstract
- There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.
|
|
| 6. |
- Lin, Zhenquan, et al.
(författare)
-
Characterization of cross-species transcription and splicing from Penicillium to Saccharomyces cerevisiae
- 2021
-
Ingår i: Journal of Industrial Microbiology and Biotechnology. - : Oxford University Press (OUP). - 1367-5435 .- 1476-5535. ; 48:9-10
-
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.
|
|
| 7. |
- Liu, Lifang, 1979, et al.
(författare)
-
Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae
- 2014
-
Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 21, s. 9-16
-
Tidskriftsartikel (refereegranskat)abstract
- Due to limitations associated with whole blood for transfusions (antigen compatibility, transmission of infections, supply and storage), the use of cell-free hemoglobin as an oxygen carrier substitute has been in the center of research interest for decades. Human hemoglobin has previously been synthesized in yeast, however the challenge is to balance the expression of the two different globin subunits, as well as the supply of the prosthetic heme required for obtaining the active hemoglobin (alpha(2)beta(2)). In this work we evaluated the expression of different combinations of alpha and beta peptides and combined this with metabolic engineering of the heme biosynthetic pathway. Through evaluation of several different strategies we showed that engineering the biosynthesis pathway can substantially increase the heme level in yeast cells, and this resulted in a significant enhancement of human hemoglobin production. Besides demonstration of improved hemoglobin production our work demonstrates a novel strategy for improving the production of complex proteins, especially multimers with a prosthetic group. Crown Copyright (C) 2013 Published by Elsevier Inc. on behalf of International Metabolic Engineering Society. All rights reserved.
|
|
| 8. |
- Liu, Lifang, 1979, et al.
(författare)
-
Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae
- 2015
-
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 15:7, s. 10-
-
Tidskriftsartikel (refereegranskat)abstract
- Oxygen is important for normal aerobic metabolism, as well as for protein production where it is needed for oxidative protein folding. However, several studies have reported that anaerobic conditions seem to be more favorable in terms of recombinant protein production. We were interested in increasing recombinant protein production under aerobic conditions so we focused on Rox1p regulation. Rox1p is a transcriptional regulator, which in oxidative conditions represses genes induced in hypoxia. We deleted ROX1 and studied the effects on the production of recombinant proteins in Saccharomyces cerevisiae. Intriguingly, we found a 100% increase in the recombinant fungal alpha-amylase yield, as well as productivity. Varied levels of improvements were also observed for the productions of the human insulin precursor and the yeast endogenous enzyme invertase. Based on the genome-wide transcriptional response, we specifically focused on the effect of UPC2 upregulation on protein production and suggested a possible mechanistic explanation.
|
|
| 9. |
- Liu, Zihe, 1984, et al.
(författare)
-
Anaerobic alpha-Amylase Production and Secretion with Fumarate as the Final Electron Acceptor in Saccharomyces cerevisiae
- 2013
-
Ingår i: Applied and Environmental Microbiology. - 1098-5336 .- 0099-2240. ; 79:9, s. 2962-2967
-
Tidskriftsartikel (refereegranskat)abstract
- In this study, we focus on production of heterologous alpha-amylase in the yeast Saccharomyces cerevisiae under anaerobic conditions. We compare the metabolic fluxes and transcriptional regulation under aerobic and anaerobic conditions, with the objective of identifying the final electron acceptor for protein folding under anaerobic conditions. We find that yeast produces more amylase under anaerobic conditions than under aerobic conditions, and we propose a model for electron transfer under anaerobic conditions. According to our model, during protein folding the electrons from the endoplasmic reticulum are transferred to fumarate as the final electron acceptor. This model is supported by findings that the addition of fumarate under anaerobic (but not aerobic) conditions improves cell growth, specifically in the alpha-amylase-producing strain, in which it is not used as a carbon source. Our results provide a model for the molecular mechanism of anaerobic protein secretion using fumarate as the final electron acceptor, which may allow for further engineering of yeast for improved protein secretion under anaerobic growth conditions.
|
|
| 10. |
- Qin, Ning, et al.
(författare)
-
Increased CO 2 fixation enables high carbon-yield production of 3-hydroxypropionic acid in yeast
- 2024
-
Ingår i: Nature Communications. - 2041-1723 .- 2041-1723. ; 15:1
-
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.
|
|
| 11. |
- Sjöström, Staffan L., et al.
(författare)
-
High-throughput screening for industrial enzyme production hosts by droplet microfluidics
- 2014
-
Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:4, s. 806-813
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 12. |
- Wang, Kai, et al.
(författare)
-
A one-carbon chemicals conversion strategy to produce precursor of biofuels with Saccharomyces cerevisiae
- 2023
-
Ingår i: Renewable Energy. - : Elsevier BV. - 0960-1481 .- 1879-0682. ; 208, s. 331-340
-
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.
|
|
| 13. |
- Wang, Kai, et al.
(författare)
-
The transition from 2G to 3G-feedstocks enabled efficient production of fuels and chemicals
- 2023
-
Ingår i: Green Energy and Environment. - 2468-0257 .- 2096-2797. ; In Press
-
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.
|
|
| 14. |
- Hou, Jin, 1982, et al.
(författare)
-
Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae
- 2012
-
Ingår i: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 14:2, s. 120-127
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 15. |
- Hou, Jin, 1982, et al.
(författare)
-
Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae
- 2012
-
Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 12:5, s. 491-510
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 16. |
- Lin, Zhenquan, et al.
(författare)
-
Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters
- 2020
-
Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media SA. - 2296-4185. ; 8
-
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.
|
|
| 17. |
- Liu, Zihe, 1984, et al.
(författare)
-
Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae
- 2013
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 97:20, s. 8955-8962
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 18. |
- Liu, Zihe, 1984, et al.
(författare)
-
Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae
- 2012
-
Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 109:5, s. 1259-1268
-
Tidskriftsartikel (refereegranskat)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.
|
|
| 19. |
- Liu, Zihe, 1984, et al.
(författare)
-
Expression of fungal biosynthetic gene clusters in S. cerevisiae for natural product discovery
- 2021
-
Ingår i: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 6:1, s. 20-22
-
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.
|
|
| 20. |
- Liu, Zihe, 1984
(författare)
-
Metabolic Engineering of Recombinant Protein Production by Saccharomyces cerevisiae
- 2012
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
|
|
| 21. |
- Liu, Zihe, 1984, et al.
(författare)
-
Synthetic Biology of Yeast
- 2019
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 58:11, s. 1511-1520
-
Forskningsöversikt (refereegranskat)abstract
- With the rapid development of DNA synthesis and next-generation sequencing, synthetic biology that aims to standardize, modularize, and innovate cellular functions, has achieved vast progress. Here we review key advances in synthetic biology of the yeast Saccharomyces cerevisiae, which serves as an important eukaryal model organism and widely applied cell factory. This covers the development of new building blocks, i.e., promoters, terminators and enzymes, pathway engineering, tools developments, and gene circuits utilization. We will also summarize impacts of synthetic biology on both basic and applied biology, and end with further directions for advancing synthetic biology in yeast.
|
|
| 22. |
- Qin, Ning, 1990, et al.
(författare)
-
Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast
- 2023
-
Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 186:4, s. 748-763.e15
-
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.
|
|
| 23. |
- Qin, Ning, et al.
(författare)
-
Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production
- 2020
-
Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 9:12, s. 3236-3244
-
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.
|
|
| 24. |
- Tyo, Keith, 1979, et al.
(författare)
-
Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress
- 2012
-
Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 10:16, s. Art. no 16-
-
Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).ResultsHAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.ConclusionsApparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
|
|
| 25. |
- Tyo, Keith, 1979, et al.
(författare)
-
Impact of protein uptake and degradation on recombinant protein secretion in yeast
- 2014
-
Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 98:16, s. 7149-7159
-
Tidskriftsartikel (refereegranskat)abstract
- Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize the protein uptake and degradation pathways of S. cerevisiae to better understand its impact on protein secretion titers. We do find that S. cerevisiae can consume significant (in the range of 1 g/L/day) quantities of whole proteins. Characterizing the physiological state and combining metabolomics and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion systems, and the current data should help formulate strategies to mitigate product loss.
|
|
