| 1. |
- Asplund-Samuelsson, Johannes, et al.
(author)
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Thermodynamic analysis of computed pathways integrated into the metabolic networks of E. coli and Synechocystis reveals contrasting expansion potential
- 2018
-
In: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 45, s. 223-236
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Journal article (peer-reviewed)abstract
- Introducing biosynthetic pathways into an organism is both reliant on and challenged by endogenous biochemistry. Here we compared the expansion potential of the metabolic network in the photoautotroph Synechocystis with that of the heterotroph E. coli using the novel workflow POPPY (Prospecting Optimal Pathways with PYthon). First, E. coli and Synechocystis metabolomic and fluxomic data were combined with metabolic models to identify thermodynamic constraints on metabolite concentrations (NET analysis). Then, thousands of automatically constructed pathways were placed within each network and subjected to a network-embedded variant of the max-min driving force analysis (NEM). We found that the networks had different capabilities for imparting thermodynamic driving forces toward certain compounds. Key metabolites were constrained differently in Synechocystis due to opposing flux directions in glycolysis and carbon fixation, the forked tri-carboxylic acid cycle, and photorespiration. Furthermore, the lysine biosynthesis pathway in Synechocystis was identified as thermodynamically constrained, impacting both endogenous and heterologous reactions through low 2-oxoglutarate levels. Our study also identified important yet poorly covered areas in existing metabolomics data and provides a reference for future thermodynamics-based engineering in Synechocystis and beyond. The POPPY methodology represents a step in making optimal pathway-host matches, which is likely to become important as the practical range of host organisms is diversified.
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| 2. |
- Borodina, I., et al.
(author)
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Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via beta-alanine
- 2015
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 27, s. 57-64
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Journal article (peer-reviewed)abstract
- Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharolnyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the beta-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of beta-alanine and its subsequent conversion into 3HP using a novel beta-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7 +/- 0.3 g L-1 with a 0.14 +/- 0.0 C-mol C-mol(-1) yield on glucose in 80 h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.
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| 3. |
- Chen, Que, et al.
(author)
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Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis : Proteorhodopsin expression increases growth rate and fitness of a Delta PSI strain of Synechocystis sp. PCC6803
- 2019
-
In: Metabolic engineering. - : Elsevier. - 1096-7176 .- 1096-7184. ; 52, s. 68-76
-
Journal article (peer-reviewed)abstract
- To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (Delta PSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the Delta PSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of Delta PSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (Delta PSI/Delta SynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing Delta PSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP(+)-reduction.
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| 4. |
- Englund, Elias, et al.
(author)
-
Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound
- 2018
-
In: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 49, s. 164-177
-
Journal article (peer-reviewed)abstract
- Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain.
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| 5. |
- Fletcher, Eugene, 1986, et al.
(author)
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Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments
- 2017
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In: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 39, s. 19-28
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Journal article (peer-reviewed)abstract
- Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.
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| 6. |
- Hing, Nathaphon Yu King, et al.
(author)
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Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803
- 2019
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In: Metabolic engineering. - : ACADEMIC PRESS INC ELSEVIER SCIENCE. - 1096-7176 .- 1096-7184. ; 56, s. 77-84
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Journal article (peer-reviewed)abstract
- Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 mu mol m(-2) s(-1)) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 mu mol m(-2) s(-1). Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wildtype cells demonstrated increases in growth rates as expected. To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.
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| 7. |
- Hu, Yating, 1991, et al.
(author)
-
Heterologous transporter expression for improved fatty alcohol secretion in yeast
- 2018
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In: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 45, s. 51-58
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Journal article (peer-reviewed)abstract
- The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.
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| 8. |
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| 9. |
- Kaczmarzyk, Danuta, et al.
(author)
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Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX
- 2018
-
In: Metabolic engineering. - : Academic Press. - 1096-7176 .- 1096-7184. ; 45, s. 59-66
-
Journal article (peer-reviewed)abstract
- Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3 mg g−1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.
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| 10. |
- Krivoruchko, Anastasia, 1984, et al.
(author)
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Microbial acetyl-CoA metabolism and metabolic engineering
- 2015
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 28, s. 28-42
-
Journal article (other academic/artistic)abstract
- Recent concerns over the sustainability of petrochemical-based processes for production of desired chemicals have fueled research into alternative modes of production. Metabolic engineering of microbial cell factories such as Saccharomyces cerevisiae and Escherichia coli offers a sustainable and flexible alternative for the production of various molecules. Acetyl-CoA is a key molecule in microbial central carbon metabolism and is involved in a variety of cellular processes. In addition, it functions as a precursor for many molecules of biotechnological relevance. Therefore, much interest exists in engineering the metabolism around the acetyl-CoA pools in cells in order to increase product titers. Here we provide an overview of the acetyl-CoA metabolism in eukaryotic and prokaryotic microbes (with a focus on S. cerevisiae and E. coli), with an emphasis on reactions involved in the production and consumption of acetyl-CoA. In addition, we review various strategies that have been used to increase acetyl-CoA production in these microbes.
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| 11. |
- Kumar, Manish, 1982, et al.
(author)
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Gut microbiota dysbiosis is associated with malnutrition and reduced plasma amino acid levels: Lessons from genome-scale metabolic modeling
- 2018
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 49, s. 128-142
-
Journal article (peer-reviewed)abstract
- Malnutrition is a severe non-communicable disease, which is prevalent in children from low-income countries. Recently, a number of metagenomics studies have illustrated associations between the altered gut microbiota and child malnutrition. However, these studies did not examine metabolic functions and interactions between individual species in the gut microbiota during health and malnutrition. Here, we applied genome-scale metabolic modeling to model the gut microbial species, which were selected from healthy and malnourished children from three countries. Our analysis showed reduced metabolite production capabilities in children from two lowincome countries compared with a high-income country. Additionally, the models were also used to predict the community-level metabolic potentials of gut microbes and the patterns of pairwise interactions among species. Hereby we found that due to bacterial interactions there may be reduced production of certain amino acids in malnourished children compared with healthy children from the same communities. To gain insight into alterations in the metabolism of malnourished (stunted) children, we also performed targeted plasma metabolic profiling in the first 2 years of life of 25 healthy and 25 stunted children. Plasma metabolic profiling further revealed that stunted children had reduced plasma levels of essential amino acids compared to healthy controls. Our analyses provide a framework for future efforts towards further characterization of gut microbial metabolic capabilities and their contribution to malnutrition.
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| 12. |
- Li, M., et al.
(author)
-
De novo production of resveratrol from glucose or ethanol by engineered Saccharomyces cerevisiae
- 2015
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 32, s. 1-11
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Journal article (peer-reviewed)abstract
- Resveratrol is a natural antioxidant compound, used as food supplement and cosmetic ingredient. Microbial production of resveratrol has until now been achieved by supplementation of expensive substrates, p-coumaric acid or aromatic amino acids. Here we engineered the yeast Saccharomyces cerevisiae to produce resveratrol directly from glucose or ethanol via tyrosine intermediate. First we introduced the biosynthetic pathway, consisting of tyrosine ammonia-lyase from Herpetosiphon aurantiacus, 4-coumaryl-CoA ligase from Arabidopsis thaliana and resveratrol synthase from Vitis vinifera, and obtained 2.73±0.05mgL-1 resveratrol from glucose. Then we over-expressed feedback-insensitive alleles of ARO4 encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate and ARO7 encoding chorismate mutase, resulting in production of 4.85±0.31mgL-1 resveratrol from glucose as the sole carbon source. Next we improved the supply of the precursor malonyl-CoA by over-expressing a post-translational de-regulated version of the acetyl-CoA carboxylase encoding gene ACC1; this strategy further increased resveratrol production to 6.39±0.03mgL-1. Subsequently, we improved the strain by performing multiple-integration of pathway genes resulting in resveratrol production of 235.57±7.00mgL-1. Finally, fed-batch fermentation of the final strain with glucose or ethanol as carbon source resulted in a resveratrol titer of 415.65 and 531.41mgL-1, respectively.
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| 13. |
- Liang, Feiyan, et al.
(author)
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Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803
- 2016
-
In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 38, s. 56-64
-
Journal article (peer-reviewed)abstract
- Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO2 assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100μmolphotonsm(-2)s(-1) light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100μmolphotonsm(-2)s(-1) light condition. Under 15μmolphotonsm(-2)s(-1) light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.
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| 14. |
- Liang, Feiyan, et al.
(author)
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Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio
- 2018
-
In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 46, s. 51-59
-
Journal article (peer-reviewed)abstract
- The Calvin-Benson-Bassham (CBB) cycle is the main pathway to fix atmospheric CO2 and store energy in carbon bonds, forming the precursors of most primary and secondary metabolites necessary for life. Speeding up the CBB cycle theoretically has positive effects on the subsequent growth and/or the end metabolite(s) production. Four CBB cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase), transketolase (TK) and aldolase (FBA) were selected to be co-overexpressed with the ethanol synthesis enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the cyanobacterium Synechocystis PCC 6803. An inducible promoter, PnrsB, was used to drive PDC and ADH expression. When PnrsB was induced and cells were cultivated at 65 µmol photons m−2 s−1, the RuBisCO-, FBP/SBPase-, TK-, and FBA-expressing strains produced 55%, 67%, 37% and 69% more ethanol and 7.7%, 15.1%, 8.8% and 10.1% more total biomass (the sum of dry cell weight and ethanol), respectively, compared to the strain only expressing the ethanol biosynthesis pathway. The ethanol to total biomass ratio was also increased in CBB cycle enzymes overexpressing strains. This study experimentally demonstrates that using the cells with enhanced carbon fixation, when the product synthesis pathway is not the main bottleneck, can significantly increase the generation of a product (exemplified with ethanol), which acts as a carbon sink.
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| 15. |
- Liu, Zhengtao, et al.
(author)
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Pyruvate kinase L/R is a regulator of lipid metabolism and mitochondrial function
- 2019
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 52, s. 263-272
-
Journal article (peer-reviewed)abstract
- The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) has been associated with altered expression of liver-specific genes including pyruvate kinase liver and red blood cell (PKLR), patatin-like phospholipase domain containing 3 (PNPLA3) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Here, we inhibited and overexpressed the expression of these three genes in HepG2 cells, generated RNA-seq data before and after perturbation and revealed the altered global biological functions with the modulation of these genes using integrated network (IN) analysis. We found that modulation of these genes effects the total triglycerides levels within the cells and viability of the cells. Next, we generated IN for HepG2 cells, identified reporter transcription factors based on IN and found that the modulation of these genes affects key metabolic pathways associated with lipid metabolism (steroid biosynthesis, PPAR signalling pathway, fatty acid synthesis and oxidation) and cancer development (DNA replication, cell cycle and p53 signalling) involved in the progression of NAFLD and HCC. Finally, we observed that inhibition of PKLR lead to decreased glucose uptake and decreased mitochondrial activity in HepG2 cells. Hence, our systems level analysis indicated that PKLR can be targeted for development efficient treatment strategy for NAFLD and HCC.
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| 16. |
- Ma, Tian, et al.
(author)
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Lipid engineering combined with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield production of lycopene
- 2019
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 52, s. 134-142
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Journal article (peer-reviewed)abstract
- Saccharomyces cerevisiae is an efficient host for natural-compound production and preferentially employed in academic studies and bioindustries. However, S. cerevisiae exhibits limited production capacity for lipophilic natural products, especially compounds that accumulate intracellularly, such as polyketides and carotenoids, with some engineered compounds displaying cytotoxicity. In this study, we used a nature-inspired strategy to establish an effective platform to improve lipid oil–triacylglycerol (TAG) metabolism and enable increased lycopene accumulation. Through systematic traditional engineering methods, we achieved relatively high-level production at 56.2 mg lycopene/g cell dry weight (cdw). To focus on TAG metabolism in order to increase lycopene accumulation, we overexpressed key genes associated with fatty acid synthesis and TAG production, followed by modulation of TAG fatty acyl composition by overexpressing a fatty acid desaturase (OLE1) and deletion of Seipin (FLD1), which regulates lipid-droplet size. Results showed that the engineered strain produced 70.5 mg lycopene/g cdw, a 25% increase relative to the original high-yield strain, with lycopene production reaching 2.37 g/L and 73.3 mg/g cdw in fed-batch fermentation and representing the highest lycopene yield in S. cerevisiae reported to date. These findings offer an effective strategy for extended systematic metabolic engineering through lipid engineering.
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| 17. |
- Marques, W. L., et al.
(author)
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Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae
- 2018
-
In: Metabolic engineering. - : Academic Press Inc.. - 1096-7176 .- 1096-7184. ; 45, s. 121-133
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Journal article (peer-reviewed)abstract
- Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h−1 (LmSPase) and 0.06 ± 0.01 h−1 (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h−1 and 0.08 ± 0.00 h−1, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.
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| 18. |
- Miao, Rui, et al.
(author)
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Protein engineering of α-ketoisovalerate decarboxylase for improved isobutanol production in Synechocystis PCC 6803
- 2018
-
In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 47, s. 42-48
-
Journal article (peer-reviewed)abstract
- Protein engineering is a powerful tool to modify e.g. protein stability, activity and substrate selectivity. Heterologous expression of the enzyme α-ketoisovalerate decarboxylase (Kivd) in the unicellular cyanobacterium Synechocystis PCC 6803 results in cells producing isobutanol and 3-methyl-1-butanol, with Kivd identified as a potential bottleneck. In the present study, we used protein engineering of Kivd to improve isobutanol production in Synechocystis PCC 6803. Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. Single replacement, either Val461 to isoleucine or Ser286 to threonine, increased the Kivd activity significantly, both in vivo and in vitro resulting in increased overall production while isobutanol production was increased more than 3-methyl-1-butanol production. Moreover, among all the engineered strains examined, the strain with the combined modification V461I/S286T showed the highest (2.4 times) improvement of isobutanol-to-3M1B molar ratio, which was due to a decrease of the activity towards 3M1B production. Protein engineering of Kivd resulted in both enhanced total catalytic activity and preferential shift towards isobutanol production in Synechocystis PCC 6803.
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| 19. |
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| 20. |
- Nilsson, Avlant, 1985, et al.
(author)
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Genome scale metabolic modeling of cancer
- 2017
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 43, s. 103-112
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Research review (peer-reviewed)abstract
- Cancer cells reprogram metabolism to support rapid proliferation and survival. Energy metabolism is particularly important for growth and genes encoding enzymes involved in energy metabolism are frequently altered in cancer cells. A genome scale metabolic model (GEM) is a mathematical formalization of metabolism which allows simulation and hypotheses testing of metabolic strategies. It has successfully been applied to many microorganisms and is now used to study cancer metabolism. Generic models of human metabolism have been reconstructed based on the existence of metabolic genes in the human genome. Cancer specific models of metabolism have also been generated by reducing the number of reactions in the generic model based on high throughput expression data, e.g. transcriptomics and proteomics. Targets for drugs and bio markers for diagnostics have been identified using these models. They have also been used as scaffolds for analysis of high throughput data to allow mechanistic interpretation of changes in expression. Finally, GEMs allow quantitative flux predictions using flux balance analysis (FBA). Here we critically review the requirements for successful FBA simulations of cancer cells and discuss the symmetry between the methods used for modeling of microbial and cancer metabolism. GEMs have great potential for translational research on cancer and will therefore become of increasing importance in the future. © 2017
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| 21. |
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| 22. |
- Pereira, Rui, 1986, et al.
(author)
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Adaptive laboratory evolution of tolerance to dicarboxylic acids in Saccharomyces cerevisiae
- 2019
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 56, s. 130-141
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Journal article (peer-reviewed)abstract
- Improving the growth phenotypes of microbes in high product concentrations is an essential design objective in the development of robust cell factories. However, the limited knowledge regarding tolerance mechanisms makes rational design of such traits complicated. Here, adaptive laboratory evolution was used to explore the tolerance mechanisms that Saccharomyces cerevisiae can evolve in the presence of inhibiting concentrations of three dicarboxylic acids: glutaric acid, adipic acid and pimelic acid. Whole-genome sequencing of tolerant mutants enabled the discovery of the genetic changes behind tolerance and most mutations could be linked to the up-regulation of multidrug resistance transporters. The amplification of QDR3, in particular, was shown to confer tolerance not only to the three dicarboxylic acids investigated, but also towards muconic acid and glutaconic acid. In addition to increased acid tolerance, QDR3 overexpression also improved the production of muconic acid in the context of a strain engineered for producing this compound.
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| 23. |
- Pfleger, B. F., et al.
(author)
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Metabolic engineering strategies for microbial synthesis of oleochemicals
- 2015
-
In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 29, s. 1-11
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Journal article (other academic/artistic)abstract
- Microbial synthesis of oleochemicals has advanced significantly in the last decade. Microbes have been engineered to convert renewable substrates to a wide range of molecules that are ordinarily made from plant oils. This approach is attractive because it can reduce a motivation for converting tropical rainforest into farmland while simultaneously enabling access to molecules that are currently expensive to produce from oil crops. In the last decade, enzymes responsible for producing oleochemicals in nature have been identified, strategies to circumvent native regulation have been developed, and high yielding strains have been designed, built, and successfully demonstrated. This review will describe the metabolic pathways that lead to the diverse molecular features found in natural oleochemicals, highlight successful metabolic engineering strategies, and comment on areas where future work could further advance the field.
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| 24. |
- Robinson, Jonathan, 1986, et al.
(author)
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An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa
- 2017
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 41, s. 67-81
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Journal article (peer-reviewed)abstract
- Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO2 ) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO3 (-)) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO -> NO3 (-)-> NO2 (-)-> NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O-2, NO2 (-), and NO3 (-) in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO3 (-), which was relieved upon O-2 depletion. As cell growth depleted dissolved O-2 levels, NO3 (-) was converted to NO2 (-) at near-stoichiometric levels, whereas NO2 (-) consumption did not coincide with NO or NO3 (-) accumulation. Assimilatory NO2 reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with Delta nirB,Delta nirS, and Delta norC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.
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| 25. |
- Rodriguez, A., et al.
(author)
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Comparison of the metabolic response to over-production of p-coumaric acid in two yeast strains
- 2017
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In: Metabolic Engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 44, s. 265-272
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Journal article (peer-reviewed)abstract
- The development of robust and efficient cell factories requires understanding of the metabolic changes triggered by the production of the targeted compound. Here we aimed to study how production of p-coumaric acid, a precursor of multiple secondary aromatic metabolites, influences the cellular metabolism of Saccharomyces cerevisiae. We evaluated the growth and p-coumaric acid production in batch and chemostat cultivations and analyzed the transcriptome and intracellular metabolome during steady state in low-and high-producers of p-coumaric acid in two strain backgrounds, S288c or CEN.PK. We found that the same genetic modifications resulted in higher production of p-coumaric acid in the CEN.PK background than in the S288c background. Moreover, the CEN.PK strain was less affected by the genetic engineering as was evident from fewer changes in the transcription profile and intracellular metabolites concentrations. Surprisingly, for both strains we found the largest transcriptional changes in genes involved in transport of amino acids and sugars, which were downregulated. Additionally, in S288c amino acid and protein biosynthesis processes were also affected. We systematically overexpressed or deleted genes with significant transcriptional changes in CEN.PK low and high-producing strains. The knockout of some of the downregulated transporters triggered a 20-50% improvement in the synthesis of p-CA in the CEN.PK high-producing strain. This study demonstrates the importance of transporters in the engineering of cell factories for production of small molecules.
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