Flux Balance Analysis Inspired Bioprocess Upgrading for Lycopene Production by a Metabolically Engineered Strain of Yarrowia lipolytica.
ABSTRACT: Genome-scale metabolic models embody a significant advantage of systems biology since their applications as metabolic flux simulation models enable predictions for the production of industrially-interesting metabolites. The biotechnological production of lycopene from Yarrowia lipolytica is an emerging scope that has not been fully scrutinized, especially for what concerns cultivation conditions of newly generated engineered strains. In this study, by combining flux balance analysis (FBA) and Plackett-Burman design, we screened chemicals for lycopene production from a metabolically engineered strain of Y. lipolytica. Lycopene concentrations of 126 and 242 mg/L were achieved correspondingly from the FBA-independent and the FBA-assisted designed media in fed-batch cultivation mode. Transcriptional studies revealed upregulations of heterologous genes in media designed according to FBA, thus implying the efficiency of model predictions. Our study will potentially support upgraded lycopene and other terpenoids production from existing or prospect bioengineered strains of Y. lipolytica and/or closely related yeast species.
Project description:Astaxanthin is a red-colored carotenoid, used as food and feed additive. Astaxanthin is mainly produced by chemical synthesis, however, the process is expensive and synthetic astaxanthin is not approved for human consumption. In this study, we engineered the oleaginous yeast Yarrowia lipolytica for de novo production of astaxanthin by fermentation. First, we screened 12 different Y. lipolytica isolates for ?-carotene production by introducing two genes for ?-carotene biosynthesis: bi-functional phytoene synthase/lycopene cyclase (crtYB) and phytoene desaturase (crtI) from the red yeast Xanthophyllomyces dendrorhous. The best strain produced 31.1 ± 0.5 mg/L ?-carotene. Next, we optimized the activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG1) and geranylgeranyl diphosphate synthase (GGS1/crtE) in the best producing strain and obtained 453.9 ± 20.2 mg/L ?-carotene. Additional downregulation of the competing squalene synthase SQS1 increased the ?-carotene titer to 797.1 ± 57.2 mg/L. Then we introduced ?-carotene ketolase (crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ) from Pantoea ananatis to convert ?-carotene into astaxanthin. The constructed strain accumulated 10.4 ± 0.5 mg/L of astaxanthin but also accumulated astaxanthin biosynthesis intermediates, 5.7 ± 0.5 mg/L canthaxanthin, and 35.3 ± 1.8 mg/L echinenone. Finally, we optimized the copy numbers of crtZ and crtW to obtain 3.5 mg/g DCW (54.6 mg/L) of astaxanthin in a microtiter plate cultivation. Our study for the first time reports engineering of Y. lipolytica for the production of astaxanthin. The high astaxanthin content and titer obtained even in a small-scale cultivation demonstrates a strong potential for Y. lipolytica-based fermentation process for astaxanthin production.
Project description:Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.
Project description:The production of lycopene in E. coli can be improved by mutated CRP.CRP is a well-known global transcription factor regulating the expression of more than 400 genes that belong to different functional groups of E. coli. During the accumulation of lycopene in E. coli, the engineered CRP alters the expression level of many genes, such as the genes belong to lycopene production pathway, which leads to the improved lycopene production. We used microarrays to detail the gene expression under the condition of engineered CRP and identified distinct classes of up-regulated and down-regulated genes. E. coli growed for 15h was selected for RNA extraction and hybridization on Affymetrix microarrays. As we know that the biggest difference in lycopene production was at 15h during cultivation, so we chose 15h as the time-point.
Project description:The codon-optimized genes crtB and crtI of Pantoea ananatis were expressed in Yarrowia lipolytica under the control of the TEF1 promoter of Y. lipolytica. Additionally, the rate-limiting genes for isoprenoid biosynthesis in Y. lipolytica, GGS1 and HMG1, were overexpressed to increase the production of lycopene. All of the genes were also expressed in a Y. lipolytica strain with POX1 to POX6 and GUT2 deleted, which led to an increase in the size of lipid bodies and a further increase in lycopene production. Lycopene is located mainly within lipid bodies, and increased lipid body formation leads to an increase in the lycopene storage capacity of Y. lipolytica. Growth-limiting conditions increase the specific lycopene content. Finally, a yield of 16 mg g(-1) (dry cell weight) was reached in fed-batch cultures, which is the highest value reported so far for a eukaryotic host.
Project description:An important goal of whole-cell computational modeling is to integrate detailed biochemical information with biological intuition to produce testable predictions. Based on the premise that prokaryotes such as Escherichia coli have maximized their growth performance along evolution, flux balance analysis (FBA) predicts metabolic flux distributions at steady state by using linear programming. Corroborating earlier results, we show that recent intracellular flux data for wild-type E. coli JM101 display excellent agreement with FBA predictions. Although the assumption of optimality for a wild-type bacterium is justifiable, the same argument may not be valid for genetically engineered knockouts or other bacterial strains that were not exposed to long-term evolutionary pressure. We address this point by introducing the method of minimization of metabolic adjustment (MOMA), whereby we test the hypothesis that knockout metabolic fluxes undergo a minimal redistribution with respect to the flux configuration of the wild type. MOMA employs quadratic programming to identify a point in flux space, which is closest to the wild-type point, compatibly with the gene deletion constraint. Comparing MOMA and FBA predictions to experimental flux data for E. coli pyruvate kinase mutant PB25, we find that MOMA displays a significantly higher correlation than FBA. Our method is further supported by experimental data for E. coli knockout growth rates. It can therefore be used for predicting the behavior of perturbed metabolic networks, whose growth performance is in general suboptimal. MOMA and its possible future extensions may be useful in understanding the evolutionary optimization of metabolism.
Project description:Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax mediterranei, a halophilic archaeon, as a new lycopene producer. H. mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.
Project description:Flux balance analysis (FBA) is an increasingly useful approach for modeling the behavior of metabolic systems. However, standard FBA modeling of genetic knockouts cannot predict drug combination synergies observed between serial metabolic targets, even though such synergies give rise to some of the most widely used antibiotic treatments. Here we extend FBA modeling to simulate responses to chemical inhibitors at varying concentrations, by diverting enzymatic flux to a waste reaction. This flux diversion yields very similar qualitative predictions to prior methods for single target activity. However, we find very different predictions for combinations, where flux diversion, which mimics the kinetics of competitive metabolic inhibitors, can explain serial target synergies between metabolic enzyme inhibitors that we confirmed in Escherichia coli cultures. FBA flux diversion opens the possibility for more accurate genome-scale predictions of drug synergies, which can be used to suggest treatments for infections and other diseases.
Project description:We present two modifications of the flux balance analysis (FBA) metabolic modeling framework which relax implicit assumptions of the biomass reaction. Our flexible flux balance analysis (flexFBA) objective removes the fixed proportion between reactants, and can therefore produce a subset of biomass reactants. Our time-linked flux balance analysis (tFBA) simulation removes the fixed proportion between reactants and byproducts, and can therefore describe transitions between metabolic steady states. Used together, flexFBA and tFBA model a time scale shorter than the regulatory and growth steady state encoded by the biomass reaction. This combined short-time FBA method is intended for integrated modeling applications to enable detailed and dynamic depictions of microbial physiology such as whole-cell modeling. For example, when modeling Escherichia coli, it avoids artifacts caused by low-copy-number enzymes in single-cell models with kinetic bounds. Even outside integrated modeling contexts, the detailed predictions of flexFBA and tFBA complement existing FBA techniques. We show detailed metabolite production of in silico knockouts used to identify when correct essentiality predictions are made for the wrong reason.
Project description:BACKGROUND:Citric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide - inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy-repeated-batch culture was applied. RESULTS:The strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm- 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm- 3 h- 1 and a yield of 0.85 g g- 1. CONCLUSIONS:The citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.
Project description:BACKGROUND: The main objective of flux balance analysis (FBA) is to obtain quantitative predictions of metabolic fluxes of an organism, and it is necessary to use an appropriate objective function to guarantee a good estimation of those fluxes. METHODOLOGY: In this study, the predictive performance of FBA was evaluated, using objective functions arising from the linear combination of different cellular objectives. This approach is most suitable for eukaryotic cells, owing to their multiplicity of cellular compartments. For this reason, Saccharomyces cerevisiae was used as model organism, and its metabolic network was represented using the genome-scale metabolic model iMM904. As the objective was to evaluate the predictive performance from the FBA using the kind of objective function previously described, substrate uptake and oxygen consumption were the only input data used for the FBA. Experimental information about microbial growth and exchange of metabolites with the environment was used to assess the quality of the predictions. CONCLUSIONS: The quality of the predictions obtained with the FBA depends greatly on the knowledge of the oxygen uptake rate. For the most of studied classifications, the best predictions were obtained with "maximization of growth", and with some combinations that include this objective. However, in the case of exponential growth with unknown oxygen exchange flux, the objective function "maximization of growth, plus minimization of NADH production in cytosol, plus minimization of NAD(P)H consumption in mitochondrion" gave much more accurate estimations of fluxes than the obtained with any other objective function explored in this study.