Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution
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ABSTRACT:
SUBMITTER: Su B
PROVIDER: S-EPMC10992865 | biostudies-literature | 2021 Jun
REPOSITORIES: biostudies-literature
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Project description:Carotenoids are a large family of health-beneficial compounds that have been widely used in the food and nutraceutical industries. There have been extensive studies to engineer Saccharomyces cerevisiae for the production of carotenoids, which already gained high level. However, it was difficult to discover new targets that were relevant to the accumulation of carotenoids. Herein, a new, ethanol-induced adaptive laboratory evolution was applied to boost carotenoid accumulation in a carotenoid producer BL03-D-4, subsequently, an evolved strain M3 was obtained with a 5.1-fold increase in carotenoid yield. Through whole-genome resequencing and reverse engineering, loss-of-function mutation of phosphofructokinase 1 (PFK1) was revealed as the major cause of increased carotenoid yield. Transcriptome analysis was conducted to reveal the potential mechanisms for improved yield, and strengthening of gluconeogenesis and downregulation of cell wall-related genes were observed in M3. This study provided a classic case where the appropriate selective pressure could be employed to improve carotenoid yield using adaptive evolution and elucidated the causal mutation of evolved strain.
2021-01-09 | GSE164470 | GEO
Project description:Quantitative physiological studies on Saccharomyces cerevisiae commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in S. cerevisiae CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution. In the present study, this laboratory strain was shown to exhibit suboptimal specific growth rates when either inositol, nicotinic acid, pyridoxine, pantothenic acid, para-aminobenzoic acid (pABA), or thiamine was omitted from SM. Subsequently, this strain was evolved in parallel serial-transfer experiments for fast aerobic growth on glucose in the absence of individual B vitamins. In all evolution lines, specific growth rates reached at least 90% of the growth rate observed in SM supplemented with a complete B vitamin mixture. Fast growth was already observed after a few transfers on SM without myo-inositol, nicotinic acid, or pABA. Reaching similar results in SM lacking thiamine, pyridoxine, or pantothenate required more than 300 generations of selective growth. The genomes of evolved single-colony isolates were resequenced, and for each B vitamin, a subset of non-synonymous mutations associated with fast vitamin-independent growth was selected. These mutations were introduced in a non-evolved reference strain using CRISPR/Cas9-based genome editing. For each B vitamin, the introduction of a small number of mutations sufficed to achieve a substantially increased specific growth rate in non-supplemented SM that represented at least 87% of the specific growth rate observed in fully supplemented complete SM.IMPORTANCE Many strains of Saccharomyces cerevisiae, a popular platform organism in industrial biotechnology, carry the genetic information required for synthesis of biotin, thiamine, pyridoxine, para-aminobenzoic acid, pantothenic acid, nicotinic acid, and inositol. However, omission of these B vitamins typically leads to suboptimal growth. This study demonstrates that, for each individual B vitamin, it is possible to achieve fast vitamin-independent growth by adaptive laboratory evolution (ALE). Identification of mutations responsible for these fast-growing phenotypes by whole-genome sequencing and reverse engineering showed that, for each compound, a small number of mutations sufficed to achieve fast growth in its absence. These results form an important first step toward development of S. cerevisiae strains that exhibit fast growth on inexpensive, fully supplemented mineral media that only require complementation with a carbon source, thereby reducing costs, complexity, and contamination risks in industrial yeast fermentation processes.
| S-EPMC7267190 | biostudies-literature
Project description:Identification of a novel metabolic engineering target for carotenoid production in Saccharomyces cerevisiae via ethanol-induced adaptive laboratory evolution
| PRJNA690797 | ENA
Project description:Backgroundβ-Caryophyllene is a plant terpenoid with therapeutic and biofuel properties. Production of terpenoids through microbial cells is a potentially sustainable alternative for production. Adaptive laboratory evolution is a complementary technique to metabolic engineering for strain improvement, if the product-of-interest is coupled with growth. Here we use a combination of pathway engineering and adaptive laboratory evolution to improve the production of β-caryophyllene, an extracellular product, by leveraging the antioxidant potential of the compound.ResultsUsing oxidative stress as selective pressure, we developed an adaptive laboratory evolution that worked to evolve an engineered β-caryophyllene producing yeast strain for improved production within a few generations. This strategy resulted in fourfold increase in production in isolated mutants. Further increasing the flux to β-caryophyllene in the best evolved mutant achieved a titer of 104.7 ± 6.2 mg/L product. Genomic analysis revealed a gain-of-function mutation in the a-factor exporter STE6 was identified to be involved in significantly increased production, likely as a result of increased product export.ConclusionAn optimized selection strategy based on oxidative stress was developed to improve the production of the extracellular product β-caryophyllene in an engineered yeast strain. Application of the selection strategy in adaptive laboratory evolution resulted in mutants with significantly increased production and identification of novel responsible mutations.
| S-EPMC8157465 | biostudies-literature
Project description:Utilising one-carbon substrates such as carbon dioxide, methane, and methanol is vital to address the current climate crisis. Methylotrophic metabolism enables growth and energy generation from methanol, providing an alternative to sugar fermentation. Saccharomyces cerevisiae is an important industrial microorganism for which growth on one-carbon substrates would be relevant. However, its ability to metabolize methanol has been poorly characterised. Here, using adaptive laboratory evolution and 13C-tracer analysis, we discover that S. cerevisiae has a native capacity for methylotrophy. A systems biology approach reveals that global rearrangements in central carbon metabolism fluxes, gene expression changes, and a truncation of the uncharacterized transcriptional regulator Ygr067cp supports improved methylotrophy in laboratory evolved S. cerevisiae. This research paves the way for further biotechnological development and fundamental understanding of methylotrophy in the preeminent eukaryotic model organism and industrial workhorse, S. cerevisiae.
| S-EPMC7643182 | biostudies-literature
Project description:l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.
| S-EPMC9769770 | biostudies-literature
Project description:Co-utilization of xylose and glucose from lignocellulosic biomass is an economically feasible bioprocess for chemical production. Many strategies have been implemented for efficiently assimilating xylose which is one of the predominant sugars of lignocellulosic biomass. However, there were few reports about engineering Saccharomyces cerevisiae for carotenoid production from xylose-glucose mixtures. Herein, we developed a platform for facilitating carotenoid production in S. cerevisiae by fermentation of xylose-glucose mixtures. Firstly, a xylose assimilation pathway with mutant xylose reductase (XYL1m), xylitol dehydrogenase (XYL2), and xylulokinase (XK) was constructed for utilizing xylose. Then, introduction of phosphoketolase (PK) pathway, deletion of Pho13 and engineering yeast hexose transporter Gal2 were conducted to improve carotenoid yields. The final strain SC105 produced a 1.6-fold higher production from mixed sugars than that from glucose in flask culture. In fed-batch fermentation with continuous feeding of mixed sugars, carotenoid production represented a 2.6-fold higher. To the best of our knowledge, this is the first report that S. cerevisiae was engineered to utilize xylose-glucose mixtures for carotenoid production with a considerable high yield. The present study exhibits a promising advantage of xylose-glucose mixtures assimilating strain as an industrial carotenoid producer from lignocellulosic biomass.
| S-EPMC7240070 | biostudies-literature
Project description:IntroductionFurfural, a main inhibitor produced during pretreatment of lignocellulose, has shown inhibitory effects on S. cerevisiae.MethodIn the present study, new strains named 12-1 with enhanced resistance to furfural were obtained through adaptive laboratory evolution, which exhibited a shortened lag phase by 36 h, and an increased ethanol conversion rate by 6.67% under 4 g/L furfural.Results and discussionTo further explore the mechanism of enhanced furfural tolerance, ADR1_1802 mutant was constructed by CRISPR/Cas9 technology, based on whole genome re-sequencing data. The results indicated that the time when ADR1_1802 begin to grow was shortened by 20 h compared with reference strain (S. cerevisiae CEN.PK113-5D) when furfural was 4 g/L. Additionally, the transcription levels of GRE2 and ADH6 in ADR1_ 1802 mutant were increased by 53.69 and 44.95%, respectively, according to real-time fluorescence quantitative PCR analysis. These findings suggest that the enhanced furfural tolerance of mutant is due to accelerated furfural degradation. Importance: Renewable carbon worldwide is vital to achieve "zero carbon" target. Bioethanol obtained from biomass is one of them. To make bioethanol price competitive to fossil fuel, higher ethanol yield is necessary, therefore, monosaccharide produced during biomass pretreatment should be effectively converted to ethanol by Saccharomyces cerevisiae. However, inhibitors formed by glucose or xylose oxidation could make ethanol yield lower. Thus, inhibitor tolerant Saccharomyces cerevisiae is important to this process. As one of the main component of pretreatment hydrolysate, furfural shows obvious impact on growth and ethanol production of Saccharomyces cerevisiae. To get furfural tolerant Saccharomyces cerevisiae and find the underlying mechanism, adaptive laboratory evolution and CRISPR/Cas9 technology were applied in the present study.
| S-EPMC10794740 | biostudies-literature
Project description:BackgroundBiofilm-immobilized continuous fermentation has the potential to enhance cellular environmental tolerance, maintain cell activity and improve production efficiency.ResultsIn this study, different biofilm-forming genes (FLO5, FLO8 and FLO10) were integrated into the genome of S. cerevisiae for overexpression, while FLO5 and FLO10 gave the best results. The biofilm formation of the engineered strains 1308-FLO5 and 1308-FLO10 was improved by 31.3% and 58.7% compared to that of the WT strain, respectively. The counts of cells adhering onto the biofilm carrier were increased. Compared to free-cell fermentation, the average ethanol production of 1308, 1308-FLO5 and 1308-FLO10 was increased by 17.4%, 20.8% and 19.1% in the biofilm-immobilized continuous fermentation, respectively. Due to good adhering ability, the fermentation broth turbidity of 1308-FLO5 and 1308-FLO10 was decreased by 22.3% and 59.1% in the biofilm-immobilized fermentation, respectively. Subsequently, for biofilm-immobilized fermentation coupled with membrane separation, the engineered strain significantly reduced the pollution of cells onto the membrane and the membrane separation flux was increased by 36.3%.ConclusionsIn conclusion, enhanced biofilm-forming capability of S. cerevisiae could offer multiple benefits in ethanol fermentation.
| S-EPMC10391976 | biostudies-literature
Project description:Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.
| S-EPMC10628280 | biostudies-literature