scholarly journals Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene

2020 ◽  
Vol 7 (4) ◽  
pp. 135
Author(s):  
Jan Niklas Bröker ◽  
Boje Müller ◽  
Dirk Prüfer ◽  
Christian Schulze Gronover
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Metabolic Flux ◽  
Mevalonate Pathway ◽  
Plant Secondary Metabolites ◽  
Product Formation ◽  
Culture Conditions ◽  
Efficient Production ◽  
Enhanced Production ◽  
Engineering Strategy

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

scholarly journals Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

2009 ◽  
Vol 75 (17) ◽  
pp. 5536-5543 ◽  
Author(s):  
Kenro Tokuhiro ◽  
Masayoshi Muramatsu ◽  
Chikara Ohto ◽  
Toshiya Kawaguchi ◽  
Shusei Obata ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Flux ◽  
Mevalonate Pathway ◽  
Fusion Gene ◽  
Pharmaceutical Products ◽  
Test Tube ◽  
Ethanol Solution ◽  
Multicopy Integration ◽  
Engineered Saccharomyces Cerevisiae ◽  
Rate Limiting

ABSTRACT (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

scholarly journals A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

2020 ◽  
Author(s):  
David C. Garcia ◽  
Jaime Lorenzo N. Dinglasan ◽  
Him Shrestha ◽  
Paul E. Abraham ◽  
Robert L. Hettich ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Cell Growth ◽  
Metabolic Flux ◽  
Selective Removal ◽  
List Type ◽  
Metabolic Potential ◽  
Metabolite Production ◽  
Genomic Engineering ◽  
Crude Extracts ◽  
Engineering Strategy

AbstractCell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell’s capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction.HighlightsA novel method of engineering crude cell lysates for enhancing specific metabolic processes is described.Multiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth.The described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells.Pooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy.

Metabolic Engineering of Saccharomyces cerevisiae for Efficient Production of Pure L-(+)-Lactic Acid

2006 ◽  
Vol 131 (1-3) ◽  
pp. 795-807 ◽  
Author(s):  
Nobuhiro Ishida ◽  
Satoshi Saitoh ◽  
Toru Ohnishi ◽  
Kenro Tokuhiro ◽  
Eiji Nagamori ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lactic Acid ◽  
Metabolic Engineering ◽  
Efficient Production

Metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin

2019 ◽  
Vol 54 ◽  
pp. 212-221 ◽  
Author(s):  
Lei Sun ◽  
Guiyou Liu ◽  
Ya Li ◽  
Dayong Jiang ◽  
Wenfeng Guo ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Efficient Production

scholarly journals Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid

2020 ◽  
Author(s):  
Pingping Zhou ◽  
Chunlei Yue ◽  
Bin Shen ◽  
Yi Du ◽  
Nannan Xu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Caffeic Acid ◽  
Acid Production ◽  
De Novo ◽  
Feedback Inhibition ◽  
Biological Activities ◽  
Acid Product ◽  
Enhanced Production ◽  
Microbial Biosynthesis

Abstract Background As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported sporadically for biosynthesis of caffeic acid via free plasmid‑mediated pathway assembly. However, the production was far from satisfactory and even relied on the addition of precursor. Results In this study, we first established a controllable caffeic acid pathway by employing a modified GAL regulatory system in S. cerevisiae and realized de novo biosynthesis of 313.8 mg/L caffeic acid from glucose. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways and overexpressing rate-limiting enzymes led to about 2.5-fold improvement in the caffeic acid production, reaching up to 769.3 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid de novo synthesized by engineered yeast. Conclusions Caffeic acid production in S. cerevisiae strain was successfully improved by adopting a glucose-regulated GAL system and comprehensive metabolic engineering strategies. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites.

scholarly journals Comprehensive metabolic engineering for fermenting glycerol efficiently in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Sadat M. R. Khattab ◽  
Takashi Watanabe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metabolic Engineering ◽  
Metabolic Flux ◽  
Plant Biomass ◽  
Industrial Applications ◽  
Cell Factory ◽  
Cell Wall Polysaccharides ◽  
Oxidative Pathway ◽  
Biomass Decomposition ◽  
Engineered Strain

ABSTRACTGlycerol is an eco-friendly solvent enhancing plant-biomass decomposition through a glycerolysis process in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae restrains many of these scenarios. Here we outline the complete strategy for the generation of efficient glycerol fermenting yeast by rewriting the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by O2-dependent dynamic shuttle while abolishing both glycerol phosphorylation and biosynthesis pathways. By following a vigorous glycerol oxidative pathway, the engineered strain demonstrated augmentation in conversion efficiency (CE) reach up to 0.49g-ethanol/g-glycerol—98% of theoretical conversion—with production rate >1 g/L-1h-1 when supplementing glycerol as a single fed-batch on a rich-medium. Furthermore, the engineered strain showed a new capability toward ferment a mixture of glycerol and glucose with producing >86 g/L of bioethanol with 92.8% of the CE. To our knowledge, this is the highest ever reported titer in this regard. Notably, this strategy flipped our ancestral yeast from non-growth on glycerol, on the minimal medium, to a fermenting strain with productivities 0.25-0.5 g/L-1h-1 and 84-78% of CE, respectively and 90% of total conversions to the products. The findings in metabolic engineering here may release the limitations of utilizing glycerol in several eco-friendly biorefinery approaches.IMPORTANCEWith the avenues for achieving efficient lignocellulosic biorefinery scenarios, glycerol gained keen attention as an eco-friendly biomass-derived solvent for enhancing the dissociation of lignin and cell wall polysaccharides during pretreatment process. Co-fermentation of glycerol with the released sugars from biomass after the glycerolysis expands the resource for ethanol production and release from the burden of component separation. Titer productivities are one of the main obstacles for industrial applications of this process. Therefore, the generation of highly efficient glycerol fermenting yeast significantly promotes the applicability of the integrated biorefineries scenario. Besides, the glycerol is an important carbon resource for producing chemicals. Hence, the metabolic flux control of yeast from glycerol contributes to generation of cell factory producing chemicals from glycerol, promoting the association between biodiesel and bioethanol industries. Thus, this study will shed light on solving the problems of global warming and agricultural wastes, leading to establishment of the sustainable society.

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