Comprehensive metabolic engineering for fermenting glycerol efficiently in Saccharomyces cerevisiae

Metabolic Engineering ◽

Metabolic Flux ◽

Plant Biomass ◽

Industrial Applications ◽

Cell Factory ◽

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.

Efficient Conversion of Glycerol to Ethanol by an Engineered Saccharomyces cerevisiae Strain

Applied and Environmental Microbiology ◽

10.1128/aem.00268-21 ◽

2021 ◽

Author(s):

Sadat M. R. Khattab ◽

Takashi Watanabe

Keyword(s):

Metabolic Flux ◽

Triosephosphate Isomerase ◽

Plant Biomass ◽

Agricultural Waste ◽

Glycerol Dehydrogenase ◽

Plant Oil ◽

Cell Factories ◽

Engineered Strain

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. Here, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O 2 -dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae D452-2 strain, as well as vigorous expression of whole genes in the DHA-pathway ( Candid utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate >1 g/L −1 h −1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/L) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from parent strain, which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g/L −1 h −1 on minimal medium under semi-aerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant-oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Co-conversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.

Metabolic engineering of Saccharomyces cerevisiae for efficient conversions of glycerol to ethanol

10.1101/2021.01.04.425180 ◽

2021 ◽

Author(s):

Sadat M. R. Khattab ◽

Takashi Watanabe

Keyword(s):

Production Rate ◽

Nicotinamide Adenine Dinucleotide ◽

Minimal Medium ◽

Plant Biomass ◽

Fine Tuning ◽

Comprehensive Strategy ◽

Oxidative Pathway ◽

Efficient Fermentation ◽

Biomass Decomposition

Glycerol is an eco-friendly solvent enhancing plant-biomass decomposition through the glycell process to bio-based chemicals. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae restrains many biorefineries-scenarios. Here, we outline a comprehensive strategy for generating efficient glycerol fermenting S. cerevisiae via rewriting the oxidation of cytosolic nicotinamide adenine dinucleotide by O2-dependent dynamic shuttle while abolishing glycerol phosphorylation and biosynthesis pathways. By following a vigorous glycerol oxidative pathway, our engineered strain demonstrated a breakthrough in conversion efficiency (CE), reaching up to 0.49g-ethanol/g-glycerol—98% of theoretical conversion—with production rate >1 gL−1h−1 on rich-medium. Interestingly, the glycerol consumption and its fermentation unrepressed during the mixing by glucose until the strain produced >86 g/L of bioethanol with 92.8% of CE. Moreover, fine-tuning of O2 boosted the production rate to >2 gL−1h−1with 82% of CE. Impressively, the strategy flipped the ancestral yeast even from non-growing on glycerol, on the minimal medium, to a fermenting strain with productivities 0.25-0.5 gL−1h−1 and 84-78% of CE, respectively. Our findings promote utlising glycerol efficiently in several eco-friendly biorefinery approaches.SummaryEfficient fermentation of glycerol in S. cerevisiae was established by comprehensive engineering of glycerol pathways and rewriting NADH pathway.

Efficient Alcoholic Conversion of Glycerol by Engineered Saccharomyces cerevisiae

10.1101/2021.05.24.445540 ◽

2021 ◽

Author(s):

Takashi Watanabe ◽

Sadat M. R. Khattab

Keyword(s):

Nicotinamide Adenine Dinucleotide ◽

Batch Fermentation ◽

Plant Biomass ◽

Glycerol Oxidation ◽

Fed Batch ◽

Fed Batch Fermentation ◽

Engineered Saccharomyces Cerevisiae ◽

Biomass Decomposition ◽

Engineered Strain

Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, the lack of efficient conversion of glycerol by natural Saccharomyces cerevisiae hinders its use in these methods. Here, we have aimed to develop a complete strategy for the generation of efficient glycerol-converting yeast by modifying the oxidation of cytosolic nicotinamide adenine dinucleotide (NADH) by an O2-dependent dynamic shuttle, while abolishing both glycerol phosphorylation and biosynthesis. By following a vigorous glycerol oxidation pathway, the engineered strain increased the conversion efficiency (CE) to up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with production rate > 1 g×L×h, when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain fermented a mixture of glycerol and glucose, producing > 86 g/L bioethanol with 92.8% CE. To our knowledge, this is the highest ever reported titer in this field. Notably, this strategy changed conventional yeast from a non-grower on minimal medium containing glycerol to a fermenting strain with productivity of 0.25-0.5 g×L×h and 84-78% CE, which converted 90% of the substrate to products. Our findings may improve the utilization of glycerol in several eco-friendly biorefinery approaches.

Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

10.1101/166678 ◽

2017 ◽

Author(s):

Chenlu Zhang ◽

Ligia Acosta-Sampson ◽

Vivian Yaci Yu ◽

Jamie H. D. Cate

Keyword(s):

Directed Evolution ◽

Plant Biomass ◽

Carbon Sources ◽

Industrial Applications ◽

Yeast Saccharomyces Cerevisiae ◽

Cellulosic Biofuel ◽

Economic Production ◽

Report Analysis ◽

Selection Medium

AbstractThe economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

Microbial Cell Factories ◽

10.1186/s12934-019-1259-x ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 5

Author(s):

Vijaydev Ganesan ◽

Michael Spagnuolo ◽

Ayushi Agrawal ◽

Spencer Smith ◽

Difeng Gao ◽

...

Keyword(s):

Metabolic Engineering ◽

Genome Editing ◽

Yarrowia Lipolytica ◽

Gene Editing ◽

Molecular Genetic ◽

Industrial Applications ◽

Cell Factory ◽

Fuel Feed ◽

Renewable Chemicals ◽

Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

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

Bioengineering ◽

10.3390/bioengineering7040135 ◽

2020 ◽

Vol 7 (4) ◽

pp. 135

Author(s):

Jan Niklas Bröker ◽

Boje Müller ◽

Dirk Prüfer ◽

Christian Schulze Gronover

Keyword(s):

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.

Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries

Cellular and Molecular Life Sciences ◽

10.1007/s00018-012-0945-1 ◽

2012 ◽

Vol 69 (16) ◽

pp. 2671-2690 ◽

Cited By ~ 263

Author(s):

Kuk-Ki Hong ◽

Jens Nielsen

Keyword(s):

Metabolic Engineering ◽

Cell Factory

Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.00712-14 ◽

2014 ◽

Vol 80 (17) ◽

pp. 5542-5550 ◽

Cited By ~ 17

Author(s):

Zihe Liu ◽

Lifang Liu ◽

Tobias Österlund ◽

Jin Hou ◽

Mingtao Huang ◽

...

Keyword(s):

Metabolic Engineering ◽

Secretory Pathway ◽

Single Point ◽

Point Mutations ◽

Model Organisms ◽

Cell Factory ◽

Amylase Secretion ◽

Content Type ◽

Inverse Metabolic Engineering

ABSTRACTThe 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 yeastSaccharomyces cerevisiaeis 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 theVTA1gene 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.

Phytochrome light receptors control metabolic flux, and their action during seedling development sets the trajectory for biomass production

10.1101/762666 ◽

2019 ◽

Author(s):

Johanna Krahmer ◽

Ammad Abbas ◽

Virginie Mengin ◽

Hirofumi Ishihara ◽

Thiago A Moraes ◽

...

Keyword(s):

Metabolic Flux ◽

Carbon Fixation ◽

Plant Biomass ◽

Growth Dynamics ◽

Biomass Accumulation ◽

Adult Plant ◽

Growth Responses ◽

Stress Metabolites ◽

Biomass Quantification

AbstractThe phytochromes (phys) photoreceptors are known to be major regulators of plastic growth responses to vegetation shade. Recent reports have begun to uncover an important role for phys in carbon resource management. Our earlier work showed that phy mutants had a distinct metabolic profile with elevated levels of metabolites including TCA intermediates, amino acids and sugars. Here we show that in seedlings phy regulates the balance between glucose and starch. Multi-allele phy mutants have excess glucose and low starch levels, which is conducive to hypocotyl elongation. 13C-CO2 labelling demonstrates that metabolic flux balance in adult plants is markedly altered in phy mutants. Phytochrome reduces synthesis rates of stress metabolites, including raffinose and proline and several typical stress-induced biosynthetic genes related to these metabolites show higher expression in phy mutants.Since growth and metabolism are typically inter-connected, we investigated why phy mutants have severely reduced biomass. Quantification of carbon fixation, biomass accumulation, and 13C labelling of cell wall polysaccharides established that relative growth rate is impaired in multi allele phy mutants for the first 2.5 weeks after germination but equivalent to the WT thereafter. Mathematical modelling predicts that the altered growth dynamics and final biomass deficit can be explained by the smaller cotyledon size of the multiple phy mutants. This indicates that the established role of phy in promoting seedling establishment has enduring effects that govern adult plant biomass.

Re-routing of Sugar Catabolism Provides a Better Insight Into Fungal Flexibility in Using Plant Biomass-Derived Monomers as Substrates

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.644216 ◽

2021 ◽

Vol 9 ◽

Author(s):

Tania Chroumpi ◽

Mao Peng ◽

Lye Meng Markillie ◽

Hugh D. Mitchell ◽

Carrie D. Nicora ◽

...

Keyword(s):

Plant Cell Wall ◽

Plant Biomass ◽

Value Added ◽

Sugar Metabolism ◽

Cell Factory ◽

Filamentous Ascomycete ◽

Extensive Metabolism ◽

Beet Pulp ◽

A Cell

The filamentous ascomycete Aspergillus niger has received increasing interest as a cell factory, being able to efficiently degrade plant cell wall polysaccharides as well as having an extensive metabolism to convert the released monosaccharides into value added compounds. The pentoses D-xylose and L-arabinose are the most abundant monosaccharides in plant biomass after the hexose D-glucose, being major constituents of xylan, pectin and xyloglucan. In this study, the influence of selected pentose catabolic pathway (PCP) deletion strains on growth on plant biomass and re-routing of sugar catabolism was addressed to gain a better understanding of the flexibility of this fungus in using plant biomass-derived monomers. The transcriptome, metabolome and proteome response of three PCP mutant strains, ΔlarAΔxyrAΔxyrB, ΔladAΔxdhAΔsdhA and ΔxkiA, grown on wheat bran (WB) and sugar beet pulp (SBP), was evaluated. Our results showed that despite the absolute impact of these PCP mutations on pure pentose sugars, they are not as critical for growth of A. niger on more complex biomass substrates, such as WB and SBP. However, significant phenotypic variation was observed between the two biomass substrates, but also between the different PCP mutants. This shows that the high sugar heterogeneity of these substrates in combination with the high complexity and adaptability of the fungal sugar metabolism allow for activation of alternative strategies to support growth.