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

Production Rate ◽

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):

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.

Comprehensive metabolic engineering for fermenting glycerol efficiently in Saccharomyces cerevisiae

10.1101/2021.02.13.430370 ◽

2021 ◽

Author(s):

Sadat M. R. Khattab ◽

Takashi Watanabe

Keyword(s):

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.

Properties of Thermobifida fusca peroxidase Tfu-1649 and its combined synergistic effects with xylanase on lignocellulose degradation

BioResources ◽

10.15376/biores.16.1.942-953 ◽

2020 ◽

Vol 16 (1) ◽

pp. 942-953

Author(s):

Wan-Yu Liao ◽

Yu-Chun Huang ◽

Wei-Lin Chen ◽

Cheng-Yu Chen ◽

Chao-Hsun Yang

Keyword(s):

Reducing Sugars ◽

Plant Biomass ◽

Synergistic Effects ◽

Culture Broth ◽

Lignocellulose Degradation ◽

Total Phenolic ◽

Thermobifida Fusca ◽

Zizania Latifolia ◽

Thermophilic Actinomycetes ◽

Biomass Decomposition

Lignocelluloses are comprised of cellulose, hemicellulose, and lignins, which constitute plant biomass. Since peroxidases can degrade lignins, the authors examined peroxidase Tfu-1649, which is secreted from the thermophilic actinomycetes, Thermobifida fusca BCRC 19214. After cultivating for 48 h, the culture broth accumulated 43.66 U/mL of peroxidase activity. The treatment of four types of lignocellulolytic byproducts, i.e., bagasse, corncob, pin sawdust, and Zizania latifolia Turcz husk, with Tfu-1649 alone increased the total phenolic compounds, with limited reducing sugars, but treatment with xylanase, Tfu-11, and peroxidase Tfu-1649 showed synergistic effects. Hence, the co-operative degradation of lignocelluloses by both peroxidase and xylanase could contribute to biomass decomposition and further applications in the agricultural and environmental industries.

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.

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 ◽

Cell Wall Polysaccharides ◽

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.

Recent Advances on Feasible Strategies for Monoterpenoid Production in Saccharomyces cerevisiae

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.609800 ◽

2020 ◽

Vol 8 ◽

Author(s):

Qiyu Gao ◽

Luan Wang ◽

Maosen Zhang ◽

Yongjun Wei ◽

Wei Lin

Keyword(s):

Plant Biomass ◽

Extraction Methods ◽

Active Components ◽

Plant Essential Oils ◽

Microbial Cell Factories ◽

Cell Factories ◽

Plant Extraction ◽

Low Efficiency ◽

Metabolic Activities

Terpenoids are a large diverse group of natural products which play important roles in plant metabolic activities. Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs. Some monoterpenoids are widely used in medicine, cosmetics and other industries, and they are mainly obtained by plant biomass extraction methods. These plant extraction methods have some problems, such as low efficiency, unstable quality, and high cost. Moreover, the monoterpenoid production from plant cannot satisfy the growing monoterpenoids demand. The development of metabolic engineering, protein engineering and synthetic biology provides an opportunity to produce large amounts of monoterpenoids eco-friendly using microbial cell factories. This mini-review covers current monoterpenoids production using Saccharomyces cerevisiae. The monoterpenoids biosynthetic pathways, engineering of key monoterpenoids biosynthetic enzymes, and current monoterpenoids production using S. cerevisiae were summarized. In the future, metabolically engineered S. cerevisiae may provide one possible green and sustainable strategy for monoterpenoids supply.

Adenine nucleotides as paracrine mediators and intracellular second messengers in immunity and inflammation

Biochemical Society Transactions ◽

10.1042/bst20180419 ◽

2019 ◽

Vol 47 (1) ◽

pp. 329-337 ◽

Cited By ~ 2

Author(s):

Ralf Fliegert ◽

Jörg Heeren ◽

Friedrich Koch-Nolte ◽

Viacheslav O. Nikolaev ◽

Christian Lohr ◽

...

Keyword(s):

Immune System ◽

T Lymphocytes ◽

Immune Responses ◽

Adenine Nucleotides ◽

Second Messengers ◽

Cellular Responses ◽

Fine Tuning ◽

Inflammatory Processes ◽

Intracellular Second Messengers

Abstract Adenine nucleotides (AdNs) play important roles in immunity and inflammation. Extracellular AdNs, such as adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD) and their metabolites, act as paracrine messengers by fine-tuning both pro- and anti-inflammatory processes. Moreover, intracellular AdNs derived from ATP or NAD play important roles in many cells of the immune system, including T lymphocytes, macrophages, neutrophils and others. These intracellular AdNs are signaling molecules that transduce incoming signals into meaningful cellular responses, e.g. activation of immune responses against pathogens.

On the Role of Menaquinone in the Reduced Nicotinamide Adenine Dinucleotide Oxidative Pathway of Bacillus brevis

Journal of General Microbiology ◽

10.1099/00221287-70-2-271 ◽

1972 ◽

Vol 70 (2) ◽

pp. 271-275

Author(s):

G. H. FYNN ◽

D. V. THOMAS ◽

B. SEDDON

Keyword(s):

Reduced Nicotinamide Adenine Dinucleotide

Nicotinamide Adenine ◽

Bacillus Brevis ◽

Oxidative Pathway ◽

Population Size Drives Industrial Saccharomyces cerevisiae Alcoholic Fermentation and Is under Genetic Control

Applied and Environmental Microbiology ◽

10.1128/aem.02547-10 ◽

2011 ◽

Vol 77 (8) ◽

pp. 2772-2784 ◽

Cited By ~ 33

Author(s):

Warren Albertin ◽

Philippe Marullo ◽

Michel Aigle ◽

Christine Dillmann ◽

Dominique de Vienne ◽

...

Keyword(s):

Population Size ◽

Production Rate ◽

Food Processing ◽

Alcoholic Fermentation ◽

Metabolic Efficiency ◽

Improvement Program ◽

Wine Production ◽

Fermentation Products ◽

Human Selection

ABSTRACTAlcoholic fermentation (AF) conducted bySaccharomyces cerevisiaehas been exploited for millennia in three important human food processes: beer and wine production and bread leavening. Most of the efforts to understand and improve AF have been made separately for each process, with strains that are supposedly well adapted. In this work, we propose a first comparison of yeast AFs in three synthetic media mimicking the dough/wort/grape must found in baking, brewing, and wine making. The fermentative behaviors of nine food-processing strains were evaluated in these media, at the cellular, populational, and biotechnological levels. A large variation in the measured traits was observed, with medium effects usually being greater than the strain effects. The results suggest that human selection targeted the ability to complete fermentation for wine strains and trehalose content for beer strains. Apart from these features, the food origin of the strains did not significantly affect AF, suggesting that an improvement program for a specific food processing industry could exploit the variability of strains used in other industries. Glucose utilization was analyzed, revealing plastic but also genetic variation in fermentation products and indicating that artificial selection could be used to modify the production of glycerol, acetate, etc. The major result was that the overall maximum CO2production rate (Vmax) was not related to the maximum CO2production rate per cell. Instead, a highly significant correlation betweenVmaxand the maximum population size was observed in all three media, indicating that human selection targeted the efficiency of cellular reproduction rather than metabolic efficiency. This result opens the way to new strategies for yeast improvement.