scholarly journals Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

2020 ◽  
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activities ◽  
Consolidated Bioprocessing ◽  
Synergistic Action ◽  
Cellulosic Biomass ◽  
Gene Copy ◽  
Cellulolytic Enzymes ◽  
Plasmid Library ◽  
Substrate Hydrolysis

Abstract Background: For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results: In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-β-D-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2 respectively (ratio of 1.6:1). Conclusions: Here we describe a synthetic biology approach to rapidly optimize gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

scholarly journals Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activities ◽  
Consolidated Bioprocessing ◽  
Synergistic Action ◽  
Cellulosic Biomass ◽  
Gene Copy ◽  
Cellulolytic Enzymes ◽  
Plasmid Library ◽  
Substrate Hydrolysis

Abstract Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-d-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Conclusions Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

scholarly journals Rapid Optimisation of Cellulolytic Enzymes Ratios in Saccharomyces Cerevisiae using in Vitro SCRaMbLE

2020 ◽  
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activities ◽  
Consolidated Bioprocessing ◽  
Synergistic Action ◽  
Cellulosic Biomass ◽  
Gene Copy ◽  
Cellulolytic Enzymes ◽  
Plasmid Library ◽  
Substrate Hydrolysis

Abstract Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes; with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene ( CEL3A ) from Saccharomycopsis fibuligera and an endoglucanase gene ( CEL5A ) from Trichoderma reesei . S. cerevisiae , transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that BPNPG5 (Megazyme®) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2 respectively (ratio of 1.6:1). Conclusions Here we describe a synthetic biology approach to rapidly optimize gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

scholarly journals Microbial Cellulose Utilization: Fundamentals and Biotechnology

2002 ◽  
Vol 66 (3) ◽  
pp. 506-577 ◽  
Author(s):  
Lee R. Lynd ◽  
Paul J. Weimer ◽  
Willem H. van Zyl ◽  
Isak S. Pretorius
Keyword(s):  
Consolidated Bioprocessing ◽  
Quantitative Description ◽  
Product Yield ◽  
Cellulose Hydrolysis ◽  
Taxonomic Diversity ◽  
Cellulosic Biomass ◽  
Limiting Factors ◽  
Cellulolytic Enzymes ◽  
Cellulase Enzymes ◽  
Microbial Cellulose

SUMMARY Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts.

scholarly journals Disruption of the Snf1 Gene Enhances Cell Growth and Reduces the Metabolic Burden in Cellulase-Expressing and Lipid-Accumulating Yarrowia lipolytica

2021 ◽  
Vol 12 ◽  
Author(s):  
Hui Wei ◽  
Wei Wang ◽  
Eric P. Knoshaug ◽  
Xiaowen Chen ◽  
Stefanie Van Wychen ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Growth ◽  
Yarrowia Lipolytica ◽  
Lipid Accumulation ◽  
Consolidated Bioprocessing ◽  
Cellulosic Biomass ◽  
Host Cells ◽  
Selection Marker ◽  
Cellulolytic Enzymes ◽  
Cellulolytic Activity

Yarrowia lipolytica is known to be capable of metabolizing glucose and accumulating lipids intracellularly; however, it lacks the cellulolytic enzymes needed to break down cellulosic biomass directly. To develop Y. lipolytica as a consolidated bioprocessing (CBP) microorganism, we previously expressed the heterologous CBH I, CBH II, and EG II cellulase enzymes both individually and collectively in this microorganism. We concluded that the coexpression of these cellulases resulted in a metabolic drain on the host cells leading to reduced cell growth and lipid accumulation. The current study aims to build a new cellulase coexpressing platform to overcome these hinderances by (1) knocking out the sucrose non-fermenting 1 (Snf1) gene that represses the energetically expensive lipid and protein biosynthesis processes, and (2) knocking in the cellulase cassette fused with the recyclable selection marker URA3 gene in the background of a lipid-accumulating Y. lipolytica strain overexpressing ATP citrate lyase (ACL) and diacylglycerol acyltransferase 1 (DGA1) genes. We have achieved a homologous recombination insertion rate of 58% for integrating the cellulases-URA3 construct at the disrupted Snf1 site in the genome of host cells. Importantly, we observed that the disruption of the Snf1 gene promoted cell growth and lipid accumulation and lowered the cellular saturated fatty acid level and the saturated to unsaturated fatty acid ratio significantly in the transformant YL163t that coexpresses cellulases. The result suggests a lower endoplasmic reticulum stress in YL163t, in comparison with its parent strain Po1g ACL-DGA1. Furthermore, transformant YL163t increased in vitro cellulolytic activity by 30%, whereas the “total in vivo newly formed FAME (fatty acid methyl esters)” increased by 16% in comparison with a random integrative cellulase-expressing Y. lipolytica mutant in the same YNB-Avicel medium. The Snf1 disruption platform demonstrated in this study provides a potent tool for the further development of Y. lipolytica as a robust host for the expression of cellulases and other commercially important proteins.

Ca-, Mg- és K-sók hatása egyes nehézfémek Saccharomyces cerevisiae törzsekre gyakorolt toxicitására

2008 ◽  
Vol 57 (1) ◽  
pp. 161-175
Author(s):  
Nikoletta Tóth ◽  
Hamuda Hosam E. A. F. Bayoumi ◽  
Attila Palágyi ◽  
Mihály Kecskés
Keyword(s):  
Saccharomyces Cerevisiae

Az utóbbi években egyre több tanulmány született a mikroorganizmusok nehézfém akkumulációjáról. A mikroszervezetek nehézfémekkel szembeni tűrőképességére és nehézfém felvételére a bioremediációs hasznosíthatóságuk miatt egyre nagyobb figyelmet fordítanak. A mikroorganizmusok tulajdonságai nagyon jól hasznosíthatóak a talajszennyezés monitorozásánál. A toxikus nehézfémek komoly ökológiai problémát jelentenek környezetünkben, ezért kiemelkedő fontosságú a nehézfémekkel szennyezett talajok tisztítása. In vitro , két S. cerevisiae törzs (NSS5099 és NSS7002) nehézfémekkel szembeni toleranciáját vizsgáltuk. A két törzs szaporodási kinetikáját olyan táptalajon tanulmányoztuk, amelyhez 50 µM koncentrációban adtunk Cu 2+ -, Pb 2+ -, Cd 2+ - vagy Ni 2+ -ionokat. A vizsgált nehézfémek élesztőtörzsekre gyakorolt toxicitása csökkenő sorrendben: Cu 2+ > Pb 2+ > Cd 2+ > Ni 2+ . A 350 µM koncentrációjú Cu 2+ , Pb 2+ vagy Cd 2+ és 450 µM koncentrációjú Ni 2+ 48 órás inkubációt követően 50%-kal csökkentette az élősejtek számát. Amikor a nehézfémek táptalajba történő adagolása előtt 50 mM Ca(HCO 3 ) 2 , 75 mM MgSO 4 , vagy 150 mM K 2 SO 4 -ot adtunk a közeghez csökkent a nehézfémek sejtekre gyakorolt toxicitása, és több sejt maradt életben. A 350 és 450 µM koncentrációban lévő nehézfémek toxicitását a fémsók 40%-kal csökkentették. A kapott eredmények alapján az NSS7002 törzs sokkal alkalmasabbnak bizonyult a nehézfémekkel szennyezett talajok tisztítására, mint az NSS5099._

In vitro Inhibition of Pancreatic Enzyme Activities by Dietary Fiber

Digestion ◽  
1982 ◽  
Vol 24 (1) ◽  
pp. 54-59 ◽  
Author(s):  
G. Isaksson ◽  
I. Lundquist ◽  
I. Ihse
Keyword(s):  
Dietary Fiber ◽  
Enzyme Activities ◽  
Pancreatic Enzyme ◽  
In Vitro Inhibition

scholarly journals Complete and efficient conversion of plant cell wall hemicellulose into high-value bioproducts by engineered yeast

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Liang Sun ◽  
Jae Won Lee ◽  
Sangdo Yook ◽  
Stephan Lane ◽  
Ziqiao Sun ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cellulosic Biomass ◽  
Hemicellulose Hydrolysate ◽  
Acetyl Coa ◽  
Engineered Yeast ◽  
Fermentation Inhibitor ◽  
Acid Lactone

AbstractPlant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.

scholarly journals Identification of a Mutant Locus by Noncomplementation of a Transposon Insertion Library in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1825-1827 ◽  
Author(s):  
Heather A Wiatrowski ◽  
Marian Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
New Approach ◽  
Transposon Insertion ◽  
Plasmid Library ◽  
Mutant Locus

Abstract We describe a new approach for identifying the gene corresponding to a mutation in Saccharomyces cerevisiae. A library of mTn-lacZ/LEU2 insertions is tested for failure to complement the mutation, and the noncomplementing insertion is used to obtain sequence. This approach offers an alternative to cloning by complementation with a plasmid library.

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