scholarly journals Consolidated Bioprocessing: Synthetic Biology Routes to Fuels and Fine Chemicals

2021 ◽  
Vol 9 (5) ◽  
pp. 1079
Author(s):  
Alec Banner ◽  
Helen S. Toogood ◽  
Nigel S. Scrutton
Keyword(s):  
Enzymatic Degradation ◽  
Consolidated Bioprocessing ◽  
Carbon Sources ◽  
Cellulolytic Enzymes ◽  
Waste Biomass ◽  
Chemical Production ◽  
Iterative Design ◽  
Biomass Feedstocks ◽  
Advanced Fuels ◽  
Design Build

The long road from emerging biotechnologies to commercial “green” biosynthetic routes for chemical production relies in part on efficient microbial use of sustainable and renewable waste biomass feedstocks. One solution is to apply the consolidated bioprocessing approach, whereby microorganisms convert lignocellulose waste into advanced fuels and other chemicals. As lignocellulose is a highly complex network of polymers, enzymatic degradation or “saccharification” requires a range of cellulolytic enzymes acting synergistically to release the abundant sugars contained within. Complications arise from the need for extracellular localisation of cellulolytic enzymes, whether they be free or cell-associated. This review highlights the current progress in the consolidated bioprocessing approach, whereby microbial chassis are engineered to grow on lignocellulose as sole carbon sources whilst generating commercially useful chemicals. Future perspectives in the emerging biofoundry approach with bacterial hosts are discussed, where solutions to existing bottlenecks could potentially be overcome though the application of high throughput and iterative Design-Build-Test-Learn methodologies. These rapid automated pathway building infrastructures could be adapted for addressing the challenges of increasing cellulolytic capabilities of microorganisms to commercially viable levels.

Pretreatment of Lignocellulosic Biomass Feedstocks for Biofuel Production

2018 ◽  
pp. 33-60 ◽  
Author(s):  
Desikan Ramesh ◽  
Iniya Kumar Muniraj ◽  
Kiruthika Thangavelu ◽  
Subburamu Karthikeyan
Keyword(s):  
Lignocellulosic Biomass ◽  
Biofuel Production ◽  
Renewable Fuels ◽  
Chemical Production ◽  
Biomass Feedstocks ◽  
Platform Chemical ◽  
Global Concern ◽  
Liquid Biofuel ◽  
Lignocellulosic Pretreatment

The shifting of dependence from conventional fuels to renewable fuels and its increased production to combat the energy, environmental, and geopolitical crises is a global concern. One of the viable and promising alternatives is liquid biofuel production using lignocellulosic biomass. Lignocellulosic biomass being the most abundant encompass cellulose, hemicellulose, and lignin.The intricate complex of hemicellulose and lignin around cellulose is the bottleneck in commercializing the biofuel process. To make the cellulose and hemicellulose more accessible for hydrolysis and valorise the underutilized lignin for platform chemical production, pretreatment becomes imperative. Various pretreatment methods such as physical, mechanical, chemical, biological, and enzymatic and their combinations are employed for the production of bioethanol. It should be stressed that each pretreatment is unique in its condition and in most cases are biomass specific. With the above view, this chapter aims at bringing out the understanding of lignocellulosic pretreatment with updated information in the field.

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 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 High throughput gene expression profiling of yeast colonies with microgel-culture Drop-seq

2018 ◽  
Author(s):  
Leqian Liu ◽  
Chiraj Dalal ◽  
Ben Heineike ◽  
Adam Abate
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Transcriptional Profiling ◽  
Cost Effective ◽  
Chemical Production ◽  
Efficient Test ◽  
Large Numbers ◽  
Design Build ◽  
Gene Expression Heterogeneity ◽  
High Throughput Gene Expression

AbstractYeasts can be engineered into “living foundries” for non-natural chemical production by reprogramming their genome using a synthetic biology “design-build-test” cycle. While methods for “design” and “build” are scalable and efficient, “test” remains a labor-intensive bottleneck, limiting the effectiveness of the genetic reprogramming results. Here we describe Isogenic Colony Sequencing (ICO-seq), a massively-parallel strategy to assess the gene expression, and thus engineered pathway efficacy, of large numbers of genetically distinct yeast colonies. We use the approach to characterize opaque-white switching in 658 C. albicans colonies. By profiling transcriptomes of 1642 engineered S. cerevisiae strains, we use it to assess gene expression heterogeneity in a protein mutagenesis library. Our approach will accelerate synthetic biology by allowing facile and cost-effective transcriptional profiling of large numbers of genetically distinct yeast strains.

Sustainable biofuel production from forestry, agricultural and waste biomass feedstocks

2017 ◽  
Vol 198 ◽  
pp. 281-283 ◽  
Author(s):  
Joann Whalen ◽  
Charles (Chunbao) Xu ◽  
Fei Shen ◽  
Amit Kumar ◽  
Mats Eklund ◽  
...  
Keyword(s):  
Biofuel Production ◽  
Waste Biomass ◽  
Biomass Feedstocks

Production of Cellulolytic Enzymes by Fungi Acrophialophora nainiana and Ceratocystis paradoxa Using Different Carbon Sources

2010 ◽  
Vol 161 (1-8) ◽  
pp. 448-454 ◽  
Author(s):  
Rodrigo R. O. Barros ◽  
Raul A. Oliveira ◽  
Leda Maria F. Gottschalk ◽  
Elba P. S. Bon
Keyword(s):  
Carbon Sources ◽  
Cellulolytic Enzymes ◽  
Ceratocystis Paradoxa ◽  
Acrophialophora Nainiana

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.

scholarly journals Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides

2017 ◽  
Author(s):  
Samuel T Coradetti ◽  
Dominic Pinel ◽  
Gina Geiselman ◽  
Masakazu Ito ◽  
Stephen Mondo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Lipid Metabolism ◽  
Functional Genomics ◽  
Lipid Accumulation ◽  
Protein Modification ◽  
Carbon Sources ◽  
Acid Synthesis ◽  
Rhodosporidium Toruloides ◽  
Chemical Production ◽  
Amino Acid Synthesis

AbstractThe basidomycete yeast Rhodosporidium toruloides (a.k.a. Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions into the R. toruloides genome. We identified 1337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We found that both mitochondrial and peroxisomal enzymes were required for growth on fatty acids, with different peroxisomal enzymes required on different fatty acids. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, as well as genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species, identify key biological processes to be further explored and optimized for production of lipid-based bioproducts, and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

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.

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