Utilization of Monosaccharides by Hungateiclostridium thermocellum ATCC 27405 through Adaptive Evolution

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

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Utilization of Monosaccharides by Hungateiclostridium thermocellum ATCC 27405 Through Adaptive Evolution

10.20944/preprints202012.0496.v1 ◽

2020 ◽

Author(s):

Dung Minh Ha-Tran ◽

Trinh Thi My Nguyen ◽

Shou-Chen Lo ◽

Chieh-Chen Huang

Keyword(s):

Sugar Transport ◽

Central Carbon Metabolism ◽

Transport Systems ◽

Crystalline Cellulose ◽

Rna Seq ◽

Genome Resequencing ◽

Adaptive Laboratory Evolution ◽

Poor Growth ◽

Genomic Screening ◽

Central Carbon

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium with a robust ability to degrade lignocellulosic biomass complexes, including crystalline cellulose components, through a multienzyme cellulosomal system. In contrast, it exhibits poor growth on simple monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on monosaccharides have been poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium on monosaccharides, and genome resequencing was used to detect the genes that had mutated during adaptation. RNA-seq data of the 1st-generation culture growing on either fructose or glucose revealed that several glycolytic genes in the EMP pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After 8 generations of culture on fructose and glucose, the evolved H. thermocellum strains grew faster and yielded greater biomass than the nonadapted strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in genes responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as targets for further metabolic engineering to improve this bacterium for bioindustrial usage.

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Laboratory evolution and reverse engineering of Clostridium thermocellum for growth on glucose and fructose

Applied and Environmental Microbiology ◽

10.1128/aem.03017-20 ◽

2021 ◽

Author(s):

Johannes Yayo ◽

Teun Kuil ◽

Daniel G. Olson ◽

Lee R. Lynd ◽

Evert K. Holwerda ◽

...

Keyword(s):

Clostridium Thermocellum ◽

Molecular Mechanisms ◽

Consolidated Bioprocessing ◽

Biofuel Production ◽

Transport Systems ◽

Good Growth ◽

Wild Type ◽

Laboratory Evolution ◽

Hexose Sugars

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate−1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate−1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, whilst wild type showed 42 ± 4 hours on glucose and >80 hours on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 hours with a growth rate of 0.11 ± 0.01 h−1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h−1. Additional introduction of a cbpAG148V mutation resulted in immediate growth on fructose at 0.32 ± 0.03 h−1. These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. Importance. C. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to i) reduce glucose accumulation, ii) study cellodextrin transport systems in vivo, iii) allow experiments at >120 g L−1 soluble substrate concentration, or iv) reduce costs for labelling studies.

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Sugar Transport Systems of Baker’s Yeast and Filamentous Fungi

Carbohydrate Metabolism in Cultured Cells ◽

10.1007/978-1-4684-7679-8_7 ◽

1986 ◽

pp. 225-244 ◽

Cited By ~ 6

Author(s):

Antonio H. Romano

Keyword(s):

Filamentous Fungi ◽

Sugar Transport ◽

Baker’S Yeast ◽

Transport Systems ◽

Baker's Yeast

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The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

Journal of Bacteriology ◽

10.1128/jb.181.12.3695-3704.1999 ◽

1999 ◽

Vol 181 (12) ◽

pp. 3695-3704 ◽

Cited By ~ 106

Author(s):

Smadar Shulami ◽

Orit Gat ◽

Abraham L. Sonenshein ◽

Yuval Shoham

Keyword(s):

Gene Cluster ◽

Transport System ◽

Bacillus Stearothermophilus ◽

Glucuronic Acid ◽

Genomic Library ◽

Regulatory Gene ◽

Sugar Transport ◽

Transcriptional Unit ◽

Transport Systems ◽

Potential Operator

ABSTRACT A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK,kdgA, uxaC, uxuA, anduxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.

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