Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae

Li, Xin; Chomvong, Kulika; Yu, Vivian Yaci; Liang, Julie M; Lin, Yuping; Cate, J.H.D.

doi:10.1186/s13068-015-0303-2

Cited by 29 publications

(29 citation statements)

References 33 publications

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“…Much like glucono-1,5-lactone, in the presence of water cellobiono-1,5-lactone might be hydrolyzed spontaneously to cellobionic acid, which would be transported to the cytoplasm. Intracellular β-glucosidase could then cleave cellobionate to glucose and gluconic acid (Li et al, 2015), two molecules easily metabolized by P. putida . Nonetheless, neither cellobionic acid formation nor its further metabolism in P. putida can be confirmed based on currently available experimental data.…”

Section: Resultsmentioning

confidence: 99%

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Dvořák

Lorenzo

2018

Preprint

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6Running title: Engineering cellobiose and xylose metabolism in P. putida 7 Abstract 1 Given its capacity to tolerate stress, NAD(P)H/ NAD(P) balance, and increased ATP levels, 2 the platform strain Pseudomonas putida EM42, a genome-edited derivative of the soil 3 bacterium P. putida KT2440, can efficiently host a suite of harsh reactions of biotechnological 4 interest. Because of the lifestyle of the original isolate, however, the nutritional repertoire of P. 5 putida EM42 is centered largely on organic acids, aromatic compounds and some hexoses 6 (glucose and fructose). To enlarge the biochemical network of P. putida EM42 to include 7 disaccharides and pentoses, we implanted heterologous genetic modules for D-cellobiose and 8 D-xylose metabolism into the enzymatic complement of this strain. Cellobiose was actively 9 transported into the cells through the ABC complex formed by native proteins PP1015-PP1018. 10The knocked-in -glucosidase BglC from Thermobifida fusca catalyzed intracellular cleavage 11 of the disaccharide to D-glucose, which was then channelled to the default central metabolism. 12Xylose oxidation to the dead end product D-xylonate was prevented by by deleting the gcd 13 gene that encodes the broad substrate range quinone-dependent glucose dehydrogenase. 14 Intracellular intake was then engineered by expressing the Escherichia coli proton-coupled 15 symporter XylE. The sugar was further metabolized by the products of E. coli xylA (xylose 16 isomerase) and xylB (xylulokinase) towards the pentose phosphate pathway. The resulting P. 17 putida strain co-utilized xylose with glucose or cellobiose to complete depletion of the sugars. 18 These results not only show the broadening of the metabolic capacity of a soil bacterium 19 towards new substrates, but also promote P. putida EM42 as a platform for plug-in of new 20 biochemical pathways for utilization and valorization of carbohydrate mixtures from 21 lignocellulose processing.22 23 24recruitment of one heterologous -glucosidase for intracellular cellobiose hydrolysis, and the 1 implementation of three enterobacterial genes (xylose transporter, isomerase and kinase) 2 sufficed to cause disaccharide and the pentose co-utilization by P. putida EM42 with 3 inactivated glucose dehydrogenase while maintaining its ability to use glucose. We also 4 demonstrate that P. putida metabolism generates more ATP when cells are grown on cellobiose 5 instead of glucose. This study expands the catalytic scope of P. putida towards utilization of 6 major components of all three lignocellulose-derived fractions. Moreover, given that the 7 cellobiose, as the bulk by-product of standard cellulose saccharification, frequently remains 8 untouched in the sugar mix (due to the inability of most microorganisms to assimilate it), our 9 results demonstrate rational tailoring of an industrially relevant microbial host to achieve a 10 specific step in such a biotechnological value chain. 11 12 13 14 6 1 Figure 1. Engineering Pseudomonas putida EM42 for co-utilization of D-cellobios...

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Section: Resultsmentioning

confidence: 99%

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Dvořák

Lorenzo

2018

Preprint

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“…Alternatively, E. coli can directly consume cellobionate as the carbon source for isobutanol production; Saccharomyces cerevisiae has been engineered to use cellobionate for ethanol production [21,35]. Furthermore, equilibrated solutions of cellobionate containing cellobiono-δlactone have significant inhibition of CDH, highlighting the importance of lactonases in whole cellulolytic systems [11,18].…”

Section: Discussionmentioning

confidence: 99%

“…The engineered N. crassa strain with multiple copies of BGLs deleted contains all of the cellulase enzymes necessary for cellulose hydrolysis to cellobiose, with CDH oxidization of cellobiose to cellobionate [19,20]. Studying the inhibition of cellulases and CDH by cellobionate is useful for optimizing the system [21]. While cellobiose inhibition has been extensively investigated, cellobionate inhibition has been minimally studied.…”

Section: Introductionmentioning

confidence: 99%

Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase

Hildebrand

Addison

Kasuga

et al. 2016

Biochemical Engineering Journal

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End-product inhibition by cellobiose and glucose is a rate-limiting factor in cellulose hydrolysis by cellulases. While cellobiose and glucose inhibition have been extensively investigated, cellobionate inhibition has been minimally studied despite the discovery that accessory proteins such as cellobiose dehydrogenase (CDH) work synergistically with cellulases to form aldonic acids. The fraction of equilibrated cellobionate in the cellobiono-δ-lactone form was determined by NMR over the range of pH 4 to 8. To better improve the understanding of cellobiono-δlactone and cellobionate inhibition of cellulases, we investigate the inhibition of Trichoderma reesei cellobiohydrolase I (CBHI), one of the most extensively studied CBHs, and Neurospora

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“…Recently, copper-dependent lytic polysaccharide monooxygenases (LPMOs) were found to be able to catalyze the oxidative cleavage of cellulose, generating oxidized cellodextrins in concert with cellobiose dehydrogenases [2]. The addition of LPMOs to cellulase enzyme cocktails to enhance cellulose degradation generates cellobionic acid and gluconic acid in addition to pentose and hexose sugars in the hydrolysate [3]. Our lab proposed an alternative route for biofuel and chemical production from cellulosic biomass in which cellobionate instead of monomeric sugars is produced as the reactive intermediate for subsequent fermentation [4].…”

Section: Introductionmentioning

confidence: 99%

Conversion of Glucose and Gluconate to Ethanol in Mineral Salts Medium using Recombinant Escherichia coli Strains

Zhang¹,

Sun²,

Lin³

et al. 2016

Ferment Technol

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Escherichia coli AH003, a derivative of E. coli KO11 with the L-lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) genes deleted and its parent strain E. coli KO11 were used as the ethanologen to convert glucose and gluconate to ethanol in M9 minimal medium. E. coli AH003 grew very poorly on glucose in M9 medium. However it achieved rapid growth when gluconate was used as the carbon source. The addition of gluconate to medium containing glucose improved the rate of glucose utilization. In contrast, E. coli KO11 grew well on both glucose and gluconate in M9 medium. The addition of gluconate to medium containing glucose did not improve the rate of glucose utilization. We believe that the deletion of the pfl gene in E. coli AH003 led to the different fermentation results. The co-fermentation of gluconate and glucose could be a useful strategy to improve the rate of glucose fermentation and decrease nutrient requirements for engineered strains lacking the pfl gene and grown under anaerobic conditions.

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Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae

Cited by 29 publications

References 33 publications

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Refactoring the upper sugar metabolism ofPseudomonas putidafor co-utilization of disaccharides, pentoses, and hexoses

Cellobionic acid inhibition of cellobiohydrolase I and cellobiose dehydrogenase

Conversion of Glucose and Gluconate to Ethanol in Mineral Salts Medium using Recombinant Escherichia coli Strains

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