Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears ...to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.
Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.
Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.
Critical features of surface morphology for LPMO synergy with cellulases are recognized.
Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.
Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid‐like C‐glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin ...based on a one‐pot coupled glycosyltransferase‐catalyzed transformation that involves perfectly selective 3′‐C‐β‐D‐glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. C‐Glucosyltransferase from Oryza sativa (rice) was used for phloretin C‐glucosylation from uridine 5′‐diphosphate (UDP)‐glucose, which was supplied continuously in situ through conversion of sucrose and UDP catalyzed by sucrose synthase from Glycine max (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 g L−1) were obtained that vastly exceed the phloretin solubility limit (5–10 mM). The intermediate UDP‐glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase‐catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (μM→mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals.
Soluble cellodextrins (linear β‐1,4‐d‐gluco‐oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom‐up synthesis from glucose is promising for bulk ...production, but to ensure a completely water‐soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α‐d‐glucose 1‐phosphate (αGlc1‐P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1‐P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg2+. Second, the product DP was controlled by the molar ratio of glucose/αGlc1‐P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1‐P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1–10 mM; 200 mM αGlc1‐P), the reaction was shifted completely towards insoluble product formation (DP ∼9–10). In summary, this study provides the basis for an efficient and product DP‐controlled biocatalytic synthesis of cellodextrins from expedient substrates.
Biocatalytic production of cellodextrins (linear β‐1,4‐d‐gluco‐oligosaccharides) from glucose using coupled cellobiose and cellodextrin phosphorylase is described. Kinetic and thermodynamic restrictions upon α‐glucose 1‐phosphate (αGlc1‐P) utilization were overcome by in situ removal of the phosphate via precipitation with Mg
2+. By varying molar ratio of glucose/αGlc1‐P, the product solubility (or degree of polymerization, DP) can be efficiently controlled. Following these strategies, the authors have achieved desired production of soluble (DP ≤ 6) or insoluble (DP 9‐10) cellodextrins.
Advanced biotransformation processes typically involve the upstream processing part performed continuously and interlinked tightly with the product isolation. Key in their development is a catalyst ...that is highly active, operationally robust, conveniently produced, and recyclable. A promising strategy to obtain such catalyst is to encapsulate enzymes as permeabilized whole cells in porous polymer materials. Here, we show immobilization of the sucrose phosphorylase from
Bifidobacterium adolescentis
(P134Q-variant) by encapsulating the corresponding
E. coli
cells into polyacrylamide. Applying the solid catalyst, we demonstrate continuous production of the commercial extremolyte 2-α-
d
-glucosyl-glycerol (2-GG) from sucrose and glycerol. The solid catalyst exhibited similar activity (≥70%) as the cell-free extract (~800 U g
−1
cell wet weight) and showed excellent in-operando stability (40 °C) over 6 weeks in a packed-bed reactor. Systematic study of immobilization parameters related to catalyst activity led to the identification of cell loading and catalyst particle size as important factors of process optimization. Using glycerol in excess (1.8 M), we analyzed sucrose conversion dependent on space velocity (0.075–0.750 h
−1
) and revealed conditions for full conversion of up to 900 mM sucrose. The maximum 2-GG space-time yield reached was 45 g L
−1
h
−1
for a product concentration of 120 g L
−1
. Collectively, our study establishes a step-economic route towards a practical whole cell-derived solid catalyst of sucrose phosphorylase, enabling continuous production of glucosides from sucrose. This strengthens the current biomanufacturing of 2-GG, but also has significant replication potential for other sucrose-derived glucosides, promoting their industrial scale production using sucrose phosphorylase.
Key points
•
Cells of sucrose phosphorylase fixed in polyacrylamide were highly active and stable.
•
Solid catalyst was integrated with continuous flow to reach high process efficiency.
•
Generic process technology to efficiently produce glucosides from sucrose is shown.
Graphical abstract
Access to chiral alcohols of high optical purity is today frequently provided by the enzymatic reduction of precursor ketones. However, bioreductions are complicated by the need for reducing ...equivalents in the form of NAD(P)H. The high price and molecular weight of NAD(P)H necessitate in situ recycling of catalytic quantities, which is mostly accomplished by enzymatic oxidation of a cheap co-substrate. The coupled oxidoreduction can be either performed by free enzymes in solution or by whole cells. Reductase selection, the decision between cell-free and whole cell reduction system, coenzyme recycling mode and reaction conditions represent design options that strongly affect bioreduction efficiency. In this paper, each option was critically scrutinized and decision rules formulated based on well-described literature examples. The development chain was visualized as a decision-tree that can be used to identify the most promising route towards the production of a specific chiral alcohol. General methods, applications and bottlenecks in the set-up are presented and key experiments required to “test” for decision-making attributes are defined. The reduction of o-chloroacetophenone to (S)-1-(2-chlorophenyl)ethanol was used as one example to demonstrate all the development steps. Detailed analysis of reported large scale bioreductions identified product isolation as a major bottleneck in process design.
In the biosynthesis of the tRNA-inserted nucleoside queuosine, the nitrile reductase QueF catalyzes conversion of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), a ...biologically unique four-electron reduction of a nitrile to an amine. The QueF mechanism involves a covalent thioimide adduct between the enzyme and preQ0 that undergoes reduction to preQ1 in two NADPH-dependent steps, presumably via an imine intermediate. Protecting a labile imine from interception by water is fundamental to QueF catalysis for proper enzyme function. In the QueF from Escherichia coli, the conserved Glu89 and Phe228 residues together with a mobile structural element composing the catalytic Cys190 form a substrate-binding pocket that secludes the bound preQ0 completely from solvent. We show here that residue substitutions (E89A, E89L, and F228A) targeted at opening up the binding pocket weakened preQ0 binding at the preadduct stage by up to +10 kJ/mol and profoundly affected catalysis. Unlike wildtype enzyme, the QueF variants, including L191A and I192A, were no longer selective for preQ1 formation. The E89A, E89L, and F228A variants performed primarily (≥90%) a two-electron reduction of preQ0, releasing hydrolyzed imine (7-formyl-7-deazaguanine) as the product. The preQ0 reduction by L191A and I192A gave preQ1 and 7-formyl-7-deazaguanine at a 4:1 and 1:1 ratio, respectively. The proportion of 7-formyl-7-deazaguanine in total product increased with increasing substrate concentration, suggesting a role for preQ0 in a competitor-induced release of the imine intermediate. Collectively, these results provide direct evidence for the intermediacy of an imine in the QueF-catalyzed reaction. They reveal determinants of QueF structure required for imine sequestration and hence for a complete nitrile-to-amine conversion by this class of enzymes.
Saccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal ...engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (Y
) and productivities (Q
) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on Y
and Q
in IBB14LA1 and IBB14LA1_5 was investigated.
In anaerobic fermentation IBB14LA1 showed a higher Y
on xylose (0.27 g g
) than on glucose (0.18 g g
). The ethanol yields (Y
, 0.15 g g
and 0.32 g g
) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on Y
was less pronounced (~ 0.80 g g
, and 0.67 g g
). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (Q
from 0.38 to 0.81 g L
h
) and IBB14LA1_5 (Q
from 0.05 to 1.77 g L
h
) at constant Y
(IBB14LA1 ~ 0.18 g g
; IBB14LA1_5 ~ 0.68 g g
). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (Y
aerobic ≤ 0.25 g g
and anaerobic ~ 0.80 g g
) at similar Q
(~ 0.04 g L
h
). Switching from aerobic to microaerophilic conditions (pO
~ 2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased Q
and Y
up to 0.11 g L
h
and 0.38 g g
, respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen.
Evidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.
Soluble proteins are often highly unstable under mixing conditions that involve dynamic contacting between the main liquid phase and a gas phase. The recombinant human growth hormone (rhGH) was ...recently shown to undergo aggregation into micrometer-sized solid particles composed of non-native (mis- or unfolded) protein, once its solutions were stirred or shaken to generate a continuously renewed air–water interface. To gain deepened understanding and improved quantification of the air–water interface effect on rhGH stability, we analyzed the protein’s aggregation rate (r agg) at controlled specific air–water surface areas (a G/L) established by stirring or bubble aeration. We show that in spite of comparable time-averaged values for a G/L (≈ 100 m2/m3), aeration gave a 40-fold higher r agg than stirring. The enhanced r agg under aeration was ascribed to faster macroscopic regeneration of free a G/L during aeration as compared to stirring. We also show that r agg was independent of the rhGH concentration in the range 0.67 – 6.7 mg/mL, and that it increased linearly dependent on the available a G/L. The nonionic surfactant Pluronic F-68, added in 1.6-fold molar excess over rhGH present, resulted in complete suppression of r agg. Foam formation was not a factor influencing r agg. Using analysis by circular dichroism spectroscopy and small-angle X-ray scattering, we show that in the presence of Pluronic F-68 under both stirring and aeration, the soluble protein retained its original fold, featuring native-like relative composition of secondary structural elements. We further provide evidence that the efficacy of Pluronic F-68 resulted from direct, probably hydrophobic protein–surfactant interactions that prevented rhGH from becoming attached to the air–water interface. Surface-induced aggregation of rhGH is suggested to involve desorption of non-native protein from the air–water interface as the key limiting step. Proteins or protein aggregates released back into the bulk liquid appear to be essentially insoluble.
Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and ...xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.
An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 +/- 0.02 h-1) in both strains.
Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.
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