One-Pot <i>De Novo</i> Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions

Fe–S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe–S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis

One-Pot <i>De Novo</i> Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions

Fe–S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe–S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis

Microalgal Polyphosphate Drives One-Pot Complete Enzymatic Generation of Flavin Adenine Dinucleotide from Adenosine and Riboflavin

Flavin adenine dinucleotide (FAD) is a universal cellular cofactor involved in biological redox and radical metabolism reactions. FAD biosynthesis from riboflavin typically proceeds through two ATP-dependent enzymatic reactions, with flavin mononucleotide (FMN) as the intermediate. Traditional in vivo methods employ microorganisms for FAD synthesis at an industrial scale; however, these approaches often suffer from complex purification processes. Considering the atomic economy and percentage yield, in vitro enzymatic FAD synthesis using enzymes could be a more efficient and sustainable alternative. While catalytically efficient, the requirements of expensive ATP (substrate) limit the industrialization of enzymatic FAD synthesis. To overcome the ATP requirements, here we develop a two-enzyme cascade for ATP regeneration from adenosine using wastewater microalgal polyphosphate as the P-donor. With the ATP regeneration system, the bifunctional riboflavin kinase/FAD synthetase and pyrophosphatase completely convert saturated riboflavin into FAD within 2 h with a titer of ∼1.2 g/L (1.5 mmol/L). Notably, orthophosphate, the only byproduct of this enzymatic process, can be recycled to synthesize polyphosphate by wastewater microalgae, which can then be fed back into the system as the P-donor in the ATP regeneration step, resulting in a FAD synthesis process with almost net-zero waste generation

Sustained dechlorination of vinyl chloride to ethene in dehalococcoides-enriched cultures grown without addition of exogenous vitamins and at low pH

© 2019 American Chemical Society. Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites

Individually Dispersed Wood-Based Cellulose Nanocrystals

Good dispersion of cellulose nanocrystals (CNCs) in the polymer matrix is one of the key factors to obtaining good properties in the resulting nanocomposites. However, the preparation of individually dispersed CNCs in solvents or in polymer matrices has been a challenge. In this study, individually dispersed wood-based CNCs have been successfully prepared in solvents, including dimethylformamide (DMF), H₂O, and a mixture of H₂O/DMF, by sonication of moisture-containing CNCs. The CNCs dispersions were characterized by dynamic light scattering (DLS). It is found that CNCs containing above about 3.8 wt % moisture is critical for achieving individually dispersed CNC in solvents. Hydrodynamic radius (<i>R</i>_h) of CNCs is smaller in H₂O/DMF co-solvent mixture than that in pure DMF or in pure H₂O under same sonication treatment conditions. Experimental results have been corroborated using molecular simulation study

An Oxygenase-Independent Cholesterol Catabolic Pathway Operates under Oxic Conditions

<div><p>Cholesterol is one of the most ubiquitous compounds in nature. The 9,10-<i>seco</i>-pathway for the aerobic degradation of cholesterol was established thirty years ago. This pathway is characterized by the extensive use of oxygen and oxygenases for substrate activation and ring fission. The classical pathway was the only catabolic pathway adopted by all studies on cholesterol-degrading bacteria. <i>Sterolibacterium denitrificans</i> can degrade cholesterol regardless of the presence of oxygen. Here, we aerobically grew the model organism with ¹³C-labeled cholesterol, and substrate consumption and intermediate production were monitored over time. Based on the detected ¹³C-labeled intermediates, this study proposes an alternative cholesterol catabolic pathway. This alternative pathway differs from the classical 9,10-<i>seco</i>-pathway in numerous important aspects. First, substrate activation proceeds through anaerobic C-25 hydroxylation and subsequent isomerization to form 26-hydroxycholest-4-en-3-one. Second, after the side chain degradation, the resulting androgen intermediate is activated by adding water to the C-1/C-2 double bond. Third, the cleavage of the core ring structure starts at the A-ring via a hydrolytic mechanism. The ¹⁸O-incorporation experiments confirmed that water is the sole oxygen donor in this catabolic pathway.</p></div

The aerobic catabolic pathways of cholesterol by bacteria.

<p>The ring identification (A–D) and carbon numbering systems (1–27) of steroids are shown in cholesterol. (A) The classical 9,10-<i>seco</i>-pathway demonstrated in <i>G. cholesterolivorans</i> DSMZ 45229. (B) The alternative 2,3-<i>seco</i>-pathway proposed in this study using <i>S. denitrificans</i> DSMZ 13999 as the model organism. 25-hydroxycholest-4-en-3-one was the last detected intermediate reported in the previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066675#pone.0066675-Chiang1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066675#pone.0066675-Chiang2" target="_blank">[33]</a>. First ring cleavage intermediates appearing in the catabolic pathways are highlighted in boxes. In this study, α,α′-D and <i>tert</i>-butyl alcohol served as the inhibitors for the 9,10-<i>seco</i>-pathway and 2,3-<i>seco</i>-pathway, respectively.</p

The structure elucidation and the investigation of the ring cleavage mechanism of compound 1 (1,17-dioxo-2,3-<i>seco</i>-androstan-3-oic acid, DSAO).

<p>(A) The interpretations of COSY and key HMBC spectra of compound <b>1.</b> (B) The chemical structure of compound <b>1</b>. *The oxygen atoms were labeled with ¹⁸O in the H₂¹⁸O-incorporation assay. (C) ESI-mass spectra (positive ion mode) of DSAO. (CI) DSAO purified from the anaerobic control assay. (CII) DSAO purified from the ¹⁸O₂-treated assay. (CIII) DSAO purified from the ¹⁸O-labeled H₂O-treated assay. For detailed NMR spectral data of compound <b>1</b>, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066675#pone.0066675.s006" target="_blank">Table S2</a>.</p

Aerobic cholesterol catabolic pathway by<i>S denitrificans</i> was inhibited by <i>tert</i>-butyl alcohol but not by α,α′-D.

<p>(A) UPLC-HRMS analysis of ethyl-acetate extracts of cholesterol-grown bacterial cells with or without α,α′-D. (AI) <i>G</i>. <i>cholesterolivorans</i> DSMZ 45229 grown with cholesterol (2 mM), (AII) <i>G</i>. <i>cholesterolivorans</i> grown with cholesterol and α,α′-D (5 mM), (AIII) <i>S. denitrificans</i> DSMZ 13999 grown with cholesterol, and (AIV) <i>S. denitrificans</i> grown with cholesterol and α,α′-D. (B) UPLC-HRMS analysis of ethyl-acetate extracts of cholesterol-grown <i>S. denitrificans</i> cells with different concentrations of <i>tert</i>-butyl alcohol. (BI) The aerobic growth without <i>tert</i>-butyl alcohol, (BII) in the presence of 2.5% (v/v) <i>tert</i>-butyl alcohol, and (BIII) in the presence of 5% <i>tert</i>-butyl alcohol. Abbreviations: TIC, total ion current; 26-OH, 26-hydroxycholest-4-en-3-one; 25-OH, 25-hydroxycholest-4-en-3-one; PCA, pregn-4-en-3-one-20-carboxylic acid; AD, androst-4-en-3,17-dione; ADD, androsta-1,4-diene-3,17-dione; *, unidentified nitrogen compounds.</p