Lignin-derived Aromatic Compounds Research Articles

Production of bio-based adipic acid using a combination of engineered Pseudomonas putida strains

Adipic acid is an industrially important dicarboxylic acid and an essential precursor in the synthesis of Nylon-6,6. Due to the environmental impact associated with the current synthetic methods for Nylon-6,6 production, greener alternatives based on renewable and bio-based feedstocks are needed. In the present study, the potential of two Pseudomonas putida strains engineered for muconic acid accumulation was used to produce adipic acid from the lignin-derived aromatic compound guaiacol. This was achieved by expressing genes enabling the conversion of guaiacol to catechol in the first strain, and the conversion of muconic acid to adipic acid via expression of the Bacillus coagulans enoate reductase gene in the second strain. Through process engineering, a 2-step production was designed in which 2.93 mM bio-adipic acid was produced, with a yield of 0.57 mol/mol. This is the first demonstration that process and strain engineering can be combined to obtain a complete bio-based route for adipic acid production from depolymerized lignin compounds using the robust P. putida chassis.

Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 μM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates’ carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

Biological Valorization of Lignin-Derived Aromatics in Hydrolysate to Protocatechuic Acid by Engineered Pseudomonas putida KT2440.

Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.

Effects of hematite on two types of dissolved organic compounds in lignocellulosic anaerobic hydrolysate: Lignin-derived aromatic compounds and denitrifying carbon sources

The utilization of anaerobic hydrolysate from agroforestry wastes is limited by dissolved lignin and aromatics, which have received insufficient attention despite their potential as excellent carbon sources for denitrification. This study aims to investigate the influence of hematite on lignin-derived aromatic compounds and denitrifying carbon sources, as well as to identify iron-reducing bacteria that utilize lignin-derived aromatic compounds as electron donors. The findings revealed that hematite facilitated the anaerobic fermentation of plant biomass, resulting in the production of small molecular organic acids. Moreover, biodegradation of lignin-derived aromatic compounds led to the formation of phenolic acids, while an increased generation of denitrifying carbon sources enhanced nitrogen removal efficiency by 13.84 %. Additionally, due to adsorption by hematite and subsequent microbial degradation, there was a significant improvement (40.32%) in color removal rate within denitrification effluent. Notably, Azonexus strains were hypothesized to be involved in Fe(Ⅲ) reduction coupled with aromatic compounds oxidation.

The catabolism of lignin-derived p-methoxylated aromatic compounds by Rhodococcus jostii RHA1.

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ΔpobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ΔpcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the β-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus, a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives.

Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Catabolic System of 5-Formylferulic Acid, a Downstream Metabolite of a β-5-Type Lignin-Derived Dimer, in Sphingobium lignivorans SYK-6.

Sphingobium lignivorans SYK-6 can assimilate various lignin-derived aromatic compounds, including a β-5-type (phenylcoumaran-type) dimer, dehydrodiconiferyl alcohol (DCA). SYK-6 converts DCA to a stilbene-type intermediate via multiple reaction steps and then to vanillin and 5-formylferulic acid (FFA). Here, we first elucidated the catabolic pathway of FFA, which is the only unknown pathway in DCA catabolism. Then, we identified and characterized the enzyme-encoding genes responsible for this pathway. Analysis of the metabolites revealed that FFA was converted to 5-carboxyferulic acid (CFA) through oxidation of the formyl group, followed by conversion to ferulic acid by decarboxylation. A comprehensive analysis of the aldehyde dehydrogenase genes in SYK-6 indicated that NAD+-dependent FerD (SLG_12800) is crucial for the conversion of FFA to CFA. LigW and LigW2, which are 5-carboxyvanillic acid decarboxylases involved in the catabolism of a 5,5-type dimer, were found to be involved in the conversion of CFA to ferulic acid, and LigW2 played a significant role. The ligW2 gene forms an operon with ferD, and their transcription was induced during growth in DCA.

Ruthenium catalysts supported on activated carbon from corncob waste for selective hydrogenolysis of C–O bonds in lignin-derived aromatic compounds

Selective hydrogenolysis of C–O bonds in lignin is regarded as an important process to produce high value-added chemicals and biofuels. Herein, high-performance Ru-based catalysts (Ru/AC) supported on activated carbon (AC) derived from corncob waste were prepared by a simple impregnation method. The optimal 5%Ru/AC possessed enormous specific surface area, well-developed porosity, high metal dispersion, small Ru particle size and strong metal-support interaction, showing a marvelous catalytic performance in the hydrogenolysis of diphenyl ether, benzyl phenyl ether and phenylethyl phenyl ether with different kinds of C–O bonds under mild conditions. Besides, various phenolic compounds were selectively hydrogenated into cycloalkanols and cycloalkanes over 5%Ru/AC, indicating the excellent catalytic activity in aromatic ring hydrogenation of this catalyst. In addition, high conversion of diphenyl ether and high selectivity of monomeric products were well maintained after employing this catalyst at least three times. The construction and application of the ingenious 5%Ru/AC catalyst highlighted a promising approach to the value-added utilization of lignin-derived aromatic compounds.

Unraveling the roles of coastal bacterial consortia in degradation of various lignocellulosic substrates.

Lignocellulose, as the most abundant natural organic carbon on earth, plays a key role in regulating the global carbon cycle, but there have been only few studies in marine ecosystems. Little information is available about the extant lignin-degrading bacteria in coastal wetlands, limiting our understanding of their ecological roles and traits in lignocellulose degradation. We utilized in situ lignocellulose enrichment experiments coupled with 16S rRNA amplicon and shotgun metagenomics sequencing to identify and characterize bacterial consortia attributed to different lignin/lignocellulosic substrates in the southern-east intertidal zone of East China Sea. We found the consortia enriched on woody lignocellulose showed higher diversity than those on herbaceous substrate. This also revealed substrate-dependent taxonomic groups. A time-dissimilarity pattern with increased alpha diversity over time was observed. Additionally, this study identified a comprehensive set of genes associated with lignin degradation potential, containing 23 gene families involved in lignin depolymerization, and 371 gene families involved in aerobic/anaerobic lignin-derived aromatic compound pathways, challenging the traditional view of lignin recalcitrance within marine ecosystems. In contrast to similar cellulase genes among the lignocellulose substrates, significantly different ligninolytic gene groups were observed between consortia under woody and herbaceous substrates. Importantly, we not only observed synergistic degradation of lignin and hemi-/cellulose, but also pinpointed the potential biological actors at the levels of taxa and functional genes, which indicated that the alternation of aerobic and anaerobic catabolism could facilitate lignocellulose degradation. Our study advances the understanding of coastal bacterial community assembly and metabolic potential for lignocellulose substrates. IMPORTANCE It is essential for the global carbon cycle that microorganisms drive lignocellulose transformation, due to its high abundance. Previous studies were primarily constrained to terrestrial ecosystems, with limited information about the role of microbes in marine ecosystems. Through in situ lignocellulose enrichment experiment coupled with high-throughput sequencing, this study demonstrated different impacts that substrates and exposure times had on long-term bacterial community assembly and pinpointed comprehensive, yet versatile, potential decomposers at the levels of taxa and functional genes in response to different lignocellulose substrates. Moreover, the links between ligninolytic functional traits and taxonomic groups of substrate-specific populations were revealed. It showed that the synergistic effect of lignin and hemi-/cellulose degradation could enhance lignocellulose degradation under alternation of aerobic and anaerobic conditions. This study provides valuable taxonomic and genomic insights into coastal bacterial consortia for lignocellulose degradation.

Stereoinversion via Alcohol Dehydrogenases Enables Complete Catabolism of β-1-Type Lignin-Derived Aromatic Isomers.

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened β-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of β-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four β-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with β-1 linkages. Therefore, elucidating the catabolic system of β-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native β-1-type dimer catabolic system.

High-level production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid as a lignin-related aromatic compound by metabolically engineered fermentation to realize industrial valorization processes of lignin

2-Pyrone-4,6-dicarboxylic acid (PDC) is a valuable building block molecule produced from lignin-derived aromatic compounds by biological funneling. This study aimed to design a fermentation process for producing PDC from vanillic acid, which could be applied at an industrial production. Metabolomic analysis revealed that a high primary metabolic activity within cells was required to improve the production efficiency. Moreover, a medium with ammonium salts and no alkali metals was advantageous because it suppressed the formation of PDC–metal complexes. Resulting optimized process yielded the highest PDC titer and productivity ever reported: 99.9 g/L and 1.69 g/L/h, respectively. Per batch, 190 g of PDC was produced per liter of initial culture media, and the final liquid volume was 1.9 L. This study demonstrates the design of fermentation processes for the advanced industrial utilization of lignin by biological funneling.

Linking soil microbial community to the chemical composition of dissolved organic matter in a boreal forest during freeze–thaw cycles

Soil dissolved organic matter (DOM) is mainly derived from the microbial degradation of organic matter. Intense freeze–thaw cycles (FTCs) elicited by warming alter the production and consumption of DOM in boreal forest soils, but little is known about the interactions between soil microbial community and DOM chemical composition. In this study, a FTCs incubation experiment was conducted to investigate the association between DOM dynamics and microbial community. DOM fingerprints were characterized using pyrolysis gas chromatography-mass spectrometry (Py-GC/MS) technology, and bacterial and fungal community composition was analyzed using high-throughput sequencing and bioinformatics. Both two and six freeze–thaw cycles (2FTCs and 6FTCs) significantly increased soil dissolved organic carbon (DOC) and NH4+-N contents at 0–10 cm and 50–60 cm depths. FTCs significantly decreased the relative abundance of aromatic components in DOM, whereas increasing the relative abundance of polysaccharides and fatty acid methyl esters (FAMEs) in DOM. The degradation of lignin-derived aromatic compounds was accelerated and microbial-derived components accumulated. Also, bacterial community responded more sensitively to FTCs than fungal community. Significant changes in bacterial community network was observed during FTCs with increased linkages, shortened average path lengths, reduced modularity and relative abundance of dominant phyla, and decreased α diversity. However, FTCs only increased the average path length of soil fungal community. The higher connectivity indicated that FTCs enhanced the interactions between microbial community and DOM chemical components. FTCs led to a shift in bacterial keystone taxa from being present in specific DOM molecules to competing for common substrates. Overall, FTCs tended to activate DOM and increase availability, which could promote DOM turnover and increase soil C stabilization. These findings provide new insights into the molecular mechanisms of DOM dynamics in the boreal forest soils under increased FTCs.

Efficient Azo Dye Biodecolorization System Using Lignin-Co-Cultured White-Rot Fungus.

The extensive use of azo dyes by the global textile industry induces significant environmental and human health hazards, which makes efficient remediation crucial but also challenging. Improving dye removal efficiency will benefit the development of bioremediation techniques for textile effluents. In this study, an efficient system for azo dye (Direct Red 5B, DR5B) biodecolorization is reported, which uses the white-rot fungus Ganoderma lucidum EN2 and alkali lignin. This study suggests that the decolorization of DR5B could be effectively enhanced (from 40.34% to 95.16%) within 48 h in the presence of alkali lignin. The dye adsorption test further confirmed that the alkali-lignin-enhanced decolorization of DR5B was essentially due to biodegradation rather than physical adsorption, evaluating the role of alkali lignin in the dye biodegradation system. Moreover, the gas chromatography/mass spectrometry analysis and DR5B decolorization experiments also indicated that alkali lignin carried an excellent potential for promoting dye decolorization and displayed a significant role in improving the activity of lignin-modifying enzymes. This was mainly because of the laccase-mediator system, which was established by the induced laccase activity and lignin-derived small aromatic compounds.

Efficient O-demethylation of lignin-derived aromatic compounds under moderate conditions.

Lignin is a potential feedstock to produce renewable aromatic chemicals. However, lignin-derived aromatics are heavily methoxylated, which affects their reactivity in some downstream valorization attempts. Herein, we report an efficient method for the demethylation of the aromatics derived from lignin depolymerization using acidic concentrated lithium bromide (ACLB) under moderate conditions (e.g., 1.5 M HCl, 110 °C, and 2 h). Aromatics with one or two methoxy groups (G-type and S-type), alkyl hydroxyl and carbonyl groups, and electron-donating and electron-withdrawing substituents were used to investigate the demethylation mechanisms. S-type aromatics were demethylated faster than their G-type analogs. Alkyl hydroxyl groups were brominated under the conditions. Carbonyl groups (aldehydes and ketones) promoted unwelcome condensation. Electron-donating substituents promoted demethylation, whereas electron-withdrawing substituents retarded the demethylation. An ortho-carboxylic group enhanced the demethylation because of the formation of a stable intermediate.

리그노셀룰로오스의 혐기성 분해에 의한 탄수화물과 방향족 화합물의 축적속도 평가

This study evaluated the degradation properties of lignocellulose in methanogenic and sulfate-reducing environment. Carbohydrates and aromatic compounds, which are lignocellulosic degradation products, were analyzed and the accumulation rates were calculated. Seeding microorganisms, as well as leachate from a model landfill reactor composed of a methanogenic and sulfate-reducing environment that hadbeen operatingfor more than three years wereused. To calculate the accumulation rates of carbohydrates and aromatic compounds, serum vials cultured for 12 days were subjected to 3% (volume ratio) toluene. Toluene was used to selectively inhibit the activity of microorganisms while maintaining the activity of the extracellular degrading enzyme. Galactose, glucose, xylose and mannose produced from holocellulose such as office paper and newspaper in the presence of 3% toluene showed a linear increase in carbohydrate accumulation with time in a sulfate-reducing environment. The accumulation rates of glucose and xylose in the sulfate-reducing environment were faster than in the methanogenic environment, indicating that holocellulose decomposition was faster in the sulfate-reducing environment than in the methanogenic environment. For the lignin-derived aromatic compounds produced in newspapers in the presence of 3% toluene, 7 aromatic compounds were identified in the sulfate-reducing environment, compared to 3 aromatic compounds identified in the methanogenic environment. Accumulation rates of benzoic acid and vanillin were faster in the sulfate-reducing environment than in the methanogenic environment, indicating that lignin degradation was faster in the sulfate-reducing environment than in the methanogenic environment.

Continuous hydrodeoxygenation of lignin to jet-range aromatic hydrocarbons

<h2>Summary</h2> Sustainable aviation fuel (SAF) is essential to decrease the carbon footprint of the aviation industry. Although many strategies have been developed to provide the linear and branched aliphatic components of SAF, few viable strategies have been demonstrated to supply the aromatic and cycloalkane fraction of the SAF at the necessary scale from bio-based feedstocks. Lignin is the largest natural source of renewable aromatic compounds; however, major challenges in deoxygenation have prevented its use as a feedstock for SAFs. Here, we report a continuous, two-stage catalytic process using molybdenum carbide to deoxygenate lignin from poplar into aromatic hydrocarbons with 87.5% selectivity toward aromatic hydrocarbons at 86% of the theoretical carbon recovery. Tier α fuel property testing indicates that the SAF-range lignin-derived aromatic compounds are likely performance-advantaged across multiple properties relative to conventional jet fuel aromatic compounds. This work demonstrates an effective approach to convert lignin into aromatic SAF blendstocks.

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for β-ketoadipate production from p-coumarate

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, β-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the βKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

Gene expression of the white-rot fungus Lenzites gibbosa during wood degradation

ABSTRACT To determine the wood degradation mechanism and its key genes and biological processes of Lenzites gibbosa, we sequenced 15 transcriptomes of mycelial samples under woody environment at 3, 5, 7, and 11 d (D3, D5, D7, and D11) and nonwoody environment (control). All the transcripts were annotated as much as possible in eight databases to determine their function. The key genes and biological processes relating to wood degradation were predicted and screened. The expression of 11 key genes during wood degradation after 5 d of sawdust treatment was detected by quantitative polymerase chain reaction (PCR). A total of 2069 differentially expressed genes (DEGs) were obtained in 10 differential groups. Comparing wood with nonwood treatment condition, the key genes were those participating in oxidation-reduction process, they were oxidoreductase and peroxidase genes and their regulator genes; these genes mainly focused on the three biological processes of carbohydrate metabolism, lignin catabolism, and secondary metabolite biosynthesis, transport, and catabolism. The mostly enriched subcategories in molecular function were oxidoreductase activity, peroxidase activity, and heme binding in Gene Ontology (GO) annotation. One cellulose and hemicellulose degradation pathway and seven pathways related to lignin-derived aromatic compound degradation or the later degradation of lignin were found. In conclusion, during the process of L. gibbosa growing on wood, gene expression at the transcriptional level indicated that lignin catabolism and hyphal growth were promoted, but the metabolism of carbon and carbohydrates including cellulose in lignocellulose in overall trend was inhibited to some extent. The results have important reference value for the study of degradation mechanism of wood white rot.

Fermenting and Lignin Degradability of a White-Rot Fungus Coriolopsis trogii Using Industrial Lignin as Substrate.

Bio-depolymerized the lignin macromolecules into low molecular lignin-derived aromatic compounds satisfies the requirement for carbon dioxide peaking and is also one of the important ways to realize lignin valorization. Coriolopsis trogii is a kind of less reported lignin-degrading white-rot fungus. The degradability of a self-isolated C. trogii TS01 on industrial lignins, including enzymatic hydrolysis lignin (EHL) and Kraft lignin (KL), was investigated in this paper. The results indicated that EHL could be used as an efficient carbon source to promote the cell growth and ligninolytic enzyme secretion of C. trogii TS01. Compared with using 2% glucose as carbon source, 1% EHL plus 1% glucose would increase the maximum cell dry weight, laccase activity, and manganese-dependent peroxidase activity of C. trogii TS01 by 24.8%, 164.1%, and 200%, respectively. However, the cell growth and ligninolytic enzyme secretion would be significantly inhibited in the case of 1% KL plus 1% glucose used as carbon source. As a result, at the 12th day of fermentation, the degradation rates of EHL and KL were 50.6% and 5.7%, respectively. The UV and FTIR analysis indicated that after been fermented by C. trogii TS01, S-unit content in EHL was decreased by 12.5% but G-unit content was increased by 53.7%. In conclusion, the research of this paper will provide a promising solution for the valorization of enzymatic hydrolysis lignin since the high biodegradation rate of lignin and high activity of ligninolytic enzymes could be achieved simultaneously.

Pseudomonas sp. NGC7 as a microbial chassis for glucose-free muconate production from a variety of lignin-derived aromatics and its application to the production from sugar cane bagasse alkaline extract

cis,cis-Muconate (ccMA) is a promising platform for use in synthesizing various polymers. A glucose-free ccMA production using Pseudomonas sp. NGC7 from hardwood lignin-derived aromatic compounds was previously reported. In that system, syringyl nucleus compounds were essential for growth. Here, it is shown that NGC7 is available for glucose-free ccMA production even from a mixture of lignin-derived aromatics that does not contain syringyl nucleus compounds. By introducing a gene set for the protocatechuate (PCA)-shunt consisting of PCA 3,4-dioxygenase and PCA decarboxylase into an NGC7-derived strain deficient in PCA 3,4-dioxygenase and ccMA cycloisomerase, it was succeeded in constructing a ccMA-producing strain that grows on a lignin-derived aromatics mixture containing no syringyl nucleus compounds. Finally, it is demonstrated that the engineered strain produced ccMA from sugar cane bagasse alkaline extract in 18.7 mol%. NGC7 is thus shown to be a promising microbial chassis for biochemicals production from lignin-derived aromatics.