Chain Elongation Pathway Research Articles

Variability in n-caprylate and n-caproate producing microbiomes in reactors with in-line product extraction.

Medium-chain carboxylates (MCCs) are used in various industrial applications. These chemicals are typically extracted from palm oil, which is deemed not sustainable. Recent research has focused on microbial chain elongation using reactors to produce MCCs, such as n-caproate (C6) and n-caprylate (C8), from organic substrates such as wastes. Even though the production of n-caproate is relatively well-characterized, bacteria and metabolic pathways that are responsible for n-caprylate production are not. Here, three 5 L reactors with continuous membrane-based liquid-liquid extraction (i.e., pertraction) were fed ethanol and acetate and operated for an operating period of 234 days with different operating conditions. Metagenomic and metaproteomic analyses were employed. n-Caprylate production rates and reactor microbiomes differed between reactors even when operated similarly due to differences in H2 and O2 between the reactors. The complete reverse β-oxidation (RBOX) pathway was present and expressed by several bacterial species in the Clostridia class. Several Oscillibacter spp., including Oscillibacter valericigenes, were positively correlated with n-caprylate production rates, while Clostridium kluyveri was positively correlated with n-caproate production. Pseudoclavibacter caeni, which is a strictly aerobic bacterium, was abundant across all the operating periods, regardless of n-caprylate production rates. This study provides insight into microbiota that are associated with n-caprylate production in open-culture reactors and provides ideas for further work.IMPORTANCEMicrobial chain elongation pathways in open-culture biotechnology systems can be utilized to convert organic waste and industrial side streams into valuable industrial chemicals. Here, we investigated the microbiota and metabolic pathways that produce medium-chain carboxylates (MCCs), including n-caproate (C6) and n-caprylate (C8), in reactors with in-line product extraction. Although the reactors in this study were operated similarly, different microbial communities dominated and were responsible for chain elongation. We found that different microbiota were responsible for n-caproate or n-caprylate production, and this can inform engineers on how to operate the systems better. We also observed which changes in operating conditions steered the production toward and away from n-caprylate, but more work is necessary to ascertain a mechanistic understanding that could be predictive. This study provides pertinent research questions for future work.

Co-digestion of solid-liquid waste from citrus agroindustrial: Effect of hydraulic retention time and organic loading rate on H2 production in a long-term continuous operation leach bed reactor

The Leach Bed Reactor (LBR) application is a promising alternative for the anaerobic digestion of solid waste and wastewater. In this study, the LBR was continuously operated for 160 days in four different phases, for co-fermentation of solid and liquid waste from the citrus agroindustry, at 37 °C, evaluating the effect of hydraulic retention time (HRT) and organic loading rate (OLR). The maximum volumetric hydrogen production (VHPR) was observed at an HRT of 12h (958.3 mLH2.L-1.d-1); however, the highest hydrogen yield (HY) was identified at an HRT of 24h (104.9 mLH2.g-1CHO). Based on the metabolic characteristics of microorganisms identified in higher relative abundance, by 16S rRNA sequencing, such as Lactobacillus, Caproicoproducens, Bifidobacterium, Olsenella, and Solobacterium, an occurrence of chain elongation pathways in the production of butyric and caproic acids was inferred, with hydrogen production resulting from the lactic acid oxidation. The maximum electricity production potential of 16.3 kWh.m-3 liquor was estimated.

A specific H2/CO2 consumption molar ratio of 3 as a signature for the chain elongation of carboxylates from brewer's spent grain acidogenesis.

Brewer's spent grain (BSG) is an undervalorized organic feedstock residue composed of fermentable macromolecules, such as proteins, starch, and residual soluble carbohydrates. It also contains at least 50% (as dry weight) of lignocellulose. Methane-arrested anaerobic digestion is one of the promising microbial technologies to valorize such complex organic feedstock into value-added metabolic intermediates, such as ethanol, H2, and short-chain carboxylates (SCC). Under specific fermentation conditions, these intermediates can be microbially transformed into medium-chain carboxylates through a chain elongation pathway. Medium-chain carboxylates are of great interest as they can be used as bio-based pesticides, food additives, or components of drug formulations. They can also be easily upgraded by classical organic chemistry into bio-based fuels and chemicals. This study investigates the production potential of medium-chain carboxylates driven by a mixed microbial culture in the presence of BSG as an organic substrate. Because the conversion of complex organic feedstock to medium-chain carboxylates is limited by the electron donor content, we assessed the supplementation of H2 in the headspace to improve the chain elongation yield and increase the production of medium-chain carboxylates. The supply of CO2 as a carbon source was tested as well. The additions of H2 alone, CO2 alone, and both H2 and CO2 were compared. The exogenous supply of H2 alone allowed CO2 produced during acidogenesis to be consumed and nearly doubled the medium-chain carboxylate production yield. The exogenous supply of CO2 alone inhibited the whole fermentation. The supplementation of both H2 and CO2 allowed a second elongation phase when the organic feedstock was exhausted, which increased the medium-chain carboxylate production by 285% compared to the N2 reference condition. Carbon- and electron-equivalent balances, and the stoichiometric ratio of 3 observed for the consumed H2/CO2, suggest an H2- and CO2-driven second elongation phase, converting SCC to medium-chain carboxylates without an organic electron donor. The thermodynamic assessment confirmed the feasibility of such elongation.

Regulation on C2-C8 carboxylic acid biosynthesis from anaerobic CO2 fermentation.

Bioconversion of CO2 into liquid fuels or chemicals, preferred medium chain carboxylic acids (caproic and caprylic acid), is an attractive CO2 utilization technology. The present study aims to investigate the effects of different ratios of H2/CO2 on regulating the distribution of C2-C8 carboxylic acid products, while the headspace pressure of 1.5bar was set to amplify the effect of different ratios. The H2/CO2 ratio of 4:1 was more suitable for preparing acetic acid, where the highest acetic acid yield was 17.5g/L. And the H2/CO2 ratio of 2:1 showed excellent chain elongation ability with the highest n-caprylic yield of 2.4g/L. Additionally, the actual H2/CO2 ratios of 4:1 reactors were higher than that in 2:1 may be course chain elongation often accompanied by H2 production. The 16S rRNA genes analysis shows that the genus Terrisporobacter and Coriobacteriales may be related to acetic acid production enriched in H2/CO2 ratio 4:1 reactors, and the genus Clostridium and Paenibacillaceae may associate with the chain elongation pathway were enriched in H2/CO2 ratio 2:1 reactors.

Caproic acid production through lactate-based chain elongation: Effect of lactate-to-acetate ratio and substrate loading

Substrate properties play a key role in promoting the caproate yield through lactate-based carbon chain elongation pathway. In the present study, the effect of lactate-to-acetate (LA/AA) carbon ratio (from 0.5 to 5.0) and substrate loading (in terms of substrate/inoculum ratio within the range 20–180 mmol-C/g-VSS) on caproate fermentation was investigated. Results showed that both caproate content and yield increased by increasing the LA/AA ratio up to 3.0, then decreased at higher ratios due to activation of acrylate pathway and dispersion of carbon flux at elevated lactate content. At the optimal LA/AA carbon ratio of 3.0, substrate loading lower than 100 mmol-C/g-VSS was beneficial for efficient substrate utilization with low caproate selectivity, while higher substrate-to-inoculums (S/I) ratio led to incomplete substrate utilization and dispersed carbon flow, which finally reduced the caproate yield. Thus, the highest caproate yield of 0.42 g-COD/g-COD and selectivity of 49.5% were recorded at LA/AA and S/I ratio ratios of 3.0 and 100 mmol-C/g-VSS, respectively. The present study further depictures the novel approach for caproate production with lactate. • Different lactate-to-acetate (LA/AA) and substrate-to-inoculums (S/I) ratios were evaluated. • Highest caproate (CA) yield was found at LA/AA ratio of 3.0 and S/I ratio of 100 mmol-C/g-VSS. • LA/AA ratios over 3.0 activate acrylate pathway and reduce the CA production. • High S/I ratios reduce substrate utilization and disperse lactate carbon. • Butyrate was the dominant intermediate during lactate-based chain elongation.

Strategic thermosonication-mediated modulation of lactic acid bacteria acidification kinetics for enhanced (post)-fermentation performance

This study explored the feasibility of thermosonication (TS)-prestressed inoculum with different fermentation patterns for regulating microbial (post)-fermentation acidification kinetics. Through a Box-Behnken design, stimulative (20 min, 400 W, 33 kHz, 25 °C) and inhibitive (10 min, 600 W, 33 kHz, 20 °C) effects on the acidification capability of Lactobacillus plantarum A3 were achieved without observing greatly activated/inactivated strains growth, further confirmed by lactose fermentation performed by Streptococcus thermophilus and Lactobacillus bulgaricus. Lactic acid was the major contributing factor responsible for TS-induced acidification modifications corresponding to the potential fluctuations of CoA biosynthesis, fatty acid degradation and chain elongation pathways to TS prestress. Microscopy observations and quantitative extracellular substance assays showed palpable stress disturbance on microbes, but causing insignificant effects on product characteristics. This investigation demonstrated the potential of controlled sonication prestress strategies to achieve dual engineering effects on microbial metabolic behavior, for alleviating post-acidification problem or enhancing process efficiencies.

Emission Patterns of Esters and Their Precursors Throughout Ripening and Senescence in ‘Redchief Delicious’ Apple Fruit and Implications Regarding Biosynthesis and Aroma Perception

The volatile profile of ‘Redchief Delicious’ apple (Malus ×domestica Borkh.) fruit was evaluated at 18 time points from 3 weeks before to 8 weeks after onset of autocatalytic ethylene production to capture the dynamics associated with development from mature green to senescent fruit. Minor amounts of ester production began several days before the onset of ethylene production. Ester production rose rapidly as internal ethylene levels increased beyond 22 nmol·L−1 (0.5 µL·L−1). Peak ester production roughly coincided with maximum ethylene synthesis, declining thereafter. Ester production was further evaluated according to the acid- (alkanoate) and alcohol- (alkyl) derived portions of the ester. The maximum rate of production for a given ester tended to occur later in development as the chain length of the alcohol-derived portion declined. The production rate for many esters paralleled the rate of emanation of their respective alcohol substrates, suggesting that availability of the alcohols limits ester production more than availability of the acid substrates. Combining production rates with sensory descriptors and human sensitivity to individual volatiles permitted approximations of aroma sensations likely engendered by the fruit throughout ripening. Overripe and alcoholic sensations are predicted to increase 2 weeks after the initiation of ripening in response to an increase in the production of ethyl esters. Acetate esters predominated, comprising 50% to 80% of esters throughout maturation and ripening, indicating that the substrate acetyl-CoA may be at saturating levels for alcohol acyl transferase (AAT) at the final step of ester formation. Acetate feeding did not enhance ester production, although label from 13C-acetate was extensively incorporated into esters. The data are consistent with the action of multiple AAT isozymes differing in activity and substrate preference. Incorporation of labeled 13C-acetate into precursors of esters, alcohols, and acids, reflected ester biosynthesis via 1- and 2-carbon chain elongation pathways in ripening ‘Redchief Delicious’ apple fruit.

Butyryl/Caproyl-CoA:Acetate CoA-transferase: cloning, expression and characterization of the key enzyme involved in medium-chain fatty acid biosynthesis.

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.

A novel cascade biorefinery approach to transform food waste into valuable chemicals and biogas through thermal pretreatment integration

A novel biorefinery platform integrating thermal pretreatment and solid-liquid separation unit is here proposed to fully exploit food waste (FW) potential for production of valuable chemicals and energy through semi-continuous anaerobic bioconversion. The liquid fraction deriving from raw or pretreated FW, was fermented into volatile fatty acids (VFAs, from acetic to caproic acid) while the residual fraction was converted into biomethane. Thermal pretreatment effectively extracted a portion of the macromolecular organics, especially starch, to the liquid phase, promoting acidogenic fermentation and chain elongation pathways (0.43 gVFA g−1VSfed and 0.58 gVFA g−1VSfed with raw and pretreated extract, respectively). In parallel, anaerobic digestion of solid residue in 10 L reactors showed process stability and higher conversion rate for the pretreated residue (0.31 against 0.26 Nm3CH4 kg−1VSfed). The mass-transfer balance coupled with the economic assessment, calculated in terms of direct gross added value, indicated promising revenues by integrating the thermal upstream treatment.

Developing Clostridia as Cell Factories for Short- and Medium-Chain Ester Production.

Short- and medium-chain volatile esters with flavors and fruity fragrances, such as ethyl acetate, butyl acetate, and butyl butyrate, are usually value-added in brewing, food, and pharmacy. The esters can be naturally produced by some microorganisms. As ester-forming reactions are increasingly deeply understood, it is possible to produce esters in non-natural but more potential hosts. Clostridia are a group of important industrial microorganisms since they can produce a variety of volatile organic acids and alcohols with high titers, especially butanol and butyric acid through the CoA-dependent carbon chain elongation pathway. This implies sufficient supplies of acyl-CoA, organic acids, and alcohols in cells, which are precursors for ester production. Besides, some Clostridia could utilize lignocellulosic biomass, industrial off-gas, or crude glycerol to produce other branched or straight-chain alcohols and acids. Therefore, Clostridia offer great potential to be engineered to produce short- and medium-chain volatile esters. In the review, the efforts to produce esters from Clostridia via in vitro lipase-mediated catalysis and in vivo alcohol acyltransferase (AAT)-mediated reaction are comprehensively revisited. Besides, the advantageous characteristics of several Clostridia and clostridial consortia for bio-ester production and the driving force of synthetic biology to clostridial chassis development are also discussed. It is believed that synthetic biotechnology should enable the future development of more effective Clostridia for ester production.

Engineering and optimization of the 2-phenylethylglucosinolate production in Nicotiana benthamiana by combining biosynthetic genes from Barbarea vulgaris and Arabidopsis thaliana.

2-Phenylethylglucosinolate (2PE) derived from homophenylalanine is present in plants of the Brassicales order as a defense compound. It is associated with multiple biological properties, including deterrent effects on pests and antimicrobial and health-promoting functions, due to its hydrolysis product 2-phenylethyl isothiocyanate, which confers 2PE as a potential application in agriculture and industry. In this study, we characterized the putative key genes for 2PE biosynthesis from Barbarea vulgaris W.T. Aiton and demonstrated the feasibility of engineering 2PE production in Nicotiana benthamiana Domin. We used different combinations of genes from B. vulgaris and Arabidopsis thaliana (L.) Heynh. to demonstrate that: (i) BvBCAT4 performed more efficiently than AtBCAT4 in biosynthesis of both homophenylalanine and dihomomethionine; (ii) MAM1 enzymes were critical for the chain-elongated profile, while CYP79F enzymes accepted both chain-elongated methionine and homophenylalanine; (iii) aliphatic but not aromatic core structure pathway catalyzed the 2PE biosynthesis; (iv) a chimeric pathway containing BvBCAT4, BvMAM1, AtIPMI and AtIPMDH1 resulted in a two-fold increase in 2PE production compared with the B. vulgaris-specific chain elongation pathway; and (v) profiles of chain-elongated products and glucosinolates partially mirrored the profiles in the gene donor plant, but were wider in N. benthamiana than in the native plants. Our study provides a strategy to produce the important homophenylalanine and 2PE in a heterologous host. Furthermore, chimeric engineering of the complex 2PE biosynthetic pathway enabled detailed understanding of catalytic properties of individual enzymes - a prerequisite for understanding biochemical evolution. The new-to-nature gene combinations have the potential for application in biotechnological and plant breeding.

In Arabidopsis thaliana Substrate Recognition and Tissue- as Well as Plastid Type-Specific Expression Define the Roles of Distinct Small Subunits of Isopropylmalate Isomerase.

In Arabidopsis thaliana, the heterodimeric isopropylmalate isomerase (IPMI) is composed of a single large (IPMI LSU1) and one of three different small subunits (IPMI SSU1 to 3). The function of IPMI is defined by the small subunits. IPMI SSU1 is required for Leu biosynthesis and has previously also been proposed to be involved in the first cycle of Met chain elongation, the first phase of the synthesis of Met-derived glucosinolates. IPMI SSU2 and IPMI SSU3 participate in the Met chain elongation pathway. Here, we investigate the role of the three IPMI SSUs through the analysis of the role of the substrate recognition region spanning five amino acids on the substrate specificity of IPMI SSU1. Furthermore, we analyze in detail the expression pattern of fluorophore-tagged IPMI SSUs throughout plant development. Our study shows that the substrate recognition region that differs between IPMI SSU1 and the other two IMPI SSUs determines the substrate preference of IPMI. Expression of IPMI SSU1 is spatially separated from the expression of IPMI SSU2 and IPMI SSU3, and IPMI SSU1 is found in small plastids, whereas IMPI SSU2 and SSU3 are found in chloroplasts. Our data show a distinct role for IMPI SSU1 in Leu biosynthesis and for IMPI SSU2 and SSU3 in the Met chain elongation pathway.

Evaluation of the feed composition for an effective medium chain carboxylic acid production in an open culture fermentation

The objective of this study was to investigate the effect of substrate composition on chain elongation pathways and on shaping reactor microbiome during open culture fermentation (OCF). The process was performed in a continuous mode in an upflow anaerobic sludge blanket (UASB) reactor fed with either fresh acid whey (AW) or AW at controlled stage of prefermentation (with controlled content of electron donors). Dosing AW with an increasing ethanol loading rate led to ethanol oxidation and short chain carboxylic acids (SCCAs) generation. Change of the feedstock composition (higher lactate and lactose content and ethanol cut off) shifted the process outcome towards medium chain carboxylic acids (MCCAs) production, with caproate as the main product. The MCCAs production rate has grown from 0.7 ± 0 to 4.12 ± 1 g/L/day (38.3 ± 5 to 212.6 ± 60 mmol C/L/day) and reached specificity of 48 ± 18% mol C. The differentiation between microbiome samples confirmed the reactor microbiome shaped according to the feed composition. The only known caproic acid producers were represented by Caproiciproducens ssp., that reached a relative OTU abundance between 3 and 7%. The developed method enables to substitute the use of fossil resources with products from the OCF of waste and wastewater. Thus, it contributes to reduce the carbon footprint and enhance the sustainability of the chemical industry.

Review on the production of medium and small chain fatty acids through waste valorization and CO2 fixation

The developing approaches in the recovery of resources from biowastes for the production of renewable value-added products and fuels, using microbial cultures as bio-catalyst have now became promising aspect. In the path of anaerobic digestion, the microorganisms are assisting transformation of a complex organic feedstock/waste to biomass and biogas. This potentiality consequently leads to the production of intermediate precursors of renewable value-added products. Particularly, a set of anaerobic pathways in the fermentation process, yields small-chain fatty acids (SCFA), and medium-chain fatty acids (MCFA) via chain elongation pathways from waste valorization and CO2 fixation. This review focuses on the production of SCFA and MCFA from CO2, synthetic substrates and waste materials. Moreover, the review introduces the metabolic engineering of Escherichia coli and Saccharomyces cerevisiae for SCFAs/MCFAs production. Furtherly, it concludes that future critical research might target progress of this promising approach as a valorization of complex organic wastes.

Characterization of Arabidopsis CYP79C1 and CYP79C2 by Glucosinolate Pathway Engineering in Nicotiana benthamiana Shows Substrate Specificity Toward a Range of Aliphatic and Aromatic Amino Acids.

Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In Arabidopsis thaliana, CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively. However, the substrate(s) for CYP79C1 and CYP79C2 were unknown. Here, we investigated the function of CYP79C1 and CYP79C2 by transiently co-expressing the genes together with three sets of remaining genes required for GLS biosynthesis in Nicotiana benthamiana. Co-expression of CYP79C2 with either the aliphatic or aromatic core structure pathways resulted in the production of primarily leucine-derived 2-methylpropyl GLS and phenylalanine-derived benzyl GLS, along with minor amounts of GLSs from isoleucine, tryptophan, and tyrosine. Co-expression of CYP79C1 displayed minor amounts of GLSs from valine, leucine, isoleucine, and phenylalanine with the aliphatic core structure pathway, and similar GLS profile (except the GLS from valine) with the aromatic core structure pathway. Additionally, we co-expressed CYP79C1 and CYP79C2 with the chain elongation and aliphatic core structure pathways. With the chain elongation pathway, CYP79C2 still mainly produced 2-methylpropyl GLS derived from leucine, accompanied by GLSs derived from isoleucine and from chain-elongated mono- and dihomoleucine, but not from phenylalanine. However, co-expression of CYP79C1 only resulted in GLSs derived from chain-elongated amino acid substrates, dihomoleucine and dihomomethionine, when the chain elongation pathway was present. This shows that CYP79 activity depends on the specific pathways co-expressed and availability of amino acid precursors, and that description of GLS core structure pathways as “aliphatic” and “aromatic” pathways is not suitable, especially in an engineering context. This is the first characterization of members of the CYP79C family. Co-expression of CYP79 enzymes with engineered GLS pathways in N. benthamiana is a valuable tool for simultaneous testing of substrate specificity against multiple amino acids.

Changing substrate specificity and iteration of amino acid chain elongation in glucosinolate biosynthesis through targeted mutagenesis of Arabidopsis methylthioalkylmalate synthase 1.

Methylthioalkylmalate synthases catalyse the committing step of amino acid chain elongation in glucosinolate biosynthesis. As such, this group of enzymes plays an important role in determining the glucosinolate composition of Brassicaceae species, including Arabidopsis thaliana. Based on protein structure modelling of MAM1 from A. thaliana and analysis of 57 MAM sequences from Brassicaceae species, we identified four polymorphic residues likely to interact with the 2-oxo acid substrate. Through site-directed mutagenesis, the natural variation in these residues and the effect on product composition were investigated. Fifteen MAM1 variants as well as the native MAM1 and MAM3 from A. thaliana were characterised by heterologous expression of the glucosinolate chain elongation pathway in Escherichia coli. Detected products derived from leucine, methionine or phenylalanine were elongated with up to six methylene groups. Product profile and accumulation were changed in 14 of the variants, demonstrating the relevance of the identified residues. The majority of the single amino acid substitutions decreased the length of methionine-derived products, while approximately half of the substitutions increased the phenylalanine-derived products. Combining two substitutions enabled the MAM1 variant to increase the number of elongation rounds of methionine from three to four. Notably, characterisation of the native MAMs indicated that MAM1 and not MAM3 is responsible for homophenylalanine production. This hypothesis was confirmed by glucosinolate analysis in mam1 and mam3 mutants of A. thaliana.

The Ca2+ Channel CNGC19 Regulates Arabidopsis Defense Against Spodoptera Herbivory.

Cellular calcium elevation is an important signal used by plants for recognition and signaling of environmental stress. Perception of the generalist insect, Spodoptera litura, by Arabidopsis (Arabidopsis thaliana) activates cytosolic Ca2+ elevation, which triggers downstream defense. However, not all the Ca2+ channels generating the signal have been identified, nor are their modes of action known. We report on a rapidly activated, leaf vasculature- and plasma membrane-localized, CYCLIC NUCLEOTIDE GATED CHANNEL19 (CNGC19), which activates herbivory-induced Ca2+ flux and plant defense. Loss of CNGC19 function results in decreased herbivory defense. The cngc19 mutant shows aberrant and attenuated intravascular Ca2+ fluxes. CNGC19 is a Ca2+-permeable channel, as hyperpolarization of CNGC19-expressing Xenopus oocytes in the presence of both cyclic adenosine monophosphate and Ca2+ results in Ca2+ influx. Breakdown of Ca2+-based defense in cngc19 mutants leads to a decrease in herbivory-induced jasmonoyl-l-isoleucine biosynthesis and expression of JA responsive genes. The cngc19 mutants are deficient in aliphatic glucosinolate accumulation and hyperaccumulate its precursor, methionine. CNGC19 modulates aliphatic glucosinolate biosynthesis in tandem with BRANCHED-CHAIN AMINO ACID TRANSAMINASE4, which is involved in the chain elongation pathway of Met-derived glucosinolates. Furthermore, CNGC19 interacts with herbivory-induced CALMODULIN2 in planta. Together, our work reveals a key mechanistic role for the Ca2+ channel CNGC19 in the recognition of herbivory and the activation of defense signaling.

Medium Chain Carboxylic Acids from Complex Organic Feedstocks by Mixed Culture Fermentation.

Environmental pressures caused by population growth and consumerism require the development of resource recovery from waste, hence a circular economy approach. The production of chemicals and fuels from organic waste using mixed microbial cultures (MMC) has become promising. MMC use the synergy of bio-catalytic activities from different microorganisms to transform complex organic feedstock, such as by-products from food production and food waste. In the absence of oxygen, the feedstock can be converted into biogas through the established anaerobic digestion (AD) approach. The potential of MMC has shifted to production of intermediate AD compounds as precursors for renewable chemicals. A particular set of anaerobic pathways in MMC fermentation, known as chain elongation, can occur under specific conditions producing medium chain carboxylic acids (MCCAs) with higher value than biogas and broader applicability. This review introduces the chain elongation pathway and other bio-reactions occurring during MMC fermentation. We present an overview of the complex feedstocks used, and pinpoint the main operational parameters for MCCAs production such as temperature, pH, loading rates, inoculum, head space composition, and reactor design. The review evaluates the key findings of MCCA production using MMC, and concludes by identifying critical research targets to drive forward this promising technology as a valorisation method for complex organic waste.

Rational design of thiolase substrate specificity for metabolic engineering applications.

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse β-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.

The functional potential and active populations of the pit mud microbiome for the production of Chinese strong-flavour liquor.

SummaryThe popular distilled Chinese strong‐flavour liquor (CSFL) is produced by solid fermentation in the ground pit. Microbes inhabiting in the pit mud (PM) on the walls of the fermentation pit are responsible for the production of caproic acid (CA) that determines the quality of CSFL to a large degree. However, little is known about the active microbial populations and metabolic potential of the PM microbiome. Here, we investigated the overall metabolic features of the PM microbiome and its active microbial components by combining metagenomics and MiSeq‐sequencing analyses of the 16S rRNA genes from DNA and RNA (cDNA). Results showed that prokaryotes were predominant populations in the PM microbiome, accounting for 95.3% of total metagenomic reads, while eukaryotic abundance was only 1.8%. The dominant prokaryotic phyla were Firmicutes, Euryarchaeota, Bacteroidetes, Actinobacteria and Proteobacteria, accounting for 48.0%, 19.0%, 13.5%, 2.5% and 2.1% of total metagenomic reads respectively. Most genes encoding putative metabolic pathways responsible for the putative CA production via chain elongation pathway were detected. This indicated that the PM microbiome owned functional potential for synthesizing CA from ethanol or lactate. Some key genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis pathways were detected in the PM metagenome, suggesting the possible occurrence of interspecies hydrogen transfer between CA‐producing bacteria and methanogens. The 16S rDNA and 16S rRNA profiles showed that the Clostridial cluster IV, Lactobacillus, Caloramator, Clostridium, Sedimentibacter, Bacteroides and Porphyromonas were active populations in situ, in which Clostridial cluster IV and Clostridium were likely involved in the CA production. This study improved our understandings on the active populations and metabolic pathways of the PM microbiome involved in the CA synthesis in the CSFL fermentation.