ABSTRACT
Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.
Subject(s)
Halomonas , Halomonas/genetics , Halomonas/metabolism , Halomonas/enzymology , Halomonas/growth & development , Metabolic Engineering , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Metabolic Networks and Pathways/genetics , Gene Deletion , Models, BiologicalABSTRACT
Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.
Subject(s)
Bile Pigments , Photoreceptors, Microbial , Photochemistry , Biliverdine , Bacterial Proteins/metabolism , Photoreceptors, Microbial/chemistry , LightABSTRACT
Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB-co-4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine.
Subject(s)
Amino Acids, Diamino , Halomonas , Halomonas/genetics , Amino Acids, Diamino/genetics , Anti-Bacterial Agents , BiopolymersABSTRACT
The use of extremophile organisms such as Halomomas spp. can eliminate the need for fermentation sterilization, significantly reducing process costs. Microbial fermentation is considered a pivotal strategy to reduce reliance on fossil fuel resources; however, sustainable processes continue to incur higher costs than their chemical industry counterparts. Most organisms require equipment sterilization to prevent contamination, a practice that introduces complexity and financial strain. Fermentations involving extremophile organisms can eliminate the sterilization process, relying instead on conditions that are conductive solely to the growth of the desired organism. This review discusses current challenges in pilot- and industrial-scale bioproduction when using the extremophile bacteria Halomomas spp. under nonsterile conditions.
Subject(s)
Halomonas , Fermentation , BacteriaABSTRACT
Polyhydroxyalkanoates (PHA) have evolved into versatile biopolymers, transcending their origins as mere bioplastics. This extensive review delves into the multifaceted landscape of PHA applications, shedding light on the diverse industries that have harnessed their potential. PHA has proven to be an invaluable eco-conscious option for packaging materials, finding use in films foams, paper coatings and even straws. In the textile industry, PHA offers a sustainable alternative, while its application as a carbon source for denitrification in wastewater treatment showcases its versatility in environmental remediation. In addition, PHA has made notable contributions to the medical and consumer sectors, with various roles ranging from 3D printing, tissue engineering implants, and cell growth matrices to drug delivery carriers, and cosmetic products. Through metabolic engineering efforts, PHA can be fine-tuned to align with the specific requirements of each industry, enabling the customization of material properties such as ductility, elasticity, thermal conductivity, and transparency. To unleash PHA's full potential, bridging the gap between research and commercial viability is paramount. Successful PHA production scale-up hinges on establishing direct supply chains to specific application domains, including packaging, food and beverage materials, medical devices, and agriculture. This review underscores that PHA's future rests on ongoing exploration across these industries and more, paving the way for PHA to supplant conventional plastics and foster a circular economy.
Subject(s)
Polyhydroxyalkanoates , Polyhydroxyalkanoates/metabolism , Biopolymers , FoodABSTRACT
The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H-transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme-substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water-mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme-substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme.
Subject(s)
Cyanobacteria , Oxidoreductases Acting on CH-CH Group Donors , Protochlorophyllide/chemistry , Light , Protons , Oxidoreductases Acting on CH-CH Group Donors/metabolism , PhotochemistryABSTRACT
The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.
ABSTRACT
The review by Christianson, published in 2017 on the twentieth anniversary of the emergence of the field, summarizes the foundational discoveries and key advances in terpene synthase/cyclase (TS) biocatalysis (Christianson, D. W. Chem Rev2017, 117 (17), 11570-11648. DOI: 10.1021/acs.chemrev.7b00287). Here, we review the TS literature published since then, bringing the field up to date and looking forward to what could be the near future of TS rational design. Many revealing discoveries have been made in recent years, building on the knowledge and fundamental principles uncovered during those initial two decades of study. We use these to explore TS reaction chemistry and see how a combined experimental and computational approach helps to decipher the complexities of TS catalysis. Revealed are a suite of catalytic motifs which control product outcome in TSs, some obvious, some more subtle. We examine each in detail, using the most recent papers and insights to illustrate how exactly this fascinating class of enzymes takes a single acyclic substrate and turns it into the many thousands of complex terpenoids found in Nature. We then explore some of the recent strategies for TS engineering, including machine learning and other data-driven approaches. From this, rational and predictive engineering of TSs, "designer terpene synthases", will begin to emerge as a realistic goal.
ABSTRACT
BACKGROUND: Production of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions. RESULTS: We describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH14CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin-Bensen-Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions. CONCLUSIONS: This proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors.
ABSTRACT
Oxygenase and peroxygenase enzymes generate intermediates at their active sites which bring about the controlled functionalization of inert C-H bonds in substrates, such as in the enzymatic conversion of methane to methanol. To be viable catalysts, however, these enzymes must also prevent oxidative damage to essential active site residues, which can occur during both coupled and uncoupled turnover. Herein, we use a combination of stopped-flow spectroscopy, targeted mutagenesis, TD-DFT calculations, high-energy resolution fluorescence detection X-ray absorption spectroscopy, and electron paramagnetic resonance spectroscopy to study two transient intermediates that together form a protective pathway built into the active sites of copper-dependent lytic polysaccharide monooxygenases (LPMOs). First, a transient high-valent species is generated at the copper histidine brace active site following treatment of the LPMO with either hydrogen peroxide or peroxyacids in the absence of substrate. This intermediate, which we propose to be a CuII-(histidyl radical), then reacts with a nearby tyrosine residue in an intersystem-crossing reaction to give a ferromagnetically coupled (S = 1) CuII-tyrosyl radical pair, thereby restoring the histidine brace active site to its resting state and allowing it to re-enter the catalytic cycle through reduction. This process gives the enzyme the capacity to minimize damage to the active site histidine residues "on the fly" to increase the total turnover number prior to enzyme deactivation, highlighting how oxidative enzymes are evolved to protect themselves from deleterious side reactions during uncoupled turnover.
Subject(s)
Copper , Histidine , Mixed Function Oxygenases , Oxidative Stress , CatalysisABSTRACT
Biocatalysis is important in the discovery, development, and manufacture of pharmaceuticals. However, the identification of enzymes for target transformations of interest requires major screening efforts. Here, we report a structure-based computational workflow to prioritize protein sequences by a score based on predicted activities on substrates, thereby reducing a resource-intensive laboratory-based biocatalyst screening. We selected imine reductases (IREDs) as a class of biocatalysts to illustrate the application of the computational workflow termed IREDFisher. Validation by using published data showed that IREDFisher can retrieve the best enzymes and increase the hit rate by identifying the top 20 ranked sequences. The power of IREDFisher is confirmed by computationally screening 1400 sequences for chosen reductive amination reactions with different levels of complexity. Highly active IREDs were identified by only testing 20 samples in vitro. Our speed test shows that it only takes 90 min to rank 85 sequences from user input and 30 min for the established IREDFisher database containing 591 IRED sequences. IREDFisher is available as a user-friendly web interface (https://enzymeevolver.com/IREDFisher). IREDFisher enables the rapid discovery of IREDs for applications in synthesis and directed evolution studies, with minimal time and resource expenditure. Future use of the workflow with other enzyme families could be implemented following the modification of the workflow scoring function.
ABSTRACT
CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes.
Subject(s)
Cold Temperature , Histidine , Ligands , Oxidation-Reduction , LightingABSTRACT
Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract.
ABSTRACT
The study of enzymes never disappoints. Despite its long history-almost 150 years following the first documented use of the word enzyme in 1878-the field of enzymology advances apace. This long journey has witnessed landmark developments that have defined modern enzymology as a broad discipline, leading to improved understanding at the molecular level, as we aspire to discover the complex relationships between enzyme structures, catalytic mechanisms and biological function. How enzymes are regulated at the gene and post-translational levels and how catalytic activity is modulated by interactions with small ligands and macromolecules, or the broader enzyme environment, are topical areas of study. Insights from such studies guide the exploitation of natural and engineered enzymes in biomedical or industrial processes; for example, in diagnostics, pharmaceuticals manufacture and processing technologies that use immobilised enzymes and enzyme reactor-based systems. In this Focus Issue, The FEBS Journal seeks to highlight breaking science and informative reviews, as well as personal reflections, to illustrate the breadth and importance of contemporary molecular enzymology research.
Subject(s)
Enzymes , Thermodynamics , Catalysis , Enzymes/genetics , Enzymes/chemistryABSTRACT
Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H4 to FMN, generating a NAD(P)H4 cation radical and anionic FMN semiquinone. This electron transfer occurs in â¼1 ps and appears to kinetically outcompete reductive quenching from aromatic residues in the active site. Time-resolved infrared measurements show that relaxation processes appear to be largely localized on the FMN and the charge-separated state is short-lived, with relaxation, presumably via back electron transfer, occurring over â¼3-30 ps. While this demonstrates the potential for non-natural photoactivity, useful photocatalysis will likely require longer-lived excited states, which may be accessible by enzyme engineering and/or a judicious choice of substrate.
Subject(s)
NAD , Oxidoreductases , Oxidoreductases/chemistry , NAD/chemistry , NADP , Oxidation-Reduction , Electrons , Flavins/chemistry , Phosphates , KineticsABSTRACT
Halomonas bluephagenesis TD1.0 was engineered to produce the biofuel propane, bioplastic poly-3-hydroxybutyrate (PHB), and biochemicals mandelate and hydroxymandelate in a single, semi-continuous batch fermentation under non-sterile conditions. Multi-product separation was achieved by segregation of the headspace gas (propane), fermentation broth ([hydroxy]mandelate) and cellular biomass (PHB). Engineering was performed by incorporating the genes encoding fatty acid photodecarboxylase (CvFAP) and hydroxymandelic acid synthase (SyHMAS) into a H. bluephagenesis hmgCAB cassette knockout to channel flux towards (hydroxy)mandelate. Design of Experiment strategies were coupled with fermentation trials to simultaneously optimize each product. Propane and mandelate titres were the highest reported for H. bluephagenesis (62 g/gDCW and 71 ± 10 mg/L respectively) with PHB titres (69% g/gDCW) comparable to other published studies. This proof-of-concept achievement of four easily separated products within one fermentation is a novel achievement probing the versatility of biotechnology, further elevating H. bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis by producing highly valued products at a reduced cost.
Subject(s)
Halomonas , Fermentation , Halomonas/genetics , Halomonas/metabolism , Biofuels , Propane , Hydroxybutyrates , Polyesters/metabolism , BiopolymersABSTRACT
To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an -NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory "cluster" models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms.
ABSTRACT
The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.
Subject(s)
Brassica , Ferrochelatase , Ferrochelatase/genetics , Heme , Brassica/genetics , Chlorophyll A , Mutation/geneticsABSTRACT
Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biological photocatalysis.