Heterologous Production Research Articles

Yeast-based heterologous production of the Colletochlorin family of fungal secondary metabolites

Transcriptomic studies have revealed that fungal pathogens of plants activate the expression of numerous biosynthetic gene clusters (BGC) exclusively when in presence of a living host plant. The identification and structural elucidation of the corresponding secondary metabolites remain challenging. The aim was to develop a polycistronic system for heterologous expression of fungal BGCs in Saccharomyces cerevisiae. Here we adapted a polycistronic vector for efficient, seamless and cost-effective cloning of biosynthetic genes using in vivo assembly (also called transformation-assisted recombination) directly in Escherichia coli followed by heterologous expression in S. cerevisiae. Two vectors were generated with different auto-inducible yeast promoters and selection markers. The effectiveness of these vectors was validated with fluorescent proteins. As a proof-of-principle, we applied our approach to the Colletochlorin family of molecules. These polyketide secondary metabolites were known from the phytopathogenic fungus Colletotrichum higginsianum but had never been linked to their biosynthetic genes. Considering the requirement for a halogenase, and by applying comparative genomics, we identified a BGC putatively involved in the biosynthesis of Colletochlorins in C. higginsianum. Following the expression of those genes in S. cerevisiae, we could identify the presence of the precursor Orsellinic acid, Colletochlorins and their non-chlorinated counterparts, the Colletorins. In conclusion, the polycistronic vectors described herein were adapted for the host S. cerevisiae and allowed to link the Colletochlorin compound family to their corresponding biosynthetic genes. This system will now enable the production and purification of infection-specific secondary metabolites of fungal phytopathogens. More widely, this system could be applied to any fungal BGC of interest.

Multicopy Chromosome Integration and Deletion of Negative Global Regulators Significantly Increased the Heterologous Production of Aborycin in Streptomyces coelicolor.

Aborycin is a type I lasso peptide with a stable interlocked structure, offering a favorable framework for drug development. The aborycin biosynthetic gene cluster gul from marine sponge-associated Streptomyces sp. HNS054 was cloned and integrated into the chromosome of S. coelicolor hosts with different copies. The three-copy gul-integration strain S. coelicolor M1346::3gul showed superior production compared to the one-copy or two-copy gul-integration strains, and the total titer reached approximately 10.4 mg/L, i.e., 2.1 times that of the native strain. Then, five regulatory genes, phoU (SCO4228), wblA (SCO3579), SCO1712, orrA (SCO3008) and gntR (SCO1678), which reportedly have negative effects on secondary metabolism, were further knocked out from the M1346::3gul genome by CRISPR/Cas9 technology. While the ΔSCO1712 mutant showed a significant decrease (4.6 mg/L) and the ΔphoU mutant showed no significant improvement (12.1 mg/L) in aborycin production, the ΔwblA, ΔorrA and ΔgntR mutations significantly improved the aborycin titers to approximately 23.6 mg/L, 56.3 mg/L and 48.2 mg/L, respectively, which were among the highest heterologous yields for lasso peptides in both Escherichia coli systems and Streptomyces systems. Thus, this study provides important clues for future studies on enhancing antibiotic production in Streptomyces systems.

Effect of autologous umbilical cord blood transfusion in the development of retinopathy of prematurity: randomized clinical trial - study protocol.

Currently, the treatment of anemia in preterm infants is based on packed red blood cell (RBC) transfusions from adult donors. Oxygen (O2) is mainly transported to the tissues bound to hemoglobin (Hb). In extremely low gestational age neonates (ELGANs), fetal hemoglobin (HbF), which has a higher affinity for O2, represents up to 95% of circulating hemoglobin. During the first month of life, the majority of ELGANs will require an adult-donor RBC transfusion causing HbF levels to rapidly drop. HbA releases 50% more oxygen in peripheral tissues than HbF. Increased release of O2 in the retina is one of the main factors related to the development of retinopathy of prematurity (ROP). Collecting umbilical cord blood and using autologous umbilical cord whole blood (UCB) transfusions would contribute to maintaining physiological HbF concentrations in newborns and avoid oxygen-in-excess derived damage. This is a randomized, double-blinded, multicenter clinical trial. ELGANs ≤28 weeks of gestational age will be randomized 1:1 to receive an autologous umbilical cord blood transfusion (intervention arm) or standard transfusion of packed RBC from an adult donor (control arm) to assess ROP development. Assuming a 50% reduction in ROP incidence, 134 patients (67 per group) will be recruited. When blood transfusion is indicated, the Blook Bank will supply UCB or RCB according to the patient's group. The primary endpoint is the incidence of any ROP. Secondary endpoints are assessessment of treatment safety, results of biomarkers related to ROP and its chronology, and urine oxidative stress markers. In addition, the cellular composition of umbilical cord blood and its relationship with prematurity-related pathologies will be analyzed. All patients will be followed-up to 24 months of corrected age to evaluate their neurodevelopment. ROP is a major cause of irreversible blindness in preterm newborns. Transfusions with adult donor blood can lead to complications, including ROP. UCB transfusions offer advantages by maintaining physiological HbF levels and potentially optimizing postnatal development. Moreover, autologous UCB transfusion could reduce risks associated with heterologous blood products, although volume collection remains challenging. UCB contains growth factors and progenitor cells that may impact ROP.

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis**

AbstractTerpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC‐like enzymes are known but are very rare. In this study, we genome‐mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A−C (1–3), that possess a 6/7/5‐fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two‐step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10‐fused bicyclic cis‐eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5‐fused tricyclic scaffold through C2−C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF‐mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis.

Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. In this study, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A-C (1-3), that possess a 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold through C2-C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF-mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.

Heterologous Production in the Synechocystis Chassis Suggests the Biosynthetic Pathway of Astaxanthin in Cyanobacteria.

Astaxanthin is a carotenoid species with the highest antioxidant capability. Its natural resource is very rare. The biosynthesis of astaxanthin from β-carotene includes a hydroxylation step and a ketolation step, for which the corresponding enzymes have been characterized in a few species. However, the sequence of these two reactions is unclear, and may vary with different organisms. In this study, we aimed to elucidate this sequence in Synechocystis, which is an ideal cyanobacterial synthetic biology chassis. We first silenced the endogenous carotene oxygenase gene SyneCrtO to avoid its possible interference in the carotenoid metabolic network. We then introduced the β-carotene ketolase gene from Haematococcus pluvialis (HpBKT) and the CrtZ-type carotene β-hydroxylase gene from Pantoea agglomerans (PaCrtZ) to this δCrtO strain. Our pigment analysis demonstrated that both the endogenous CrtR-type carotene hydroxylase SyneCrtR and HpBKT have the preference to use β-carotene as their substrate for hydroxylation and ketolation reactions to produce zeaxanthin and canthaxanthin, respectively. However, the endogenous SyneCrtR is not able to further catalyze the 3,3'-hydroxylation of canthaxanthin to generate astaxanthin. From our results, a higher accumulation of canthaxanthin and a much lower level of astaxanthin, as confirmed using liquid chromatography-tandem mass spectrometry analysis, were detected in our transgenic BKT+/CrtZ+/δCrtO cells. Therefore, we proposed that the bottleneck for the heterologous production of astaxanthin in Synechocystis might exist at the hydroxylation step, which requires a comprehensive screening or genetic engineering for the corresponding carotene hydroxylase to enable the industrial production of astaxanthin.

Product Profiles of Promiscuous Enzymes Can be Altered by Controlling In Vivo Spatial Organization.

Enzyme spatial organization is an evolved mechanism for facilitating multi-step biocatalysis and can play an important role in the regulation of promiscuous enzymes. The latter function suggests that artificial spatial organization can be an untapped avenue for controlling the specificity of bioengineered metabolic pathways. A promiscuous terpene synthase (nerolidol synthase) is co-localized and spatially organized with the preceding enzyme (farnesyl diphosphate synthase) in a heterologous production pathway, via translational protein fusion and/or co-encapsulation in a self-assembling protein cage. Spatial organization enhances nerolidol production by ≈11- to ≈62-fold relative to unorganized enzymes. More interestingly, striking differences in the ratio of end products (nerolidol and linalool) are observed with each spatial organization approach. This demonstrates that artificial spatial organization approaches can be harnessed to modulate the product profiles of promiscuous enzymes in engineered pathways in vivo. This extends the application of spatial organization beyond situations where multiple enzymes compete for a single substrate to cases where there is competition among multiple substrates for a single enzyme.

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes—isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.

Heterologous Production and Biosynthesis of Threonine-16:0dioic acids with a Hydroxamate Moiety.

Dereplication and genome mining in Streptomyces aureus LU18118 combined with heterologous expression of selected biosynthetic gene clusters (BGCs) led to the discovery of various threonine-16:0dioic acids named lipothrenins. Lipothrenins consist of the core elements l-Thr, d-allo-Thr, or Dhb, which are linked to hexadecanedioic acid by an amide bond. The main compound lipothrenin A (1) carries the N-hydroxylated d-allo form of threonine and expresses a siderophore activity. The lipothrenin BGC was analyzed by a series of deletion experiments. As a result, a variety of interesting genes involved in the recruitment and selective activation of linear 16:0dioic acids, amide bond formation, and the epimerization of l-Thr were revealed. Furthermore, a diiron N-oxygenase was identified that may be directly involved in the monooxygenation of the amide bond. This is divergent from the usual hydroxamate formation mechanism in siderophores, which involves hydroxylation of the free amine prior to amide bond formation. Siderophore activity was observed for all N-hydroxylated lipothrenins by application of the CAS assay method.

A temperature dependent pilin promoter for production of thermostable enzymes in Thermus thermophilus

BackgroundEnzymes from thermophiles are of great interest for research and bioengineering due to their stability and efficiency. Thermophilic expression hosts such as Thermus thermophilus [T. thermophilus] can overcome specific challenges experienced with protein production in mesophilic expression hosts, such as leading to better folding, increased protein stability, solubility, and enzymatic activity. However, available inducible promoters for efficient protein production in T. thermophilus HB27 are limited.ResultsIn this study, we characterized the pilA4 promoter region and evaluated its potential as a tool for production of thermostable enzymes in T. thermophilus HB27. Reporter gene analysis using a promoterless β-glucosidase gene revealed that the pilA4 promoter is highly active under optimal growth conditions at 68 °C and downregulated during growth at 80 °C. Furthermore, growth in minimal medium led to significantly increased promoter activity in comparison to growth in complex medium. Finally, we proved the suitability of the pilA4 promoter for heterologous production of thermostable enzymes in T. thermophilus by producing a fully active soluble mannitol-1-phosphate dehydrogenase from Thermoanaerobacter kivui [T. kivui], which is used in degradation of brown algae that are rich in mannitol.ConclusionsOur results show that the pilA4 promoter is an efficient tool for gene expression in T. thermophilus with a high potential for use in biotechnology and synthetic biology applications.

Production of (hydroxy)benzoate-derived polyketides by engineered Pseudomonas with in situ extraction

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3′,4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives.

Heterologous Biosynthesis of Complex Bacterial Natural Products in Burkholderia gladioli.

Bacterial natural products (NPs) are an indispensable source of drugs and biopesticides. Heterologous expression is an essential method for discovering bacterial NPs and the efficient biosynthesis of valuable NPs, but the chassis for Gram-negative bacterial NPs remains inadequate. In this study, we built a Burkholderiales mutant Burkholderia gladioli Δgbn::attB by introducing an integrated site (attB) to inactivate the native gladiolin (gbn) biosynthetic gene cluster, which stabilizes large foreign gene clusters and reduces the native metabolite profile. The growth and successful heterologous production of high-value NPs such as phylogenetically close Burkholderiales-derived antitumor polyketides (PKs) rhizoxins, phylogenetically distant Gammaproteobacteria-derived anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotics WAP-8294As, and Deltaproteobacteria-derived antitumor PKs disorazols demonstrate that this strain is a potential chassis for Gram-negative bacterial NPs. We further improved the yields of WAP-8294As through promoter insertions and precursor pathway overexpression based on heterologous expression in this strain. This study provides a robust bacterial chassis for genome mining, efficient production, and molecular engineering of bacterial NPs.

Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks

Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4–4.5 g/L, representing 35–40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production.Key points• Process-related fermentation stresses influence heterologous protein production.• Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP.• CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.

Green synthesis of gold nanoparticles by engineered Escherichia coli for photodegradation of p-nitrophenol

Gold nanoparticles (AuNPs) are widely employed in the environmental, energy, and biomedical fields. The green synthesis of AuNPs is significant for reducing the hazardous chemicals and toxic byproducts of traditional synthesis procedures. In this study, engineered Escherichia coli (E. coli-GolR) capable of heterologous production of the gold-reducing protein (GolR) were constructed. Uniform and stable loading of AuNPs on E. coli-GolR were demonstrated by characterization. Experiments using live-dead cell labeling showed that E. coli-GolR was able to withstand the toxicity of AuIII. Within 5 min, 3 mM of 4-nitrophenol (4-NP) could be totally broken down by the acquired AuNPs@E. coli-GolR. More importantly, the AuNPs@E. coli-GolR could be recycled at least ten times, significantly decreasing the cost of their application. This work broadens the possibilities for using AuNPs by offering fresh insights into the microbial production of AuNPs.

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker’s yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants’ heterologous microbial production and could impact the breeding of industrial and ornamental plants.

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5–7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Production of Potential Substitutes for Conventional Plastics Using Metabolically Engineered Acetobacterium woodii

Increasing greenhouse gas emissions and decreasing fossil fuel supplies necessitate the development of alternative methods for producing petroleum-based commodities. Plastics are also primarily petroleum-based goods with rising demand, thus there is growing interest in plastic substitutes. Polyhydroxyalkanoates (PHAs) are naturally produced biopolymers that are utilized by microorganisms as a source of energy and carbon storage. Poly-3-hydroxybutyrate (PHB) is a member of the PHA family and is considered the most promising candidate to replace polyethylene (PE). PHB is naturally produced by Cupriavidus necator, but recombinant production has also been recently established. This study is the first to investigate the heterologous production of PHB with recombinant Acetobacterium woodii using CO2 + H2 as a carbon and energy source. The introduction of a synthetic PHB production pathway resulted in the production of 1.23 g/L CDW and 1.9% PHB/cell dry weight (CDW), which corresponds to a production of 23.5 mg/L PHB. PHB quantification was simplified using LipidGreen2 fluorescence measurements.

CYP72D19 from Tripterygium wilfordii catalyzes C-2 hydroxylation of abietane-type diterpenoids.

CYP72D19, the first functional gene of the CYP72D subfamily, catalyzes the C-2 hydroxylation of abietane-type diterpenoids. The abietane-type diterpenoids, e.g., triptolide, tripdiolide, and 2-epitripdiolide, are the main natural products for the anti-tumor, anti-inflammatory, and immunosuppressive activities of Tripterygium wilfordii, while their biosynthetic pathways are not resolved. Here, we cloned and characterized the CYP72D19-catalyzed C-2 hydroxylation of dehydroabietic acid, a compound that has been proven to be a biosynthetic intermediate in triptolide biosynthesis. Through molecular docking and site-directed mutagenesis, L386, L387, and I493 near the active pocket were found to have an important effect on the enzyme activity, which also indicates that steric hindrance of residues plays an important role in function. In addition, CYP72D19 also catalyzed a variety of abietane-type diterpenoids with benzene ring, presumably because the benzene ring of the substrate molecule stabilized the C-ring, allowing the protein and the substrate to form a relatively stable spatial structure. This is the first demonstration of CYP72D subfamily gene function. Our research provides important genetic elements for the structural modification of active ingredients and the heterologous production of other 2-hydroxyl abietane-type natural products.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies.