HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Metabolic flux enhancement from the translational fusion of terpene synthases is linked to terpene synthase accumulation.

The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), corresponding with increases of a similar magnitude in terpene synthase levels. Our data demonstrate that increased in vivo enzyme levels - resulting from improved expression and/or improved protein stability - is a major driver of catalytic enhancement from enzyme fusion.

Characterisation of acetogen formatotrophic potential using Eubacterium limosum.

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280 ± 56 mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO2 (150 ± 11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. KEY POINTS: First Eubacterium limosum steady-state formatotrophic growth omics dataset High formate specific uptake rate, however carbon dioxide was the major product Formate may be the cause of intracellular stress and biofilm formation.

Redox controls metabolic robustness in the gas-fermenting acetogen Clostridium autoethanogenum.

Living biological systems display a fascinating ability to self-organize their metabolism. This ability ultimately determines the metabolic robustness that is fundamental to controlling cellular behavior. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cultures exhibit rhythmic oscillatory behavior in high cell-density continuous cultures. Oscillatory behavior provides a unique opportunity for quantitating the robustness of metabolism, as cells respond to changes by inherently compromising metabolic efficiency. Here, we quantify the limits of metabolic robustness in self-oscillating autotrophic continuous cultures of the gas-fermenting acetogen Clostridium autoethanogenum Online gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular byproducts synchronized with biomass levels. The data show initial growth on CO, followed by growth on CO and H2 Growth on CO and H2 results in an accelerated growth phase, after which a downcycle is observed in synchrony with a loss in H2 uptake. Intriguingly, oscillations are not linked to translational control, as no differences were observed in protein expression during oscillations. Intracellular metabolomics analysis revealed decreasing levels of redox ratios in synchrony with the cycles. We then developed a thermodynamic metabolic flux analysis model to investigate whether regulation in acetogens is controlled at the thermodynamic level. We used endo- and exo-metabolomics data to show that the thermodynamic driving force of critical reactions collapsed as H2 uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in acetogen gas fermentation are controlled at the thermodynamic level.

Glyceryl triacetate feeding in mice increases plasma acetate levels but has no anticonvulsant effects in acute electrical seizure models.

INTRODUCTION: Acetate has been shown to have neuroprotective and anti-inflammatory effects. It is oxidized by astrocytes and can thus provide auxiliary energy to the brain in addition to glucose. Therefore, we hypothesized that it may protect against seizures, which is investigated here by feeding glyceryl triacetate (GTA), to provide high amounts of acetate without raising sodium or acid levels. METHOD: CD1 male mice were fed controlled diets with or without GTA for up to three weeks. Body weights, blood glucose levels, plasma short-chain fatty acid levels, and other hematological parameters were monitored. Seizure thresholds were determined in 6 Hz and maximal electroshock seizure threshold (MEST) tests. Antioxidant capacities were evaluated in the cerebral cortex and plasma using a ferric reducing antioxidant power (FRAP) assay and Trolox equivalent antioxidant capacity assay. RESULTS: Body weight gain was similar with both diets with and without GTA in two experiments. Glyceryl triacetate-fed groups showed 2-3- and 1.6-fold increased acetate and propionate levels in plasma, respectively. Glucose levels were unaltered in blood collected from the tail tip but increased in trunk blood. No differences were found in the activity of cerebral cortex acetyl-CoA synthetase. In the 6 Hz threshold test, seizure thresholds were lower by 3 mA and 2.4 mA after 8 and 14 days, respectively, in the GTA compared to the control diet-fed group, but showed no difference on day 16, showing that GTA has small, but inconsistent proconvulsant effects in this model. In MEST tests, a slightly increased seizure threshold (1 mA) was found on day 19 in the GTA-fed group, but not in another experiment on day 21. There were no differences in antioxidant capacity in plasma or cortex between the two groups. CONCLUSION: Glyceryl triacetate feeding showed no antioxidant effects nor beneficial changes in acute electrical seizure threshold mouse models, despite its ability to increase plasma acetate levels.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

Production of human vitronectin in Nicotiana benthamiana using the INPACT hyperexpression platform.

Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum-free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol-inducible AlcR:alcA gene switch. hVN expression was maximal 4-5 days postactivation of the INPACT platform with a dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of 643 ± 78 mg/kg fresh weight. A three-stage purification protocol was developed using heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant-made hVN product of >90% purity. Storage conditions for plant-made hVN were identified that maximized the capacity of the recombinant protein to promote cell adhesion. Critically, plant-made hVN was shown to be functionally equivalent to commercial, plasma-derived hVN at promoting one-half maximal attachment of murine fibroblast cells (BALB-C/3T3) in serum-free medium at <0.1 µg/cm2 to tissue culture plasticware. The INPACT platform represents an attractive means of producing large quantities of functional, animal-free hVN for in vitro applications.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

A squalene synthase protein degradation method for improved sesquiterpene production in Saccharomyces cerevisiae.

Sesquiterpenes are C15 isoprenoids with utility as fragrances, flavours, pharmaceuticals, and potential biofuels. Microbial fermentation is being examined as a competitive approach for bulk production of these compounds. Competition for carbon allocation between synthesis of endogenous sterols and production of the introduced sesquiterpene limits yields. Achieving balance between endogenous sterols and heterologous sesquiterpenes is therefore required to achieve economical yields. In the current study, the yeast Saccharomyces cerevisiae was used to produce the acyclic sesquiterpene alcohol, trans-nerolidol. Nerolidol production was first improved by enhancing the upstream mevalonate pathway for the synthesis of the precursor farnesyl pyrophosphate (FPP). However, excess FPP was partially directed towards squalene by squalene synthase (Erg9p), resulting in squalene accumulation to 1% biomass; moreover, the specific growth rate declined. In order to re-direct carbon away from sterol production and towards the desired heterologous sesquiterpene, a novel protein destabilisation approach was developed for Erg9p. It was shown that Erg9p is located on endoplasmic reticulum and lipid droplets through a C-terminal ER-targeted transmembrane peptide. A PEST (rich in Pro, Glu/Asp, Ser, and Thr) sequence-dependent endoplasmic reticulum-associated protein degradation (ERAD) mechanism was established to decrease cellular levels of Erg9p without relying on inducers, repressors or specific repressing conditions. This improved nerolidol titre by 86% to ~100mgL-1. In this strain, squalene levels were similar to the wild-type control strain, and downstream ergosterol levels were slightly decreased relative to the control, indicating redirection of carbon away from sterols and towards sesquiterpene production. There was no negative effect on cell growth under these conditions. Protein degradation is an efficient mechanism to control carbon allocation at flux-competing nodes in metabolic engineering applications. This study demonstrates that an engineered ERAD mechanism can be used to balance flux competition between the endogenous sterol pathway and an introduced bio-product pathways at the FPP node. The approach of protein degradation in general might be more widely applied to improve metabolic engineering outcomes.

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum.

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO2 and H2). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (tD~4h) compared to growth on yeast extract (tD~9h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase.

BACKGROUND: Propionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed. RESULTS: Propionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation. CONCLUSIONS: The native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway was shown to be functional under anaerobic and aerobic conditions.

Systems biology and metabolic modelling unveils limitations to polyhydroxybutyrate accumulation in sugarcane leaves; lessons for C4 engineering.

In planta production of the bioplastic polyhydroxybutyrate (PHB) is one important way in which plant biotechnology can address environmental problems and emerging issues related to peak oil. However, high biomass C4 plants such as maize, switch grass and sugarcane develop adverse phenotypes including stunting, chlorosis and reduced biomass as PHB levels in leaves increase. In this study, we explore limitations to PHB accumulation in sugarcane chloroplasts using a systems biology approach, coupled with a metabolic model of C4 photosynthesis. Decreased assimilation was evident in high PHB-producing sugarcane plants, which also showed a dramatic decrease in sucrose and starch content of leaves. A subtle decrease in the C/N ratio was found which was not associated with a decrease in total protein content. An increase in amino acids used for nitrogen recapture was also observed. Based on the accumulation of substrates of ATP-dependent reactions, we hypothesized ATP starvation in bundle sheath chloroplasts. This was supported by mRNA differential expression patterns. The disruption in ATP supply in bundle sheath cells appears to be linked to the physical presence of the PHB polymer which may disrupt photosynthesis by scattering photosynthetically active radiation and/or physically disrupting thylakoid membranes.

Increased sensitivity to tryptophan bioavailability is a positive adaptation by the human strains of Chlamydia pneumoniae.

One of the most significant activities induced by interferon-gamma against intracellular pathogens is the induction of IDO (indoleamine 2,3-dioxygenase) expression, which subsequently results in the depletion of tryptophan. We tested the hypothesis that human strains of Chlamydia pneumoniae are more sensitive to tryptophan limitation than animal C. pneumoniae strains. The human strains were significantly more sensitive to IFN-γ than the animal strains in a lung epithelia cell model (BEAS-2B), with exposure to 1 U ml(-1) IFN-γ resulting in complete loss of infectious yield of human strains, compared to the animal strains where reductions in infectious progeny were around 3.5-4.0 log. Strikingly, the IFN-γ induced loss of ability to form infectious progeny production was completely rescued by removal of the IFN-γ and addition of exogenous tryptophan for the human strains, but not the animal strains. In fact, a human heart strain was more capable of entering a non-infectious, viable persistent stage when exposed to IFN-γ and was also more effectively rescued, compared to a human respiratory strain. Exquisite susceptibility to IFN-γ, specifically due to tryptophan availability appears to be a core adaptation of the human C. pneumoniae strains, which may reflect the chronic nature of their infections in this host.

The use of an acetoacetyl-CoA synthase in place of a ß-ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells.

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing ß-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the ß-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.

Circulating TMAO, the gut microbiome and cardiometabolic disease risk: an exploration in key precursor disorders.

BACKGROUND: Elevations in the gut metabolite trimethylamine-N-oxide (TMAO) have been linked to cardiovascular and metabolic diseases. Whether elevated TMAO levels reflect early mechanistic involvement or a sequela of evolving disease awaits elucidation. The purpose of this study was to further explore these potential associations. METHODS: We investigated relationships between circulating levels of TMAO and its pre-cursor substrates, dietary factors, gut microbiome profiles and disease risk in individuals with a Healthy BMI (18.5 < BMI < 25, n = 41) or key precursor states for cardiometabolic disease: Overweight (25 < BMI < 30 kg/m2, n = 33), Obese (BMI > 30, n = 27) and Metabolic Syndrome (MetS; ≥ 3 ATPIII report criteria, n = 39). RESULTS: Unexpectedly, plasma [TMAO] did not vary substantially between groups (means of 3-4 µM; p > 0.05), although carnitine was elevated in participants with MetS. Gut microbial diversity and Firmicutes were also significantly reduced in the MetS group (p < 0.05). Exploratory analysis across diverse parameters reveals significant correlations between circulating [TMAO] and seafood intake (p = 0.007), gut microbial diversity (p = 0.017-0.048), and plasma [trimethylamine] (TMA; p = 0.001). No associations were evident with anthropometric parameters or cardiometabolic disease risk. Most variance in [TMAO] within and between groups remained unexplained. CONCLUSIONS: Data indicate that circulating [TMAO] may be significantly linked to seafood intake, levels of TMA substrate and gut microbial diversity across healthy and early disease phenotypes. However, mean concentrations remain < 5 µM, with little evidence of links between TMAO and cardiometabolic disease risk. These observations suggest circulating TMAO may not participate mechanistically in cardiometabolic disease development, with later elevations likely a detrimental sequela of extant disease.

Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae.

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L-1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause─the chromosome integration of a 2µ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L-1 nerolidol and ∼0.96 g L-1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2µ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

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.

High methanol-to-formate ratios induce butanol production in Eubacterium limosum.

Unlike gaseous C1 feedstocks for acetogenic bacteria, there has been less attention on liquid C1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW-1 day-1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h-1 ) and cell density (maximum 1.2 ± 0.043 gDCW l-1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases - growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin.

Microbial Production, Extraction, and Quantitative Analysis of Isoprenoids.

Isoprenoids, also known as terpenes or terpenoids, are compounds made of one or more isoprene (C5H8) moieties and constitute the largest class of natural products. They play diverse roles in biology and have broad industrial uses as flavors, fragrances, biofuels, polymers, agricultural chemicals, and medicines. Most isoprenoids are secondary plant metabolites and only produced in very low amounts. To make these valuable compounds economically accessible, significant efforts in the culture and engineering of microbial cells for isoprenoid biosynthesis have been made in the last decades. The protocols presented here describe lab-scale cultivation of microbes, either naturally producing or engineered, for isoprenoid production, the extraction of products and their quantification by high-performance liquid chromatography. Examples of isoprenoids covered in this chapter include (C10) mono-, (C15) sesqui-, (C20) di-, (C30) tri-, and (C40) tetraterpenoids. We focus on yeast and cyanobacteria as production systems, but the protocols can be adapted for other organisms.

Molecular characterization of cyanobacterial short-chain prenyltransferases and discovery of a novel GGPP phosphatase.

Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short-chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type-II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C20 ) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C15 ) and geranyl pyrophosphate (GPP, C10 ); whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co-encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain-length terpenoids in cyanobacteria.