Mark P. Hodson

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post‐translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra‐ and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC‐MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism. Biotechnol. Bioeng. 2012; 109:1404–1414.

Flux balance analysis of CHO cells before and after a metabolic switch from lactate production to consumption

Mammalian cell cultures typically exhibit an energy inefficient phenotype characterized by the consumption of large quantities of glucose and the concomitant production of large quantities of lactate. Under certain conditions, mammalian cells can switch to a more energy efficient state during which lactate is consumed. Using a metabolic model derived from a mouse genome scale model we performed flux balance analysis of Chinese hamster ovary cells before and after a metabolic switch from lactate production (in the presence of glucose) to lactate consumption (after glucose depletion). Despite a residual degree of freedom after accounting for measurements, the calculated flux ranges and associated errors were narrow enough to enable investigation of metabolic changes across the metabolic switch. Surprisingly, the fluxes through the lower part of the TCA cycle from oxoglutarate to malate were very similar (around 60 µmol/gDW/h) for both phases. A detailed analysis of the energy metabolism showed that cells consuming lactate have an energy efficiency (total ATP produced per total C-mol substrate consumed) six times greater than lactate producing cells.

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly.

A novel anticonvulsant mechanism via inhibition of complement receptor C5ar1 in murine epilepsy models

The role of complement system-mediated inflammation is of key interest in seizure and epilepsy pathophysiology, but its therapeutic potential has not yet been explored. We observed that the pro-inflammatory C5a receptor, C5ar1, is upregulated in two mouse models after status epilepticus; the pilocarpine model and the intrahippocampal kainate model. The C5ar1 antagonist, PMX53, was used to assess potential anticonvulsant actions of blocking this receptor pathway. PMX53 was found to be anticonvulsant in several acute models (6Hz and corneal kindling) and one chronic seizure model (intrahippocampal kainate model). The effects in the 6Hz model were not found in C5ar1-deficient mice, or with an inactive PMX53 analogue suggesting that the anticonvulsant effect of PMX53 is C5ar1-specific. In the pilocarpine model, inhibition or absence of C5ar1 during status epilepticus lessened seizure power and protected hippocampal neurons from degeneration as well as halved SE-associated mortality. C5ar1-deficiency during pilocarpine-induced status epilepticus also was accompanied by attenuation of TNFα upregulation by microglia, suggesting that C5ar1 activation results in TNFα release contributing to disease. Patch clamp studies showed that C5a-induced microglial K(+) outward currents were also inhibited with PMX53 providing a potential mechanism to explain acute anticonvulsant effects. In conclusion, our data indicate that C5ar1 activation plays a role in seizure initiation and severity, as well as neuronal degeneration following status epilepticus. The widespread anticonvulsant activity of PMX53 suggests that C5ar1 represents a novel target for improved anti-epileptic drug development which may be beneficial for pharmaco-resistant patients.

Discovering the recondite secondary metabolome spectrum of Salinispora species: a study of inter-species diversity.

Patterns of inter-species secondary metabolite production by bacteria can provide valuable information relating to species ecology and evolution. The complex nature of this chemical diversity has previously been probed via directed analyses of a small number of compounds, identified through targeted assays rather than more comprehensive biochemical profiling approaches such as metabolomics. Insights into ecological and evolutionary relationships within bacterial genera can be derived through comparative analysis of broader secondary metabolite patterns, and this can also eventually assist biodiscovery search strategies for new natural products. Here, we investigated the species-level chemical diversity of the two marine actinobacterial species Salinispora arenicola and Salinispora pacifica, isolated from sponges distributed across the Great Barrier Reef (GBR), via their secondary metabolite profiles using LC-MS-based metabolomics. The chemical profiles of these two species were obtained by UHPLC-QToF-MS based metabolic profiling. The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. We found a high level of inter-species diversity in strains from these two bacterial species. We also found rifamycins and saliniketals were produced exclusively by S. arenicola species, as the main secondary metabolites differentiating the two species. Furthermore, the discovery of 57 candidate compounds greatly increases the small number of secondary metabolites previously known to be produced by these species. In addition, we report the production of rifamycin O and W, a key group of ansamycin compounds, in S. arenicola for the first time. Species of the marine actinobacteria harbour a much wider spectrum of secondary metabolites than suspected, and this knowledge may prove a rich field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest

Significance Engineered cardiac muscle can be used to promote the structural and functional maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). However, previous studies have not yet produced cardiac tissues with metabolic and proliferative maturation. Here, we develop a 96-well screening platform and screen for cardiac maturation conditions in engineered cardiac muscle. We found that simulating the postnatal switch in metabolic substrates from carbohydrates to fatty acids promoted a switch in metabolism, DNA damage response, and cell cycle arrest in hPSC-CM. Our study shows that this mechanism can be harnessed to enhance the maturation of human hPSC-CM and cardiac tissues, which has major implications for stem cell sciences, drug discovery, and regenerative medicine. The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

LC-MS-Based Metabolomics Study of Marine Bacterial Secondary Metabolite and Antibiotic Production in Salinispora arenicola

An LC-MS-based metabolomics approach was used to characterise the variation in secondary metabolite production due to changes in the salt content of the growth media as well as across different growth periods (incubation times). We used metabolomics as a tool to investigate the production of rifamycins (antibiotics) and other secondary metabolites in the obligate marine actinobacterial species Salinispora arenicola, isolated from Great Barrier Reef (GBR) sponges, at two defined salt concentrations and over three different incubation periods. The results indicated that a 14 day incubation period is optimal for the maximum production of rifamycin B, whereas rifamycin S and W achieve their maximum concentration at 29 days. A “chemical profile” link between the days of incubation and the salt concentration of the growth medium was shown to exist and reliably represents a critical point for selection of growth medium and harvest time.

Alterations of hippocampal glucose metabolism by even versus uneven medium chain triglycerides

Medium chain triglycerides (MCTs) are used to treat neurologic disorders with metabolic impairments, including childhood epilepsy and early Alzheimers disease. However, the metabolic effects of MCTs in the brain are still unclear. Here, we studied the effects of feeding even and uneven MCTs on brain glucose metabolism in the mouse. Adult mice were fed 35% (calories) of trioctanoin or triheptanoin (the triglycerides of octanoate or heptanoate, respectively) or a matching control diet for 3 weeks. Enzymatic assays and targeted metabolomics by liquid chromatography tandem mass spectrometry were used to quantify metabolites in extracts from the hippocampal formations (HFs). Both oils increased the levels of β-hydroxybutyrate, but no other significant metabolic alterations were observed after triheptanoin feeding. The levels of glucose 6-phosphate and fructose 6-phosphate were increased in the HF of mice fed trioctanoin, whereas levels of metabolites further downstream in the glycolytic pathway and the pentose phosphate pathway were reduced. This indicates that trioctanoin reduces glucose utilization because of a decrease in phosphofructokinase activity. Trioctanoin and triheptanoin showed similar anticonvulsant effects in the 6 Hz seizure model, but it remains unknown to what extent the anticonvulsant mechanism(s) are shared. In conclusion, triheptanoin unlike trioctanoin appears to not alter glucose metabolism in the healthy brain.

Inflammatory-induced hibernation in the fetus: priming of fetal sheep metabolism correlates with developmental brain injury.

Prenatal inflammation is considered an important factor contributing to preterm birth and neonatal mortality and morbidity. The impact of prenatal inflammation on fetal bioenergetic status and the correlation of specific metabolites to inflammatory-induced developmental brain injury are unknown. We used a global metabolomics approach to examine plasma metabolites differentially regulated by intrauterine inflammation. Preterm-equivalent sheep fetuses were randomized to i.v. bolus infusion of either saline-vehicle or LPS. Blood samples were collected at baseline 2 h, 6 h and daily up to 10 days for metabolite quantification. Animals were killed at 10 days after LPS injection, and brain injury was assessed by histopathology. We detected both acute and delayed effects of LPS on fetal metabolism, with a long-term down-regulation of fetal energy metabolism. Within the first 3 days after LPS, 121 metabolites were up-regulated or down-regulated. A transient phase (4–6 days), in which metabolite levels recovered to baseline, was followed by a second phase marked by an opposing down-regulation of energy metabolites, increased pO2 and increased markers of inflammation and ADMA. The characteristics of the metabolite response to LPS in these two phases, defined as 2 h to 2 days and at 6–9 days, respectively, were strongly correlated with white and grey matter volumes at 10 days recovery. Based on these results we propose a novel concept of inflammatory-induced hibernation of the fetus. Inflammatory priming of fetal metabolism correlated with measures of brain injury, suggesting potential for future biomarker research and the identification of therapeutic targets.

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.