Esteban Marcellin

Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

The molecular weight of hyaluronan is important for its rheological and biological function. The molecular mechanisms underlying chain termination and hence molecular weight control remain poorly understood, not only for hyaluronan synthases but also for other β-polysaccharide synthases, e.g. cellulose, chitin, and 1,3-betaglucan synthases. In this work, we manipulated metabolite concentrations in the hyaluronan pathway by overexpressing the five genes of the hyaluronan synthesis operon in Streptococcus equi subsp. zooepidemicus. Overexpression of genes involved in UDP-glucuronic acid biosynthesis decreased molecular weight, whereas overexpression of genes involved in UDP-N-acetylglucosamine biosynthesis increased molecular weight. The highest molecular mass observed was at 3.4 ± 0.1 MDa twice that observed in the wild-type strain, 1.8 ± 0.1 MDa. The data indicate that (a) high molecular weight is achieved when an appropriate balance of UDP-N-acetylglucosamine and UDP-glucuronic acid is achieved, (b) UDP-N-acetylglucosamine exerts the dominant effect on molecular weight, and (c) the wild-type strain has suboptimal levels of UDP-N-acetylglucosamine. Consistent herewith molecular weight correlated strongly (ρ = 0.84, p = 3 × 10−5) with the concentration of UDP-N-acetylglucosamine. Data presented in this paper represent the first model for hyaluronan molecular weight control based on the concentration of activated sugar precursors. These results can be used to engineer strains producing high molecular weight hyaluronan and may provide insight into similar polymerization mechanisms in other polysaccharides.

Engineering and adaptive evolution of Escherichia coli for D-lactate fermentation reveals GatC as a xylose transporter.

Despite the abundance of xylose in nature, the production of chemicals from C5 sugars remains challenging in metabolic engineering. By deleting xylFGH genes and using adaptive evolution, an efficient E. coli strain capable of producing D-lactate from xylose was engineered. Quantitative proteomics and genome sequencing were used to understand the new phenotype and the metabolic limitations of xylose conversion to D-lactate. Proteomics identified major changes in enzyme concentration in the glycolytic and tricarboxylic acid pathways. Whole genome sequencing of the evolved strain identified a point mutation in the gatC gene, which resulted in a change from serine to leucine at position 184 of the GatC protein. The knockout of gatC in a number of strains and the insertion of the mutation in the non-evolved strain confirmed its activity as a xylose transporter and demonstrated that the mutation is responsible for the high xylose consumption phenotype in the evolved strain. The newly found xylose transporter is a candidate for future strain engineering for converting C5-C6 syrups into valuable chemicals.

High-Antibody-Producing Chinese Hamster Ovary Cells Up-Regulate Intracellular Protein Transport and Glutathione Synthesis

Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.

Saccharopolyspora erythraea’s genome is organised in high-order transcriptional regions mediated by targeted degradation at the metabolic switch

BackgroundActinobacteria form a major bacterial phylum that includes numerous human pathogens. Actinobacteria are primary contributors to carbon cycling and also represent a primary source of industrial high value products such as antibiotics and biopesticides. Consistent with other members of the actinobacterial phylum, Saccharopolyspora erythraea undergo a transitional switch. This switch is characterized by numerous metabolic and morphological changes.ResultsWe performed RNA sequencing to analyze the transcriptional changes that occur during growth of Saccharopolyspora erythraea in batch culture. By sequencing RNA across the fermentation time course, at a mean coverage of 4000X, we found the vast majority of genes to be prominently expressed, showing that we attained close to saturating sequencing coverage of the transcriptome. During the metabolic switch, global changes in gene expression influence the metabolic machinery of Saccharopolyspora erythraea, resetting an entirely novel gene expression program. After the switch, global changes include the broad repression of half the genes regulated by complex transcriptional mechanisms. Paralogous transposon clusters, delineate these transcriptional programs. The new transcriptional program is orchestrated by a bottleneck event during which mRNA levels are severely restricted by targeted mRNA degradation.ConclusionsOur results, which attained close to saturating sequencing coverage of the transcriptome, revealed unanticipated transcriptional complexity with almost one third of transcriptional content originating from un-annotated sequences. We showed that the metabolic switch is a sophisticated mechanism of transcriptional regulation capable of resetting and re-synchronizing gene expression programs at extraordinary speed and scale.

Quantitative analysis of intracellular sugar phosphates and sugar nucleotides in encapsulated streptococci using HPAEC‐PAD

Metabolomics is a powerful tool for the study of biological systems. Besides analytical techniques, cell harvest and extraction are critical steps, especially when studying encapsulated streptococci. We have compared four different harvesting techniques for biomass from liquid culture of the hyaluronic acid (HA)‐producing bacterium Streptococcus zooepidemicus. The best method for cell separation was quick (2 min) centrifugation, which allowed efficient medium removal and enabled quantification of the broadest range of sugar metabolites. Unlike observations for other microbes, changes in metabolite pools due to a delay of extraction by the centrifugation were not observed, so metabolite levels accurately reflected the metabolome at the point of cell harvest. A hypothesis is that the capsule itself isolates the cells from the surroundings and still supports it with nutrients during the harvest. Quantification of sugar phosphates and nucleotide sugars was performed using high‐performance anion exchange chromatography combined with pulsed amperometric detection, achieving limits of quantification of 2.5 pmol for sugar phosphates and 5 pmol on column for nucleotide sugars. Intracellular pool sizes for intermediates of the HA pathway under production conditions ranged from 0.2 to 0.5 μmol/g cell dry weight.

Understanding plasmid effect on hyaluronic acid molecular weight produced by Streptococcus equi subsp. zooepidemicus.

Hyaluronic acid is a biopolymer with valuable applications in the pharmaceutical and cosmetic industries. Streptococcus equi subspecies zooepidemicus cells transformed with a nisin-inducible, empty plasmid control displayed higher molecular weight. This increase in molecular weight is independent of the nisin promoter or antibiotic resistance. Using 2D DIGE followed by mass spectrometry, we identified up-regulation of the last step in UDP-N-acetyl-glucosamine biosynthesis (GlmU) and down-regulation of the first step in peptidoglycan biosynthesis (MurA) as possible mechanism for the plasmid effect. Over-expression of GlmU to further increase activity had no effect on UDP-N-acetyl-glucosamine levels or molecular weight, while over-expression of MurA reduced UDP-N-acetyl-glucosamine levels and molecular weight. Global transcriptional analysis revealed that differential regulation of GlmU and MurA activity was not reflected in transcription levels. This results, suggest that regulation is at a translational or post-translational level. Differential expression of two clp proteases may explain this effect as well as the small but significant changes in transcription levels of nearly 300 genes.

Insight into hyaluronic acid molecular weight control

Hyaluronic acid (HA) is a ubiquitous polysaccharide found in humans, animals, bacteria, algae and molluscs. Simple yet sophisticated, HA demonstrates unique and valuable rheological properties. In solution, HA behaves as a stiffened random coil and the resultant behaviour, even at low concentrations, is far from Newtonian or ‘ideal’. These rheological properties are heavily influenced by molecular weight (MW), so it is not surprising that many of the biological functions of HA are dependent on molecular size. The current billion dollar market for HA continues to grow rapidly, both in gross production and the number of applications for its use. Increasing demand, in conjunction with a reticence to use animal-derived HA, has revitalised the market for HA produced by bacterial fermentation. Although the genes and pathways involved in bacterial production of HA are well characterised, the mechanisms that underlie HA MW control are less well understood. By performing a thorough analysis of the proposed mechanisms of MW control in bacterial fermentation, this mini-review tries to elucidate the challenges and future directions for bacterial HA biosynthesis.

Temporal Dynamics of the Saccharopolyspora erythraea Phosphoproteome

Actinomycetes undergo a dramatic reorganization of metabolic and cellular machinery during a brief period of growth arrest (“metabolic switch”) preceding mycelia differentiation and the onset of secondary metabolite biosynthesis. This study explores the role of phosphorylation in coordinating the metabolic switch in the industrial actinomycete Saccharopolyspora erythraea. A total of 109 phosphopeptides from 88 proteins were detected across a 150-h fermentation using open-profile two-dimensional LC-MS proteomics and TiO2 enrichment. Quantitative analysis of the phosphopeptides and their unphosphorylated cognates was possible for 20 pairs that also displayed constant total protein expression. Enzymes from central carbon metabolism such as putative acetyl-coenzyme A carboxylase, isocitrate lyase, and 2-oxoglutarate dehydrogenase changed dramatically in the degree of phosphorylation during the stationary phase, suggesting metabolic rearrangement for the reutilization of substrates and the production of polyketide precursors. In addition, an enzyme involved in cellular response to environmental stress, trypsin-like serine protease (SACE_6340/NC_009142_6216), decreased in phosphorylation during the growth arrest stage. More important, enzymes related to the regulation of protein synthesis underwent rapid phosphorylation changes during this stage. Whereas the degree of phosphorylation of ribonuclease Rne/Rng (SACE_1406/NC_009142_1388) increased during the metabolic switch, that of two ribosomal proteins, S6 (SACE_7351/NC_009142_7233) and S32 (SACE_6101/NC_009142_5981), dramatically decreased during this stage of the fermentation, supporting the hypothesis that ribosome subpopulations differentially regulate translation before and after the metabolic switch. Overall, we show the great potential of phosphoproteomic studies to explain microbial physiology and specifically provide evidence of dynamic protein phosphorylation events across the developmental cycle of actinomycetes.

Cyclic‐di‐AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis

The second messenger cyclic‐di‐adenosine monophosphate (c‐di‐AMP) plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c‐di‐AMP synthesizing diadenylate cyclase (CdaA); however, little is known about signals and effectors controlling CdaA activity and c‐di‐AMP levels. In this study, a genetic screen was employed to identify components which affect the c‐di‐AMP level in Lactococcus. We characterized suppressor mutations that restored osmoresistance to spontaneous c‐di‐AMP phosphodiesterase gdpP mutants, which contain high c‐di‐AMP levels. Loss‐of‐function and gain‐of‐function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c‐di‐AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM, which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c‐di‐AMP level and a reduction in the key peptidoglycan precursor UDP‐N‐acetylglucosamine in L. lactis. C‐di‐AMP synthesis by CdaA was shown to be inhibited by GlmMI154F more than GlmM and GlmMI154F was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c‐di‐AMP level modulating protein and provide a direct connection between c‐di‐AMP synthesis and peptidoglycan biosynthesis.

The role of hyaluronic acid precursor concentrations in molecular weight control in Streptococcus zooepidemicus

The biosynthetic pathway responsible for the production of hyaluronic acid (HA) has been thoroughly studied; however, many aspects remain elusive regarding the mechanisms that control molecular weight (MW). Previously, we demonstrated a positive correlation between MW and the concentration of the HA precursor sugar UDP-N acetylglucosamine (UDP-GlcNAc). To further investigate the role of UDP-GlcNAc in MW control, we increased the intracellular concentration of this monomer using both feeding strategies and genetic engineering approaches. Feeding cells glucosamine dramatically increased intracellular levels of UDP-GlcNAc, but unexpectedly, produced HA of a lower MW. This was subsequently attributed to an equally dramatic decrease in the level of the other HA precursor sugar UDP-glucuronic acid (UDP-GlcUA). Feeding cells a mixture of glucose and GlcNAc addressed this imbalance of precursor sugars, leading to an increase in both UDP-GlcNAc and UDP-GlcUA; however, no significant increase in MW was observed. Despite the increase in UDP-sugars, RNA sequencing identified no increase in the expression of the genes involved in production of HA. Returning to genetic engineering approaches to examine UDP-GlcNAc and MW, genes known to contribute to the production of UDP-GlcNAc were over-expressed, both individually and together. Using this approach, UDP-GlcNAc and MW increased. At lower levels of UDP-GlcNAc, the positive correlation between UDP-GlcNAc levels and MW was maintained, however this relationship stalled at higher concentrations of UDP-GlcNAc. Taken together, these results suggest that while optimising HA precursor levels using feeding or genetic engineering approaches can improve HA MW, there is a point at which excess availability of precursors is no longer advantageous. Once precursor concentrations are addressed, it would seem that other uncharacterised factor(s) (e.g. rate of HA synthesis) also contribute to HA MW control.