Supplementation of critical amino acids improves glycerol and lactose uptake and enhances recombinant protein production in Escherichia coli.

BACKGROUND: Lactose-based induction strategy in E. coli cultivation has several advantages over IPTG as it is cheap, does not impart metabolic stress to cells, and is non-toxic to cells. However, complexity of lactose as an inducer limits its application in fed-batch cultivation. A mixed glycerol-lactose based induction strategy is generally opted during fed-batch cultivation of E. coli. However, slow growth of E. coli in glycerol and lactose results in slower induction of heterologous protein. MAIN METHODS AND MAJOR RESULTS: In this study, initially we have demonstrated supplementation of critical amino acids (AAs) improves uptake rate of glycerol and lactose in wildtype E. coli BL21(DE3) in defined medium. A feeding strategy of mixed glycerol-lactose feed along with supplement of critical AAs enhances recombinant production of pramlintide multimer (rPramlintide). High cell density cultivation of E. coli using mixed glycerol-lactose feed and critical AAs supplement resulted in final cell density of 52.2 ± 0.90 g L-1  and rPramlintide titer of 7.8 g L-1 . RT-qPCR analysis of genes involved in glycerol and lactose metabolism of recombinant culture showed upregulation with AAs supplementation. CONCLUSIONS AND IMPLICATIONS: We hypothesize that supplementation of critical AAs serves dual purpose: (i) faster assimilation of carbon sources, and (ii) combating metabolic stress arises due to AAs starvation. The substrate uptake and gene expression profiles demonstrate that AAs addition enhances glycerol and lactose assimilation due to overall improvement in their metabolism governed by global regulators of carbon metabolism.

Genomics and transcriptomics analysis reveals the mechanism of isobutanol tolerance of a laboratory evolved Lactococcus lactis strain.

Isobutanol, in spite of its significant superiority over ethanol as a biofuel, remains commercially non-viable due to the non-availability of a suitable chassis which can handle the solvent toxicity associated with its production. To meet this challenge, we chose Lactococcus lactis which is known for its ability to handle environmental stress and carried out Adaptive laboratory evolution (ALE) in a continuous stirred tank reactor (CSTR) to evolve an isobutanol tolerant strain. The strain was grown for more than 60 days (> 250 generations) while gradually increasing the selection pressure, i.e. isobutanol concentration, in the feed. This led to the evolution of a strain that had an exceptionally high tolerance of up to 40 g/l of isobutanol even though a scanning electron microscope (SEM) study as well as analysis of membrane potential revealed only minor changes in cellular morphology. Whole genome sequencing which was done to confirm the strain integrity also showed comparatively few mutations in the evolved strain. However, the criticality of these mutations was reflected in major changes that occurred in the transcriptome, where gene expression levels from a wide range of categories that involved membrane transport, amino acid metabolism, sugar uptake and cell wall synthesis were significantly altered. Analysing the synergistic effect of these changes that lead to the complex phenotype of isobutanol tolerance can help in the construction of better host platforms for isobutanol production.

Amino acid supplementation for enhancing recombinant protein production in E. coli.

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent-like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth-promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

Microaerobic fermentation alters lactose metabolism in Escherichia coli.

Microaerobic fermentation has been shown to improve lactose transport and recombinant protein production in Escherichia coli. Mechanistic correlation between lactose and dissolved oxygen has been studied and it has been demonstrated that E. coli can switch its genetic machinery upon fluctuations in dissolved oxygen levels and thereby impact lactose transport, resulting in product formation. Continuous induction of lactose in microaerobic fermentation led to a 3.3-fold improvement in product titre of rLTNF oligomer and a 1.8-fold improvement in product titre of rSymlin oligomer as compared with traditional aerobic fermentation. Transcriptome profiling indicated that ribosome synthesis, lactose transport and amino acid synthesis genes were upregulated during microaerobic fermentation. Besides, novel lactose transporter setB was examined and it was observed that lactose uptake rate was 1.4-fold higher in microaerobic fermentation. The results indicate that microaerobic fermentation can offer a superior alternative for industrial production of recombinant therapeutics, industrial enzymes and metabolites in E. coli. KEY POINTS: • Microaerobic fermentation results in significantly improved protein production • Lactose transport, ribosome synthesis and amino acid synthesis are enhanced • Product titre improves by 1.8-3.3-fold.

Philopatry and dispersal patterns in tiger (Panthera tigris).

BACKGROUND: Tiger populations are dwindling rapidly making it increasingly difficult to study their dispersal and mating behaviour in the wild, more so tiger being a secretive and solitary carnivore. METHODS: We used non-invasively obtained genetic data to establish the presence of 28 tigers, 22 females and 6 males, within the core area of Pench tiger reserve, Madhya Pradesh. This data was evaluated along with spatial autocorrelation and relatedness analyses to understand patterns of dispersal and philopatry in tigers within this well-managed and healthy tiger habitat in India. RESULTS: We established male-biased dispersal and female philopatry in tigers and reiterated this finding with multiple analyses. Females show positive correlation up to 7 kms (which corresponds to an area of approximately 160 km(2)) however this correlation is significantly positive only upto 4 kms, or 50 km(2) (r  = 0.129, p<0.0125). Males do not exhibit any significant correlation in any of the distance classes within the forest (upto 300 km(2)). We also show evidence of female dispersal upto 26 kms in this landscape. CONCLUSIONS: Animal movements are important for fitness, reproductive success, genetic diversity and gene exchange among populations. In light of the current endangered status of tigers in the world, this study will help us understand tiger behavior and movement. Our findings also have important implications for better management of habitats and interconnecting corridors to save this charismatic species.