Microbial production of 1,3-propanediol: Recent developments and emerging opportunities

1,3-Propanediol, a valuable bifunctional molecule, can be produced from renewable resources using microorganisms. It has several promising properties for many synthetic reactions, particularly for polymer and cosmetic industries. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,3-propanediol. Various strategies for the microbial production of 1,3-propanediol are reviewed and compared in this article with their promises and constraints. Furthermore, genetic and metabolic engineering could significantly improve product yields and overcome the limitations of fermentation technology. Present review gives an overview on 1,3-propanediol production by wild and recombinant strains. It also attempts to encompass the various issues concerned in utilization of crude glycerol for 1,3-propanediol production, with particular emphasis laid on biodiesel industries. This review also summarizes the present state of strategies studied for the downstream processing and purification of biologically produced 1,3-propanediol. The future prospect of 1,3-propanediol and its potential as a major bulk chemical are discussed under the light of the current research.     

A statistical method for enhancing the production of succinic acid from Escherichia coli under anaerobic conditions

The most influential parameters for succinic acid production obtained through one at a time method were sucrose, tryptone, magnesium carbonate, inoculum size and incubation period. These resulted in the production of 7.0 g L(-1) of succinic acid in 60 h from Escherichia coli W3110 under anaerobic conditions. Based on these results, a statistical method, face centered central composite design (FCCCD) falling under response surface method (RSM) was employed for further enhancing the succinic acid production and to monitor the interactive effect of these parameters, which resulted in a twofold increase in yield (14.3 g L(-1) in 48 h). The analysis of variance (ANOVA) showed the adequacy of the model and the verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using conditions optimized through RSM, 24.2 g L(-1) of succinic acid was obtained in 30 h. This clearly indicated that the model stood valid even on large-scale. Thus, the statistical optimization strategy led to a 3.5-fold increase in the yield of succinic acid. This is the first report on the use of FCCCD to improve succinic acid production from E. coli.     

An improved procedure of mapping a quantitative trait locus via the EM algorithm using posterior probabilities

Mapping a locus controlling a quantitative genetic trait (e.g. blood pressure) to a specific genomic region is of considerable contemporary interest. Data on the quantitative trait under consideration and several codominant genetic markers with known genomic locations are collected from members of families and statistically analysed to estimate the recombination fraction, θ, between the putative quantitative trait locus and a genetic marker. One of the major complications in estimating θ for a quantitative trait in humans is the lack of haplotype information on members of families. We have devised a computationally simple two-stage method of estimation of θ in the absence of haplotypic information using the expectation-maximization (EM) algorithm. In the first stage, parameters of the quantitative trait locus (QTL) are estimated on the basis of data of a sample of unrelated individuals and a Bayes’s rule is used to classify each parent into a QTL genotypic class. In the second stage, we have proposed an EM algorithm for obtaining the maximum-likelihood estimate of θ based on data of informative families (which are identified upon inferring parental QTL genotypes performed in the first stage). The purpose of this paper is to investigate whether, instead of using genotypically ‘classified’ data of parents, the use of posterior probabilities of QT genotypes of parents at the second stage yields better estimators. We show, using simulated data, that the proposed procedure using posterior probabilities is statistically more efficient than our earlier classification procedure, although it is computationally heavier.     

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.     

LucY: A Versatile New Fluorescent Reporter Protein

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.