Genome mining, in silico validation and phase selection of a novel aldo-keto reductase from Candida glabratea for biotransformation

Previously, we published cloning, over-expression, characterization and subsequent exploitation of a carbonyl reductase (cr) gene, belonging to general family aldo-keto reductase from Candida glabrata CBS138 to convert keto ester (COBE) to a chiral alcohol (ethyl-4-chloro-3-hydroxybutanoate or CHBE). Exploiting global transcription factor CRP, rDNA and transporter engineering, we have improved batch production of CHBE by trinomial bioengineering. Herein, we present the exploration of cr gene in Candida glabrata CBS138 through genome miningapproach, in silico validation of its activity and selection of its biocatalytic phase.For exploration of the gene under investigation, three template genes were chosen namelySaccharomyces cerevisae YDR541c, YGL157w and YOL151w. The CR showed significant homology match, overlapping of substrate binding site and NADPH binding sitewith the template proteins. The binding affinity of COBE towards CR (−4.6 Kcal/ mol) was found higher than that of the template proteins (−3.5 to −4.5 Kcal/ mol). Biphasic biocatalysis with cofactor regeneration improved product titer 4∼5 times better than monophasic biotransformation. Currently we are working on DNA Shuffling as a next level of strain engineering and we demonstrate this approach herein as a future strategy of biochemical engineering

Isolation and characterization of acid and base degradation products in Atenolol and Hydrochlorothiazide and a validated selective stability-indicating HPLCUV method for their quantification

Atenolol (( RS )-2-{4-[2-Hydroxy-3-(propan-2-ylamino)propoxy]phenyl}acetamide) and Hydrochlorothiazide (6-chloro-1,1-dioxo-3,4-dihydro-2 H -1,2,4-benzothiadiazine-7-sulfonamide) are beta 1 (? 1 ) receptor blocker and diuretic drug respectively; however the combination dosage regime are used for cardiovascular therapy. Thus a forced degradation study was carried out upon this combination drug regime under acidic and basic environment in order to deconvolute the possible degradation product under specified stressed conditions. Under acidic conditions atenolol and hydrochlorothiazide were cleaved into 2-(4-(3-amino-2- oxopropoxy) phenyl) acetamide and 6-sulphamido benzothiazide. However, under basic conditions, the drugs were spliced into 2-(4-(2-hydroxypropoxy) phenyl) acetamide and 2-chloro 4-amino 1, 6-dihydro benzene sulphonamide respectively. The degradant peaks were elucidated by HPLC using C18 column with methanol: phosphate buffer (70:30 v/v) with a flow rate of 0.5ml/min (UV detection at 226nm). For quantitative method validation, linearity was observed over product concentration range 2g/ml - 100 g/ml (r 2 0.9992) with regression equation y=43432x. The products were first identified by LC-MS and further confirmed by FT-IR and 1 H 1 NMR. A specific and sensitive stability-indicating assay method for the simultaneous determination of the drugs, its process related impurities and degradation products was developed

Improving E.coli performance under stress by rewiring its global regulator Camp Receptor Protein (CRP)

Strain engineering tools are usually adopted to improve microorganism tolerance towards stressful environments in bioindustries. However, classical strain engineering techniques of using UV/chemical mutagens are often both labor- and time- intensive. In recent years, transcriptional engineering approach has started to attract attention in strain engineering. The better efficiency of this method has made it more preferable over the classical methods. Here in this thesis, I would adopt error-prone PCR technique to engineer global transcription factor cAMP receptor protein (CRP), which can regulate more than 400 genes in Escherichia coli, to enhance its tolerance against organic solvents, oxidative stress, and low pH. E. coli DH5α-∆crp strain was transformed with the plasmid-containing crp mutants, which were generated from error-prone PCR, followed by a selection under various stresses. All selected mutants exhibited much better tolerance than the wild type (WT) against respective stress. For example, the best toluene tolerant mutant showed growth even in 0.23% (v/v) toluene 0.51 h-1 whereas the growth of WT was completely inhibited. The best oxidative stress mutant demonstrated viability in 12 mM H2O2, while WT growth was halted at 6 mM H2O2. The best mutant identified under various stresses were then subjected to various characterizations, including cross-tolerance check, DNA microarray analysis, quantitative real time PCR and enzyme assay. For instance, toluene-tolerant mutant was also able to have improved growth against n-hexane, cyclohexane and p-xylene while oxidative-stress-tolerant mutant also exhibits thermotolerance. DNA microarray analysis and qRT-PCR results have demonstrated that the modifications to CRP would not only bring differential expression in CRP-regulated genes but also those non-CRP-regulated genes. In conclusion, random mutagenesis of CRP can provide an efficient alternative for E. coli strain engineering.DOCTOR OF PHILOSOPHY (SCBE

Enhancing <em>E. coli</em> Tolerance towards Oxidative Stress via Engineering Its Global Regulator cAMP Receptor Protein (CRP)

<div><p>Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve <em>E. coli</em> performance under oxidative stress <em>via</em> engineering its global regulator cAMP receptor protein (CRP), which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ∼400 genes in <em>E. coli</em>. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1∼OM3) were identified with improved tolerance <em>via</em> H₂O₂ enrichment selection. The best mutant OM3 could grow in 12 mM H₂O₂ with the growth rate of 0.6 h⁻¹, whereas the growth of wild type was completely inhibited at this H₂O₂ concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS), which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H₂O₂ treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold), but also RpoS- and OxyR-regulated genes (up to 7.7-fold). qRT-PCR data and enzyme activity assay suggested that catalase (<em>katE</em>) could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving <em>E. coli</em> oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for <em>E. coli</em>.</p> </div

Error-prone PCR of global transcription factor cyclic AMP receptor protein for enhanced organic solvent (toluene) tolerance

Organic solvents are often applied in biphasic biocatalysis or involved in bioremediation, but native microorganisms usually have low tolerance toward these organic solvents and cannot survive in the stressful environment. Here, we aimed to improve the organic solvent tolerance of Escherichia coli by engineering its global transcription factor cAMP receptor protein (CRP). Toluene was chosen as model organic solvent. The mutated crp genes were generated via error-prone PCR and three toluene-tolerant mutants M1–M3 were isolated from random mutagenesis libraries by enrichment selection. All mutants exhibited much better growth than the wild type upon exposure to 0.2–0.23% (v/v) toluene, with M2 the best. When toluene concentration was at 0.2%, M1–M3 shared similar growth rate at 0.68 h−1 despite the elongated 20-h lag phase, with WT exhibiting null growth. In 0.23% toluene, the growth rate of M2 was found to be 0.51 h−1 while the growth of WT was completely inhibited. M2 also demonstrated excellent growth in other organic solvents such as n-hexane, cyclohexane and p-xylene as compared to the wild type. Our quantitative real-time reverse transcription PCR analysis with organic-solvent stress associated genes indicated that amino acid mutations in CRP would lead to changes in the expression level of genes that are not regulated by CRP. The field emission scanning electron microscopy images revealed that both wild type and M2, though combating toluene stress, did not have any significant changes in their cellular size and shape

Cell growth in the absence or presence of H₂O₂ (A) 0 mM H₂O₂, (B) 8 mM H₂O₂, (C) 12 mM H₂O₂,

<p>Cells were cultured in LB-kanamycin medium at 37°C, 200 rpm. Each data point is the mean of three replicates.</p

DNA microarray data of certain genes in OM3 after H₂O₂ treatment (<i>p</i><0.05, Log₂ Fold Change>2.0).

*<p>- Analyzed by qRT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051179#pone.0051179.s011" target="_blank">Table S4</a>).</p

Cell growth profile after the introduction of 12 mM H₂O₂ during mid log phase (OD₆₀₀ 0.65).

<p>Each data point is the mean of three replicates.</p

Intracellular ROS level in OM3 and WT with cells treated with or without 4 mM H₂O₂.

<p>Mid exponential phase grown cells (OD₆₀₀ 0.6) were incubated with 10 µm H₂DCFDA (dissolved in dimethyl sulfoxide) at 30°C, 200 rpm. The oxidized fluorophore was quantified using excitation wavelength 485 nm and emission wavelength 528 nm. Each data point is the mean of five independent observations.</p