Genome-scale metabolic models (GEMs) have been developed and used in guiding systems' metabolic engineering strategies for strain design and development. This strategy has been used in fermentative production of bio-based industrial chemicals and fuels from alternative carbon sources. However, computer-aided hypotheses building using established algorithms and software platforms for biological discovery can be integrated into the pipeline for strain design strategy to create superior strains of microorganisms for targeted biosynthetic goals. Here, I described an integrated workflow strategy using GEMs for strain design and biological discovery. Specific case studies of strain design and biological discovery using Escherichia coli genome-scale model are presented and discussed. The integrated workflow presented herein, when applied carefully would help guide future design strategies for high-performance microbial strains that have existing and forthcoming genome-scale metabolic models.
Over the last 20 years in biotechnology, the production of recombinant proteins has been a crucial bioprocess in both biopharmaceutical and research arena in terms of human health, scientific impact and economic volume. Although logical strategies of genetic engineering have been established, protein overexpression is still an art. In particular, heterologous expression is often hindered by low level of production and frequent fail due to opaque reasons. The problem is accentuated because there is no generic solution available to enhance heterologous overexpression. For a given protein, the extent of its solubility can indicate the quality of its function. Over 30% of synthesized proteins are not soluble. In certain experimental circumstances, including temperature, expression host, etc., protein solubility is a feature eventually defined by its sequence. Until now, numerous methods based on machine learning are proposed to predict the solubility of protein merely from its amino acid sequence. In spite of the 20 years of research on the matter, no comprehensive review is available on the published methods.
Microbial strain optimization focuses on improving technological properties of the strain of microorganisms. However, the complexities of the metabolic networks, which lead to data ambiguity, often cause genetic modification on the desirable phenotypes difficult to predict. Furthermore, vast number of reactions in cellular metabolism lead to the combinatorial problem in obtaining optimal gene deletion strategy. Consequently, the computation time increases exponentially with the increase in the size of the problem. Hence, we propose an extension of a hybrid of Bees Algorithm and Flux Balance Analysis (BAFBA) by integrating OptKnock into BAFBA to validate the result. This paper presents a number of computational experiments to test on the performance and capability of BAFBA. Escherichia coli, Bacillus subtilis and Clostridium thermocellum are the model organisms in this paper. Also included is the identification of potential reactions to improve the production of succinic acid, lactic acid and ethanol, plus the discussion on the changes in the flux distribution of the predicted mutants. BAFBA shows potential in suggesting the non-intuitive gene knockout strategies and a low variability among the several runs. The results show that BAFBA is suitable, reliable and applicable in predicting optimal gene knockout strategy.
Consumption of probiotics has been associated with decreased risk of colon cancer and reported to have antimutagenic/ anti-carcinogenic properties. One possible mechanism for this effect involves physical binding of the mutagenic compounds, such as heterocyclic amines (HCAs), to the bacteria. Therefore, the objective of this study was to examine the binding capacity of bifidobacterial strains of human origin on mutagenic heterocyclic amines which are suspected to play a role in human cancers. In vitro binding of the mutagens Trp-p-2, IQ, MeIQx, 7,8DiMeIQx and PhIP by three bacterial strains in two media of different pH was analysed using high performance liquid chromatography. Bifidobacterium pseudocatenulatum G4 showed the highest decrease in the total HCAs content, followed by Bifidobacterium longum, and Escherichia coli. pH affects binding capacity; the highest binding was obtained at pH 6.8. Gram-positive tested strains were found to be consistently more effective than the gram-negative strain. There were significant decreases in the amount of HCAs in the presence of different cell concentrations of B. pseudocatenulatum G4; the highest decrease was detected at the concentration of 10(10) cfu/ml. The results showed that HCAs were able to bind with all bacterial strains tested in vitro, thus it may be possible to decrease their absorption by human intestine and increase their elimination via faeces.
Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.
Three transition metal derivatives (Zn, Cu, and Ni) of 2-[2-bromoethyliminomethyl]-4-[ethoxymethyl]phenol (L) were synthesized by the reaction of the metal salts with the Schiff base ligand in one pot. In the crystal structure of [Zn(L)Br], the Schiff base ligand binds to the metal center through its phenolate oxygen and imine nitrogen, and adopts a distorted tetrahedral geometry. These compounds were found to inhibit topoisomerase I (topo I) activity, induce DNA cleavage and show DNA binding activity. Moreover, these compounds were found to be cytotoxic towards several cancer cell lines (A2780, MCF-7, HT29, HepG2, A549, PC3, LNCaP) and prevent metastasis of PC3. Collectively, Cu(II) complex 2 shows superior activity relative to its Zn(II) and Ni(II) analogs.
E. coli has been engineered to produce xylitol, but the production faces bottlenecks in terms of production yield and cell viability. In this study, recombinant E. coli (rE. coli) was immobilized on untreated and treated multiwalled carbon nanotubes (MWCNTs) for xylitol production. The immobilized rE. coli on untreated MWCNTs gave the highest xylitol production (5.47 g L-1) and a productivity of 0.22 g L-1 h-1. The doubling time for the immobilized cells increased up to 20.40 h and was higher than that of free cells (3.67 h). Cell lysis of the immobilized cells was reduced by up to 73 %, and plasmid stability improved by up to 17 % compared to those of free cells. Xylitol production using the optimum parameters (pH 7.4, 0.005 mM and 29 °C) achieved a xylitol production and productivity of 6.33 g L-1 and 0.26 g L-1 h-1, respectively. A seven-cycle repeated batch fermentation was carried out for up to 168 h, which showed maximum xylitol production of 7.36 g L-1 during the third cycle. Hence, this new adsorption immobilization system using MWCNTs is an alternative to improve the production of xylitol.
Ethanol was produced via the simultaneous saccharification and fermentation (SSF) of dilute sodium hydroxide treated corn stover. Saccharification was achieved by cultivating either Phanerochaete chrysosporium or Gloeophyllum trabeum on the treated stover, and fermentation was then performed by using either Saccharomyces cerevisiae or Escherichia coli K011. Ethanol production was highest on day 3 for the combination of G. trabeum and E. coli K011 at 6.68 g/100g stover, followed by the combination of P. chrysosporium and E. coli K011 at 5.00 g/100g stover. SSF with S. cerevisiae had lower ethanol yields, ranging between 2.88 g/100g stover at day 3 (P. chrysosporium treated stover) and 3.09 g/100g stover at day 4 (G. trabeum treated stover). The results indicated that mild alkaline pretreatment coupled with fungal saccharification offers a promising bioprocess for ethanol production from corn stover without the addition of commercial enzymes.
This paper presents a study on gene knockout strategies to identify candidate genes to be knocked out for improving the production of succinic acid in Escherichia coli. Succinic acid is widely used as a precursor for many chemicals, for example production of antibiotics, therapeutic proteins and food. However, the chemical syntheses of succinic acid using the traditional methods usually result in the production that is far below their theoretical maximums. In silico gene knockout strategies are commonly implemented to delete the gene in E. coli to overcome this problem. In this paper, a hybrid of Ant Colony Optimization (ACO) and Minimization of Metabolic Adjustment (MoMA) is proposed to identify gene knockout strategies to improve the production of succinic acid in E. coli. As a result, the hybrid algorithm generated a list of knockout genes, succinic acid production rate and growth rate for E. coli after gene knockout. The results of the hybrid algorithm were compared with the previous methods, OptKnock and MOMAKnock. It was found that the hybrid algorithm performed better than OptKnock and MOMAKnock in terms of the production rate. The information from the results produced from the hybrid algorithm can be used in wet laboratory experiments to increase the production of succinic acid in E. coli.
Comparative whole-genome sequencing enables the identification of specific mutations during adaptation of bacteria to new environments and allelic replacement can establish their causality. However, the mechanisms of action are hard to decipher and little has been achieved for epistatic mutations, especially at the metabolic level. Here we show that a strain of Escherichia coli carrying mutations in the rpoC and glpK genes, derived from adaptation in glycerol, uses two distinct metabolic strategies to gain growth advantage. A 27-bp deletion in the rpoC gene first increases metabolic efficiency. Then, a point mutation in the glpK gene promotes growth by improving glycerol utilization but results in increased carbon wasting as overflow metabolism. In a strain carrying both mutations, these contrasting carbon/energy saving and wasting mechanisms work together to give an 89% increase in growth rate. This study provides insight into metabolic reprogramming during adaptive laboratory evolution for fast cellular growth.
Pseudogenes are considered to be nonfunctional genes that lack a physiological role. By screening 3985 Escherichia coli mutants using chemochromic membranes, we found four pseudogenes involved in hydrogen metabolism. Knockouts of pseudogenes ydfW and ypdJ had a defective hydrogen phenotype on glucose and formate, respectively. Also, the knockout of pseudogene yqiG formed hydrogen from formate but not from glucose. For the yqiG mutant, 100% hydrogen recovery was obtained by the complementation of YqiG via a plasmid. The knockout of pseudogene ylcE showed hydrogen deficiency in minimal media which suggested that the role of YlcE is associated with cell growth. Hence, the products of these four pseudogenes play an important physiological role in hydrogen production in E. coli.
Successful gene delivery into mammalian cells using bactofection requires entry of the bacterial vector via cell surface integrin receptors followed by release of plasmid DNA into the cellular environment. We show, for the first time, that addition of the DNA transfection reagent Lipofectamine improves entry of invasive Escherichia coli into HeLa cells and enhances up to 2.8-fold green fluorescent protein (GFP) expression from a reporter plasmid. The addition of Lipofectamine may be applicable to other bacterial vectors to increase their DNA delivery efficiency into mammalian cells.
Quorum sensing controls the virulence determinants in most proteobacteria. In this work, the hexane, chloroform and methanol extracts of an Ayurveda spice, namely clove (Syzygium aromaticum), shown anti-quorum sensing activity. Hexane and methanol extracts of clove inhibited the response of C. violaceum CV026 to exogenously supplied N-hexanoylhomoserine lactone, in turn preventing violacein production. Chloroform and methanol extracts of clove significantly reduced bioluminescence production by E. coli [pSB1075] grown in the presence of N-(3-oxododecanoyl)-L-homoserine lactone. We demonstrated that clove extract inhibited quorum sensing-regulated phenotypes in Pseudomonas aeruginosa PA01, including expression of lecA::lux (by hexane extract), swarming (maximum inhibition by methanol extract), pyocyanin (maximum inhibition by hexane extract). This study shows that the presence of natural compounds that exhibit anti-quorum sensing activity in the clove extracts may be useful as the lead of anti-infective drugs.
Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.
Reporter gene activity under the regulation of the oil palm metallothionein-like gene, MT3-A promoter was assessed in prokaryotes. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuronidase (GUS) gene in pCAMBIA 1304 vector were produced. 5'-rapid amplification of cDNA ends (RACE) using mRNA isolated from Escherichia coli and Agrobacterium tumefaciens harboring W1MT3-A confirmed that fusion transcripts of MT3-A 5'-UTR-GUS were successfully produced in both bacteria. Competitive PCR and GUS fluorometric assay showed changes in the level of GUS gene transcripts and enzyme activity in response to increasing concentrations of Cu²+ and Zn²+. The application of Cu²+ increased GUS activity and GUS mRNA level in both bacteria. In E. coli, a high level of GUS activity driven by W1MT3-A and W2MT3-A was observed in treatment with 25 μM Cu²+ resulting in an increase in the GUS mRNA level to 7.2 and 7.5 x 10⁻⁴ pmol/μl respectively, compared to the control (5.1 x 10⁻⁴ pmol/μl). The lowest GUS activity and GUS mRNA level were obtained for W1MT3-A and W2MT3-A in the presence of 100 μM Cu²+ in both bacteria compared to the control (without Cu²+). The application of different Zn²+ concentrations resulted in a strong decrease in the GUS activity and GUS mRNA level in E. coli and A. tumefaciens. These findings showed that the oil palm MT3-A promoter is functional in prokaryotes and produced detectable GUS transcripts and enzyme activities. This promoter may potentially be used in prokaryotic systems which require metal inducible gene expression.
The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.
HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active.
Succinic acid is an important platform chemical that has broad applications and is been listed as one of the top twelve bio-based chemicals produced from biomass by the US Department of Energy. The metabolic role of Escherichia coli formate dehydrogenase-O (fdoH) under anaerobic conditions in relation to succinic acid production remained largely unspecified. Herein we report, what are to our knowledge, the first metabolic fdoH gene knockout that have enhanced succinate production using glucose and glycerol substrates in E. coli. Using the most recent E. coli reconstruction iJO1366, we engineered its host metabolism to enhance the anaerobic succinate production by deleting the fdoH gene, which blocked H(+) conduction across the mutant cell membrane for the enhanced succinate production. The engineered mutant strain BMS4 showed succinate production of 2.05 g l(-1) (41.2-fold in 7 days) from glycerol and .39 g l(-1) (6.2-fold in 1 day) from glucose. This work revealed that a single deletion of the fdoH gene is sufficient to increase succinate production in E. coli from both glucose and glycerol substrates.
The nucleocapsid protein (NP) of Newcastle disease virus expressed in E. coli assembled as ring- and herringbone-like particles. In order to identify the contiguous NP sequence essential for assembly of these particles, 11 N- or C-terminally deleted NP mutants were constructed and their ability to self-assemble was tested. The results indicate that a large part of the NP N-terminal end, encompassing amino acids 1 to 375, is required for proper folding to form a herringbone-like structure. In contrast, the C-terminal end covering amino acids 376 to 489 was dispensable for the formation of herringbone-like particles. A region located between amino acids 375 to 439 may play a role in regulating the length of the herringbone-like particles. Mutants with amino acid deletions further from the C-terminal end (84, 98, 109 and 114 amino acids) tended to form longer particles compared to mutants with shorter deletions (25 and 49 amino acids).