Productivity improvement of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentation via robust optimization

A nonlinear model of a recombinant Escherichia coli producing porcine growth hormone (pGH) fermentation was developed. The model was used to calculate a glucose feeding and temperature strategy to optimize the production of pGH. Simulations showed that the implementation of optimal feed and temperature profiles was sensitive to the maximum specific growth rate, and a mismatch could result in excessive acetate production and a significant reduction in pGH yield. An optimization algorithm was thus developed, using feedback control, to counter the effects of uncertainty in the specific growth rate and thus determine an optimal operating strategy for pGH production. This policy was experimentally implemented in a 10 L fermenter and resulted in a 125% increase in productivity over the previous best experimental result with this system—in spite of significant plant-model mismatch.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of <Emphasis Type="Italic">cra</Emphasis> gene knockout together with <Emphasis Type="Italic">edd</Emphasis> and <Emphasis Type="Italic">iclR</Emphasis> genes knockout on the metabolism in <Emphasis Type="Italic">Escherichia coli</Emphasis>

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved. Dayanidhi Sarkar and Khandaker Al Zaid Siddiquee have contributed equally.     

Cloning of a xylanase gene <Emphasis Type="Italic">xyn2A</Emphasis> from rumen fungus <Emphasis Type="Italic">Neocallimastix</Emphasis> sp. GMLF2 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its partial characterization

Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.     

Improving heterologous polyketide production in <Emphasis Type="Italic">Escherichia coli</Emphasis> by overexpression of an <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l⁻¹ . In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> malate dehydrogenase,a novel solubility enhancer for heterologous proteins synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.     

Challenges Associated with Heterologous Expression of <Emphasis Type="Italic">Flavobacterium psychrophilum</Emphasis> Proteins in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production of F. psychrophilum recombinant proteins.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)     

Design and construction of a synthetic <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry4Aa gene: Hyperexpression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is, therefore, of great interest for developing a bioinsecticide to control mosquitoes. However, the expression of Cry4Aa in Escherichia coli is relatively low, which is a major disadvantage in its development as a bioinsecticide. In this study, to establish an effective production system, a 1,914-bp modified gene (cry4Aa-S1) encoding Cry4Aa was designed and synthesized in accordance with the G + C content and codon preference of E. coli genes without altering the encoded amino acid sequence. The cry4Aa-S1 gene allowed a significant improvement in expression level, over five-fold, compared to that of the original cry4Aa gene. The product of the cry4Aa-S1 gene showed the same level of insecticidal activity against Culex pipiens larvae as that from cry4Aa. This suggested that unfavorable codon usage was one of the reasons for poor expression of cry4Aa in E. coli, and, therefore, changing the cry4Aa codons to accord with the codon usage in E. coli led to efficient production of Cry4Aa. Efficient production of Cry4Aa in E. coli can be a powerful measure to prepare a sufficient amount of Cry4Aa protein for both basic analytical and applied researches.     

Sucrose and overexpression of trehalose biosynthetic genes (<Emphasis Type="Italic">otsBA</Emphasis>) increase desiccation tolerance of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Survival after desiccation was highest for recombinant strains of E. coli engineered to produce ethanol (KO11 and LY163) and lactate (TG106) when sucrose was provided as the fermentable sugar. Desiccation tolerance was lower with glucose and xylose. Further improvements in desiccation tolerance with sucrose were obtained by combining this with increased expression of otsBA genes encoding trehalose biosynthesis, removal of products from metabolism by resuspending in fresh medium, and harvesting cells prior to the end of log phase. With sucrose and otsBA expression, survivals of 20%–80% were readily achieved. Fermentation tests with EM2L, a derivative of LY163 expressing ostBA, demonstrated that ethanol production from seed fermentations begun with desiccated cells is equivalent to that of an undesiccated control.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

Effect of the chitin binding domain deletion from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> chitinase Chi255 on its stability in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Bacillus thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene, chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed in the native chitinase, and for improving its activity.     

Construction of an Effective Protein Expression System Using the <Emphasis Type="Italic">tpl</Emphasis> Promoter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An effective protein expression system was constructed in Escherichia coli using the promoter of the tyrosine phenol-lyase (tpl) gene of Erwinia herbicola. This system involves a mutant form of the TyrR protein with an enhanced ability to activate tpl and the TutB protein with an ability to transport L-tyrosine (an inducer of Tpl). The highest expression level obtained for this system was more than twice that obtained for the tac system, although it was lower than the level obtained for the T7 system, as revealed with the lac-reporter assay and SDS-polyacrylamide gel electrophoresis.     

Expression of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> butanol synthetic genes in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), β-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.     

The Evolutionary History of <Emphasis Type="Italic">Shigella</Emphasis> and Enteroinvasive <Emphasis Type="Italic">Escherichia coli</Emphasis> Revised

In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical star phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.