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.     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

Redirecting carbon flux through <Emphasis Type="Italic">pgi</Emphasis>-deficient and heterologous transhydrogenase toward efficient succinate production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is particularly known for its potentiality in succinate production. We engineered C. glutamicum for the production of succinate. To enhance C₃–C₄ carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain NC-4. To increase intracellular NADH pools, the pntAB gene from Escherichia coli, encoding for transhydrogenase, was chromosomally integrated into NC-4, leading to strain NC-5. Furthermore, we deleted pgi gene in strain NC-5 to redirect carbon flux to the pentose phosphate pathway (PPP). To solve the drastic reduction of PTS-mediated glucose uptake, the ptsG gene from C. glutamicum, encoding for the glucose-specific transporter, was chromosomally integrated into pgi-deficient strain resulted in strain NC-6. In anaerobic batch fermentation, the production of succinate in pntAB-overexpressing strain NC-5 increased by 14% and a product yield of 1.22 mol/mol was obtained. In anaerobic fed-batch process, succinic acid concentration reached 856 mM by NC-6. The yields of succinate from glucose were 1.37 mol/mol accompanied by a very low level of by-products. Activating PPP and transhydrogenase in combination led to a succinate yield of 1.37 mol/mol, suggesting that they exhibited a synergistic effect for improving succinate yield.     

Functional analysis of arabinofuranosidases and a xylanase of <Emphasis Type="Italic">Corynebacterium alkanolyticum</Emphasis> for arabinoxylan utilization in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k _cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

<Emphasis Type="Italic">In vitro</Emphasis> Blastogenesis inhibited by <Emphasis Type="Italic">Erwinia carotovora</Emphasis><Emphasis Type="SmallCaps">L</Emphasis>-Asparaginase

Escherichia coliL-asparaginase, an antileukaemic agent in man¹, inhibits in vitro mitogen or antigen-induced blastogenesis in man^2,3 and in animals (M. Bennett, E. G. Mayhew and T. Han, unpublished data) and suppresses bone-marrow derived antibody precursor cells in the mouse⁴. We now report that another L-asparaginase preparation—from Erwinia carotovora—also possesses antileukaemic activity^5,6 and has a more pronounced immunosuppressive effect on in vitro blastogenesis than the E. coli enzyme.     

Anaerobic biosynthesis of intermediates of reductive branch of tricarboxylic acids cycle by <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with inactivated <Emphasis Type="Italic">frdAB</Emphasis> and <Emphasis Type="Italic">sdhAB</Emphasis> genes

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.     

The <Emphasis Type="Italic">yajC</Emphasis> gene from <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> and its role in ethanol tolerance

The yajC gene (Lbuc_0921) from Lactobacillus buchneri NRRL B-30929 was identified from previous proteomics analyses in response to ethanol treatment. The YajC protein expression was increased by 15-fold in response to 10 % ethanol vs 0 % ethanol. The yajC gene encodes the smaller subunit of the preprotein translocase complex, which interacts with membrane protein SecD and SecF to coordinate protein transport and secretion across cytoplasmic membrane in Escherichia coli. The YajC protein was linked to sensitivity to growth temperatures in E. coli, involved in translocation of virulence factors during Listeria infection, and stimulating a T cell-mediated response of Brucella abortus. In this study, the L. buchneri yajC gene was over-expressed in E. coli. The strain carrying pET28byajC that produces YajC after isopropyl β-d-1-thiogalactopyranoside induction showed tolerance to 4 % ethanol in growth media, compared to the control carrying pET28b. This is the first report linking YajC to ethanol stress and tolerance.     

Improved acid-stress tolerance of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> NZ9000 and <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21 by overexpression of the anti-acid component <Emphasis Type="Italic">recT</Emphasis>

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.     

Participation of <Emphasis Type="Italic">SRM5/CDC28</Emphasis>, <Emphasis Type="Italic">SRM8/NET1</Emphasis>, and <Emphasis Type="Italic">SRM12/HFI1</Emphasis> genes in checkpoint control in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G₂/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.     

Metabolism of the thermophilic bacterium <Emphasis Type="Italic">Oceanithermus profundus</Emphasis>

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurred via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> W3110 for the production of <Emphasis Type="SmallCaps">l</Emphasis>-methionine

In this study, we constructed an l-methionine-producing recombinant strain from wild-type Escherichia coli W3110 by metabolic engineering. To enhance the carbon flux to methionine and derepression met regulon, thrBC, lysA, and metJ were deleted in turn. Methionine biosynthesis obstacles were overcome by overexpression of metA ^Fbr (Fbr, Feedback resistance), metB, and malY under control of promoter pN25. Recombinant strain growth and methionine production were further improved by attenuation of metK gene expression through replacing native promoter by metK84p. Blocking the threonine pathway by deletion of thrBC or thrC was compared. Deletion of thrC showed faster growth rate and higher methionine production. Finally, metE, metF, and metH were overexpressed to enhance methylation efficiency. Compared with the original strain E. coli W3110, the finally obtained Me05 (pETMA^Fbr-B-Y/pKKmetH) improved methionine production from 0 to 0.65 and 5.62 g/L in a flask and a 15-L fermenter, respectively.     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.