Characterization of salt-tolerant mutant for enhancement of l-threonine production in Escherichia coli

 Escherichia coli strain HS3, metabolically engineered to have Met^–, AHV^r, Ile^L and AEC^r characteristics, produced 58.0 g/l of l-threonine, but it was neither salt-tolerant nor osmotolerant; and the growth and threonine production of the strain were severely inhibited both by the addition of NaCl with a concentration higher than 2% and by the presence of glucose with a concentration higher than 10%. Therefore, salt-tolerant mutants were isolated. The salt-tolerant mutants, HS454 and HS528 which were derived from strain HS3, were both tolerant to salt (2%) and hyperproductive. The growth and l-threonine production by the mutant strain HS454 were almost unaffected by a glucose concentration lower than 10%, but gradually reduced with increasing glucose concentration, up to 15%. However, the mutant strain HS528 showed slightly enhanced growth and l-threonine production with increasing glucose concentration, up to 10–12.5%. Strains HS454 and HS528 produced 69.8 g/l and 74.0 g/l of l-threonine, respectively in a 5-l jar fermentor. Received: 21 January 2000 / Received revision: 31 March 2000 / Accepted: 5 May 2000     

Immune response induced by ppGpp-defective <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Gallinarum in chickens

To protect chickens from typhoid caused by Salmonella enterica serovar Gallinarum (S. Gallinarum), the attenuated 9R strain has been used in the field as a vaccine. However, safety concerns have been raised because the mutations in 9R are undefined while its efficacy is still a question under debate. A global regulator, ppGpp, synthesized by RelA and SpoT, has been shown to induce various virulence genes in S. Gallinarum (Jeong et al., 2008). In this study, two mutant strains defective in ppGpp-synthesis were constructed in wild-type S. Gallinarum (ΔppGpp) and 9R strain (9R-ΔppGpp) backgrounds and tested as live vaccines in chickens. After oral inoculation, the LD₅₀ values of ΔppGpp and 9R-ΔppGpp were approximately 5×10¹⁰ colony forming unit (CFU) similarly as 9R strain, which was ∼10⁵-fold higher than that of the wildtype S. Gallinarum strain. Immunological analyses revealed immunization with either of the two attenuated ppGpp-defective strains induced significant antibody responses, the production of antibody-secreting B cells in blood, proliferation of CD4+ and CD8+ T cells in the spleen, and splenic expression of proinflammatory cytokines, such as IFN-γ and TGF-β4, at levels comparable to the 9R strain. Chickens immunized with the mutants (1×10⁸ CFU) were 80% protected against oral challenge with 1×10⁹ wild-type virulent bacteria (4,000-fold LD₅₀ dose), similar to the level of protection achieved by 9R immunization. Based on these data, live attenuated ΔppGpp-defective strains may serve as novel vaccines to control fowl typhoid in chickens.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

Effects of the amplification of the genes coding for the L-threonine biosynthetic enzymes on the L-threonine production from methanol by a gram-negative obligate methylotroph,Methylobacillus glycogenes

We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.     

Characterization of a sodium-regulated glutaminase from cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K _m value of the recombinant protein for glutamine was 26.6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na⁺. Moreover, the K _m value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na⁺. These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na⁺ through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Δslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Δslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Δslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Δslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Δslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Δslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.     

Effects of <Emphasis Type="Italic">dapA</Emphasis> gene deletion on coronatine biosynthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">glycinea</Emphasis> PG4180

Coronatine (COR) is a structural and functional analogue of jasmonic acid that might be employed in agriculture to elicit plant resistance against various aggressors. However, the yield of COR is low both in chemosynthesis and biosynthesis, so broad investigation of COR is difficult. Coronatine combines two distinct components: coronafacic acid (CFA) and coronamic acid (CMA). Synthesis of both CMA and CFA is involved in l-isoleucine metabolism, so the objective of this work was to investigate if COR production can be improved by regulating amino acid biosynthesis in P. syringae pv. glycinea. Inhibition of dihydrodipicolinate synthase was achieved by removing the dapA gene via homologous recombination, which resulted in a COR yield by the dapA ⁻ mutant of about 1.5-fold greater than the wild strain. Thus, regulation of amino acid metabolism is a feasible way to increase COR production, which could be a more effective method than adding substrates into culture medium.     

Asporogenic recombinant producer of anthrax protective antigen

An asporogenic recombinant strain Bacillus anthracis 55ΔTPA-1(Spo⁻) producing anthrax protective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon pUB110PA-1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity. The level of PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than the values determined for vaccine strains B. anthracis STI-1 and B. anthracis 55. The strain preserves asporogenicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically active PA was isolated from the constructed strain B. anthracis 55ΔTPA-1(Spo⁻). Double immunization of rabbits with 50 μg of the purified recombinant product provides their 100% protection from infection with 50 LD₅₀ of a highly virulent anthrax strain.     

An efficient succinic acid production process in a metabolically engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> strain

A Corynebacterium glutamicum strain (ΔldhA-pCRA717) that overexpresses the pyc gene encoding pyruvate carboxylase while simultaneously exhibiting a disrupted ldhA gene encoding l-lactate dehydrogenase was investigated in detail for succinic acid production. Succinic acid was shown to be efficiently produced at high-cell density under oxygen deprivation with intermittent addition of sodium bicarbonate and glucose. Succinic acid concentration reached 1.24 M (146 g l⁻¹) within 46 h. The yields of succinic acid and acetic acid from glucose were 1.40 mol mol⁻¹ (0.92 g g⁻¹) and 0.29 mol mol⁻¹ (0.10 g g⁻¹), respectively. The succinic acid production rate and yield depended on medium bicarbonate concentration rather than glucose concentration. Consumption of bicarbonate accompanied with succinic acid production implied that added bicarbonate was used for succinic acid synthesis.     

Efficient induction of formate hydrogen lyase of aerobically grown <Emphasis Type="Italic">Escherichia coli</Emphasis> in a three-step biohydrogen production process

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA ⁺⁺, ΔhycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 ± 5.0 mmol h⁻¹ g⁻¹ dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 ΔldhA ΔfrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 ± 6.9 mmol h⁻¹ g⁻¹. Furthermore, a maximum hydrogen production rate of 144.2 mmol h⁻¹ g⁻¹ was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h⁻¹ was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.     

Enhanced expression of heterologous inulinase in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> by disruption of <Emphasis Type="Italic">hap1</Emphasis> gene

Inulinase gene (Kcinu) derived from Kluyveromyces cicerisporus was expressed extracellularly in Kluyveromyces lactis using an episomal vector directed by Kcinu promoter. The influence of hap1 gene disruption on the expression of inulinase was studied. Inulinase activity in the supernatant of the recombinant Klhap1Δ strain was 391 U ml⁻¹ after cultured 120 h, which was 2.2-fold that of the wild type host. The relative inulinase mRNA level of the Klhap1Δ strain was 11.3-fold that of the wild type strain, and the expression plasmid was more stable in the mutant host. Based on these results, the disruption of hap1 facilitated the high and stable expression of inulinase controlled by Kcinu promoter in K. lactis.     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

Production of threonine byBrevibacterium flavum containing threonine biosynthesis genes fromEscherichia coli

Genes of the threonine operon ofEscherichia coli were used for the construction of aBrevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Km^r/Nm^r), various plasmids carryingE. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment ofB. flavum carrying these recombinant plasmids. A mutant ofB. flavum CCM 351 carrying the cloned genesthrA andthrB accumulated 12 g/L of threonine after 48 h of cultivation.     

An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L⁻¹) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis.     

Improvement of milbemycin-producing <Emphasis Type="Italic">Streptomyces bingchenggensis</Emphasis> by rational screening of ultraviolet- and chemically induced mutants

Milbemycin antibiotics are produced by Streptomyces hygroscopicus subsp. aureolacrimosus and a newly isolated Streptomyces bingchenggensis, respectively. According to the biosynthetic pathway of milbemycins generated by S. hygroscopicus subsp. aureolacrimosus, a rational screening procedure with UV irradiation and N-methyl-N′-nitroso-N-nitrosoguanidine (NTG) mutation was performed to obtain high milbemycin-producing S. bingchenggensis. Aminoacetic acid (Glycine)-resistant mutants (AA^r), propionate-resistant mutants (PRP^r), streptomycin-resistant mutants (STR^r) and 2-deoxy-d-glucose-resistant mutants (DOG^r) were selected successively. A strain S. bingchenggensis BC-109-6 with AA^r, PRPr, STR^r and DOG^r was obtained and its production of milbemycin A3 and A4 reached 1,450 μg/ml, which was 80% higher than that of the ancestral strain S. bingchenggensis BC-101-4. The subculture experiments indicated that the hereditary characteristic of high productivity of S. bingchenggensis BC-109-6 was stable. The production of milbemycin A3 and A4 by S. bingchenggensis BC-109-6 in a 50-l fermentor could reach 1,380 μg/ml after 360 h batch fermentation.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.