<Emphasis Type="Italic">glyA</Emphasis> gene knock-out in <Emphasis Type="Italic">Escherichia coli</Emphasis> enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C₁ units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C₁ supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA ^Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.     

Volatile compounds from beneficial or pathogenic bacteria differentially regulate root exudation,transcription of iron transporters,and defense signaling pathways in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Key message

Our results show that Sorghum bicolor is able to recognize bacteria through its volatile compounds and differentially respond to beneficial or pathogens via eliciting nutritional or defense adaptive traits.

Abstract

Plants establish beneficial, harmful, or neutral relationships with bacteria. Plant growth promoting rhizobacteria (PGPR) emit volatile compounds (VCs), which may act as molecular cues influencing plant development, nutrition, and/or defense. In this study, we compared the effects of VCs produced by bacteria with different lifestyles, including Arthrobacter agilis UMCV2, Bacillus methylotrophicus M4-96, Sinorhizobium meliloti 1021, the plant pathogen Pseudomonas aeruginosa PAO1, and the commensal rhizobacterium Bacillus sp. L2-64, on S. bicolor. We show that VCs from all tested bacteria, except Bacillus sp. L2-64, increased biomass and chlorophyll content, and improved root architecture, but notheworthy A. agilis induced the release of attractant molecules, whereas P. aeruginosa activated the exudation of growth inhibitory compounds by roots. An analysis of the expression of iron-transporters SbIRT1, SbIRT2, SbYS1, and SbYS2 and genes related to plant defense pathways COI1 and PR-1 indicated that beneficial, pathogenic, and commensal bacteria could up-regulate iron transporters, whereas only beneficial and pathogenic species could induce a defense response. These results show how S. bicolor could recognize bacteria through their volatiles profiles and highlight that PGPR or pathogens can elicit nutritional or defensive traits in plants.

     

Induction of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Resistance to Pathogenic Bacteria by Lipopolysaccharide and Salicylic Acid

The effects of combined treatment with an elicitor (lipopolysaccharide) and a signaling molecule (salicylic acid) on the disease resistance of wild-type (Col-0) and mutant Arabidopsis thaliana L. plants have been compared. The mutant lines used were jin1 (with impaired jasmonate signaling), npr1 (lacking expression of pathogen-dependent PR genes), and NahG (expressing an active bacterial salicylate hydroxylase transgene). The lipopolysaccharide was isolated from a saprophytic strain (8614) of Pseudomonas aeruginosa bacteria. Treatment of A. thaliana seeds with a composite preparation (lipopolysaccharide and salicylic acid–SA) increased the resistance of seedlings to a subsequent infection by the pathogenic 9096 strain of P. aeruginosa bacteria. The protective effect was more pronounced in jin1 mutant seedlings, which was indicative of the possible compensation of jasmonate signaling impairment due to activation of the SA-dependent signaling pathway. We concluded that a preparation composed of an elicitor and a signaling molecule could affect regulatory mechanism functioning in a plant cell and, in particular, compensate for the absence of a certain signaling pathway by activating another.     

Efficient inhibition of some multi-drug resistant pathogenic bacteria by bioactive metabolites from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> S<Subscript>5</Subscript>I<Subscript>4</Subscript> isolated from archaeological soil in Egypt

Sixty-eight bacterial cultures were isolated from 5 archaeological soils in Egypt. It is necessary to characterize bacteria from ancient temples to develop protection programs for such archaeological places. Purified bacterial cultures were then tested for their capability to inhibit some multi-drug resistant (MDR) pathogenic bacteria including Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli and Klebsiella pneumoniae. Among the most active 10 antibacterial isolates, only one isolate designated as S₅I₄ was selected, characterized and identified as belonging to Bacillus amyloliquefaciens. The strain identification was confirmed by amplification of its 16S rRNA gene. The partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted in GenBank with accession number AB813716. The physical and nutritional parameters were optimized to improve the production of antimicrobial agents by the B. amyloliquefaciens S₅I₄. The maximum antagonistic effect of this strain against the tested MDR pathogenic bacteria was achieved in presence of 1% galactose and 0.5% yeast extract at 37°C and pH 7.0 after 48 h incubation. The antibacterial compounds of B. amyloliquefaciens S₅I₄ were extracted, purified and characterized using spectroscopic analysis (IR, UV, proton NMR and MS). The compound having inhibitory activity was identified as butanedioic acid, octadecyl,1(1carboxy1methylethyl) 4octyl ester.     

Identification of the di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate-biodegrading strains and the biodegradation pathway in strain LMB-1

DNA isolated from a greenhouse soil (Nanjing, Jiangsu Province, China) was suitable for PCR amplification of gene segment coding for the 16S rRNA. Diverse PCR products were characterized by cloning and sequencing, and analysis of bacterial colonies showed the presence over 26 phyla. The most bacteria belonged to Proteobacteria, Actinobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetes. Furthermore, after the enrichment procedure of DBP-degrading microorganisms, 4 strains were isolated from the soil sample with di-n-butyl phthalate (DBP) biodegradability, and they were identified to be Rhizobium sp., Streptomyces sp., Pseudomonas sp. and Acinetobacter sp. Analysis of the degradation products by LC-MS led to identification of metabolites of DBP in strain LMB-1 (identified as Rhizobium sp.) which suggests that DBP was degraded through β-oxidation, demethylation, de-esterification and cleavage of aromatic ring.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SNP0614 and its Effect on Biodegradation of Petroleum

An efficient biosurfactant-producing strain was isolated and cultured from Dagang oil field (China) using crude oil as sole source of carbon. Based on partial sequenced 16S rDNA analysis, the isolated strain was identified as Pseudomonas aeruginosa SNP0614. The bacterium P. aeruginosa SNP0614 produced a type of biosurfactant with excessive foam-forming properties. After microbial cultivation at 37°C and 150 rpm for 12 h, the produced biosurfactant was found to reduce the surface tension to 25.4 mN/m with critical micelle concentration (CMC) of 45.0 mg/L. After 20 days of incubation, the biosurfactant exhibited 90% emulsification activity (E₂₄) on crude oil. FTIR spectroscopy of extracted biosurfactant indicated the biosurfactant as lipopeptide. The significant synergistic effect between P. aeruginosa SNP0614 and the mixed oildegrading bacteria resulted in increasing n-alkanes degradation rate by 30%. The strain P. aeruginosa SNP0614 represented as a promising biosurfactant producer and could be applied in a variety of biotechnological and industrial processes, particularly in microbial enhanced oil recovery and the bioremediation of oil pollution.     

Growth of <Emphasis Type="Italic">Dehalococcoides mccartyi</Emphasis> species in an autotrophic consortium producing limited acetate

The dechlorinating Dehalococcoides mccartyi species requires acetate as carbon source, but little is known on its growth under acetate limiting conditions. In this study, we observed growth and dechlorination of a D. mccartyi-containing mixed consortium in a fixed-carbon-free medium with trichloroethene in the aqueous phase and H₂/CO₂ in the headspace. Around 4 mM formate was produced by day 40, while acetate was constantly below 0.05 mM. Microbial community analysis of the consortium revealed dominance by D. mccartyi and Desulfovibrio sp. (57 and 22% 16S rRNA gene copies, respectively). From this consortium, Desulfovibrio sp. strain F1 was isolated and found to produce formate and acetate (1.2 mM and 48 µM, respectively, by day 24) when cultivated alone in the above mentioned medium without trichloroethene. An established co-culture of strain F1 and D. mccartyi strain 195 demonstrated that strain 195 could grow and dechlorinate using acetate produced by strain F1; and that acetate was constantly below 25 µM in the co-culture. To verify that such low level of acetate is utilizable by D. mccartyi, we cultivated strain 195 alone under acetate-limiting conditions and found that strain 195 consumed acetate to below detection (5 µM). Based on the acetate consumption and cell yield of D. mccartyi, we estimated that on average 1.2?×?10⁸ acetate molecules are needed to supply carbon for one D. mccartyi cell. Our study suggests that Desulfovibrio may supply a steady but low amount of fixed carbon to dechlorinating bacteria, exhibiting important implications for natural bio-attenuation when fixed carbon is limited.     

Enhancement of 5-aminolevulinic acid production by metabolic engineering of the glycine biosynthesis pathway in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To construct a strain of Corynebacterium glutamicum capable of efficiently producing 5-aminolevulinic acid (5-ALA) via the C4 pathway by modification of serine and glycine pathway using glucose as sole carbon source.

Results

The recombinant C. glutamicum strain AP2 harboring a codon-optimized hemA gene from Rhodobacter sphaeroides was used as host strain for 5-ALA production. A plasmid harboring the serine operon, which contained serB, serC and the site-specific mutant serA ^Δ197 , was constructed and introduced into C. glutamicumAP2, leading to an increase of 70% in 5-ALA production. Further overexpression of the glyA gene increased production of 5-ALA by 150% over the control. 5-ALA production was thus significantly enhanced by engineering the glycine biosynthetic pathway. C.glutamicum AG3 produced 3.4 ± 0.2 g 5-ALA/l in shake-flask cultures in CGIIIM medium with the addition of 7.5 g glycine/l.

Conclusion

This is the first report of remodeling the serine and glycine biosynthetic pathway to improve the production of 5-ALA in C. glutamicum.

     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

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.     

Anaerobic degradation of 1-methylnaphthalene by a member of the <Emphasis Type="Italic">Thermoanaerobacteraceae</Emphasis> contained in an iron-reducing enrichment culture

An anaerobic culture (1MN) was enriched with 1-methylnaphthalene as sole source of carbon and electrons and Fe(OH)₃ as electron acceptor. 1-Naphthoic acid was produced as a metabolite during growth with 1-methylnaphthalene while 2-naphthoic acid was detected with naphthalene and 2-methylnaphthalene. This indicates that the degradation pathway of 1-methylnaphthalene might differ from naphthalene and 2-methylnaphthalene degradation in sulfate reducers. Terminal restriction fragment length polymorphism and pyrosequencing revealed that the culture is mainly composed of two bacteria related to uncultured Gram-positive Thermoanaerobacteraceae and uncultured gram-negative Desulfobulbaceae. Stable isotope probing showed that a ¹³C-carbon label from ¹³C₁₀-naphthalene as growth substrate was mostly incorporated by the Thermoanaerobacteraceae. The presence of putative genes involved in naphthalene degradation in the genome of this organism was confirmed via assembly-based metagenomics and supports that it is the naphthalene-degrading bacterium in the culture. Thermoanaerobacteraceae have previously been detected in oil sludge under thermophilic conditions, but have not been shown to degrade hydrocarbons so far. The second member of the community belongs to the Desulfobulbaceae and has high sequence similarity to uncultured bacteria from contaminated sites including recently proposed groundwater cable bacteria. We suggest that the gram-positive Thermoanaerobacteraceae degrade polycyclic aromatic hydrocarbons while the Desulfobacterales are mainly responsible for Fe(III) reduction.     

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.     

Reducing diacetyl production of wine by overexpressing <Emphasis Type="Italic">BDH1</Emphasis> and <Emphasis Type="Italic">BDH2</Emphasis> in <Emphasis Type="Italic">Saccharomyces uvarum</Emphasis>

As a byproduct of yeast valine metabolism during fermentation, diacetyl can produce a buttery aroma in wine. However, high diacetyl concentrations generate an aromatic off-flavor and poor quality in wine. 2,3-Butanediol dehydrogenase encoded by BDH1 can catalyze the two reactions of acetoin from diacetyl and 2,3-butanediol from acetoin. BDH2 is a gene adjacent to BDH1, and these genes are regulated reciprocally. In this study, BDH1 and BDH2 were overexpressed in Saccharomyces uvarum to reduce the diacetyl production of wine either individually or in combination. Compared with those in the host strain WY1, the diacetyl concentrations in the recombinant strains WY1-1 with overexpressed BDH1, WY1-2 with overexpressed BDH2 alone, and WY1-12 with co-overexpressed BDH1 and BDH2 were decreased by 39.87, 33.42, and 46.71%, respectively. BDH2 was only responsible for converting diacetyl into acetoin, but not for the metabolic pathway of acetoin to 2,3-butanediol in S. uvarum. This study provided valuable insights into diacetyl reduction in wine.     

A dual approach for improving homogeneity of a human-type N-glycan structure in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae.     

<Emphasis Type="Italic">Hymenobacter terrigena</Emphasis> sp. nov., isolated from soil

A Gram-stain-negative, non-motile, non-spore-forming, rodshaped, aerobic bacterial strain, designated S1-2-2-5^T, was isolated from the Jeollabuk-do province, Republic of Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-2-5^T belonged to the family Cytophagaceae in phylum Bacteroidetes, and was most closely related to Hymenobacter terrae DG7A^T (98.2%), Hymenobacter rubidus DG7B^T (98.0%), Hymenobacter soli PB17^T (97.7%), Hymenobacter daeguensis 16F3Y-2^T (97.2%) and Hymenobacter saemangeumensis GSR0100^T (97.0%). The G + C content of the genomic DNA of strain S1-2-2-5^T was 59.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone, a fatty acid profile with summed feature 3 (C_16:1ω7c/C_16:1ω6c; 32.0%), C_15:0 iso (19.0%), and C_15:0 anteiso (15.0%) as the major components, and a polar lipid profile with phosphatidylethanolamine as the major component supported the affiliation of strain S1-2-2-5^T to the genus Hymenobacter. The DNA-DNA relatedness between strain S1-2-2-5^T and H. terrae KCTC 32554^T, H. rubidus KCTC 32553^T, H. soli KCTC 12607^T, H. daeguensis KCTC 52537^T, and H. saemangeumensis KACC 16452^T were 49.5, 48.2, 34.1, 28.1, and 31.8% respectively, clearly showing that the isolate is not related to them at the species level. Strain S1-2-2-5^T could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain S1-2-2-5^T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter terrigena sp. nov. is proposed. The type strain is S1-2-2-5^T (= KCTC 52737^T = JCM 32195^T).     

Isolation,characterization and virulence of bacteria from <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

Isolation and identification of cultivated bacteria associated with soybeans and their biocontrol activity against <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

One hundred bacteria, isolated from rhizospheric soil and rhizoplane of healthy soybean plants, were assayed for antifungal activity against six Phytophthora sojae isolates. Nine of the tested bacteria inhibited the hyphal growth of P. sojae in vitro. They were subsequently evaluated for their in vitro traits and identified using the 16S rRNA gene sequences. Four of them (Paenibacillus sp.,—S1; Streptomyces sp.,—S9, S10 and S11) were further selected on the basis of their strongest antagonistic activity in vitro against P. sojae race 4, the predominant race in most growing soybean areas in Canada, and tested for their beneficial effects on soybean plants in the greenhouse. Results showed that application of bacterial strain S11 as seed coating reduced the disease severity by 57.1% and increased the root and shoot weight by and 140 and 108% respectively, in comparison to the diseased control. Overall, a positive correlation was recorded between the in vitro and in planta effects of the selected bacteria. This is promising for further application as select environmentally safe biological control agents in the protection of soybean against root rot diseases.     

Phosphoenolpyruvate:glucose phosphotransferase system modification increases the conversion rate during <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli FB-04(pta1), a recombinant l-tryptophan production strain, was constructed in our laboratory. However, the conversion rate (l-tryptophan yield per glucose) of this strain is somewhat low. In this study, additional genes have been deleted in an effort to increase the conversion rate of E. coli FB-04(pta1). Initially, the pykF gene, which encodes pyruvate kinase I (PYKI), was inactivated to increase the accumulation of phosphoenolpyruvate, a key l-tryptophan precursor. The resulting strain, E. coli FB-04(pta1)ΔpykF, showed a slightly higher l-tryptophan yield and a higher conversion rate in fermentation processes. To further improve the conversion rate, the phosphoenolpyruvate:glucose phosphotransferase system (PTS) was disrupted by deleting the ptsH gene, which encodes the phosphocarrier protein (HPr). The levels of biomass, l-tryptophan yield, and conversion rate of this strain, E. coli FB-04(pta1)ΔpykF/ptsH, were especially low during fed-batch fermentation process, even though it achieved a significant increase in conversion rate during shake-flask fermentation. To resolve this issue, four HPr mutations (N12S, N12A, S46A, and S46N) were introduced into the genomic background of E. coli FB-04(pta1)ΔpykF/ptsH, respectively. Among them, the strain harboring the N12S mutation (E. coli FB-04(pta1)ΔpykF-ptsHN12S) showed a prominently increased conversion rate of 0.178 g g^?1 during fed-batch fermentation; an increase of 38.0% compared with parent strain E. coli FB-04(pta1). Thus, mutation of the genomic of ptsH gene provided an alternative method to weaken the PTS and improve the efficiency of carbon source utilization.