<Superscript>15</Superscript>N–{<Superscript>1</Superscript>H} NOE experiment at high magnetic field strengths

The heteronuclear ¹⁵N–{¹H} NOE values are typically determined by taking the ratio of ¹⁵N signal intensities recorded in the presence and absence of ¹H saturation prior to evolution of ¹⁵N magnetization. Since the intensity ratio of two independent experiments is taken, complete recovery of ¹⁵N magnetization during the scan repetition delay is critical to obtain reliable NOE values. Because it may not be practical to wait for the complete recovery of magnetization at high magnetic fields, Solomon equations may be used to correct measured NOE values. Here, based on experiments and simulations, we show that since the cross-correlation between ¹H–¹⁵N dipole and ¹⁵N chemical shift anisotropy becomes significant at high fields for small or deuterated proteins, measured NOE values can not be accurately corrected based on the Solomon equations. We also discuss ranges of rotational correlation times and proton spin-flip rate, in which the NOE values can be corrected by the equations.     

Practical considerations for investigation of protein conformational dynamics by <Superscript>15</Superscript>N <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript> relaxation dispersion

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by ¹⁵N R _1ρ relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R _1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.     

Does sequence polymorphism of <Emphasis Type="Italic">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.     

Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

Microsecond motions probed by near-rotary-resonance <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript><Superscript>15</Superscript>N MAS NMR experiments: the model case of protein overall-rocking in crystals

F_oF₁-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H⁺ translocation in membranes. The F_o c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state ¹³C NMR (ssNMR) signals of the F_o c-subunit ring of thermophilic Bacillus PS3 (TF_o c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly ¹³C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C^α(i+1)–C′C^α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue ¹³C–¹³C chemical shift correlations, 97% of C^α, 97% of C^β and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF_o c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF_o c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.     

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">15</Emphasis></Subscript>, in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Key message

A novel rust resistance gene, R ₁₅ , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R ₁₅ were identified, facilitating marker-assisted selection of resistance genes.

Abstract

The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F₂ individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R ₁₅ ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R ₁₅ was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

<Emphasis Type="Italic">δ</Emphasis><Superscript>13</Superscript>C values and crassulacean acid metabolism in <Emphasis Type="Italic">Clusia</Emphasis> species from Panama

The genus Clusia is notable in that it contains arborescent crassulacean acid metabolism (CAM) plants. As part of a study of CAM in Clusia, titratable acidities were measured in 25 species and ¹³C values were measured for 38 species from Panamá, including seven undescribed species, and 11 species from Colombia, Costa Rica and Honduras. CAM was detected in 12 species. Clusia flava, C. rosea and C. uvitana exhibited ¹³C values or diurnal fluctuations in acidity indicative of strong CAM. In C. croatii, C. cylindrica, C. fructiangusta, C. lineata, C. odorata, C. pratensis, C. quadrangula, C. valerioi and C. sp. D diurnal fluctuations in acidity were consistent with weak CAM but the ¹³C values were C₃-like. All of the species that exhibited strong or weak CAM were in the C. flava or C. minor species groups. CAM was not detected in any member of the C. multiflora species group. Strong CAM species were not collected at altitudes above 680 m a.s.l. On the basis of ¹³C values, the expression of CAM was similar in terrestrial, hemi-epiphytic and epiphytic species and did not differ between individuals of the same species that exhibited different life-forms. This study indicates that phylogenetic affiliation may be a predictor of an ability to exhibit CAM in Clusia species from the Panamanian region, and that weak CAM is probably a common photosynthetic option in many Clusia species. ¹³C value is not a particularly good indicator of a potential of Clusia species growing in the field to exhibit CAM because it appears that the contribution in most species of CAM to carbon gain is generally rather small when integrated over the life-time of leaves.     

An <Emphasis Type="Italic">R</Emphasis><Subscript>0</Subscript> theory for source–sink dynamics with application to <Emphasis Type="Italic">Dreissena</Emphasis> competition

Source–sink dynamics may be ubiquitous in ecology. We developed a theory for source–sink dynamics using spatial extensions of the net reproductive value, R ₀, which has been used elsewhere to define fitness, disease eradication, population growth, and invasion risk. R ₀ decomposes into biologically meaningful components—lifetime reproductive output, survival, and dispersal—that are widely adaptable and easily interpreted. The theory provides a general quantitative means for relating fundamental niche, biotic interactions, dispersal, and species distributions. We applied the methods to Dreissena and found a resolution to a paradox in invasion biology—competitive coexistence between quagga (Dreissena bugensis) and zebra (D. polymorpha) mussels among lakes despite extensive niche overlap within lakes. Source–sink dynamics within lakes between deepwater and shallow habitats, which favor quagga and zebra mussels, respectively, yield a metacommunity distribution where quagga mussels dominate large lakes and zebra mussels dominate small lakes. The source–sink framework may also be useful in spatial competition theory, habitat conservation, marine protected areas, and ecological responses to climate change.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

XH/<Emphasis Type="Italic">π</Emphasis> interactions with the <Emphasis Type="Italic">π</Emphasis> system of porphyrin ring in porphyrin-containing proteins

Searching structures of porphyrin-containing proteins from the Protein Data Bank revealed that the π system of every porphyrin ring is involved in XH/π interactions, with most of the porphyrins having several interactions. Both five-membered pyrrole rings and six-membered chelate rings are involved in XH/π interactions; the number of interactions with five-membered rings is larger than the number of interactions with six-membered rings. We found interactions with C–H and N–H groups as hydrogen-atom donors; however, the number of CH/π interactions is much larger than the number of NH/π interactions. The amino acids involved in the interactions show a high conservation score. Our results that every porphyrin is involved in XH/π interactions and that amino acids involved in these interactions are highly conserved demonstrate that XH/π interactions play an important role in porphyrin–protein stability. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insoluble but enzymatically active <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

The gene encoding thermostable α-amylase from Bacillus licheniformis consisting of 483 amino acid residues (mature protein) was cloned and expressed in Escherichia coli under the control of T7 promoter. The analysis of the soluble and insoluble fractions after lyzing the host cells revealed that recombinant α-amylase was produced in insoluble aggregates. Despite being produced in the insoluble aggregates the recombinant enzyme was highly active with a specific activity of 408 U/mg.     

Gene–environment interactions between <Emphasis Type="Italic">ERCC2</Emphasis>, <Emphasis Type="Italic">ERCC3</Emphasis>, <Emphasis Type="Italic">XRCC1</Emphasis> and cadmium exposure in nasal polyposis disease

Gene–environment interactions have long been known to play an important role in complex disease aetiology, such as nasal polyposis (NP). The present study supports the concept that DNA repair gene polymorphisms play critical roles in modifying individual susceptibility to environmental diseases. In fact, we investigated the role of polymorphisms in DNA repair genes and cadmium as risk factors for Tunisian patients with NP. To the best of our knowledge, this is the first report on the impact of combined effects of cadmium and ERCC3 7122 A>G (rs4150407), ERCC2 Lys751Gln (rs13181) and XRCC1 Arg399Gln (rs25487) genes in the susceptibility to NP disease. Significant associations between the risk of developing NP disease and ERCC2 [odds ratio (OR)?=?2.0, 95 % confidence interval (CI)?=?1.1–3.7, p?=?0.023] and ERCC3 (OR?=?2.2, 95 % CI?=?1.2–4.1, p?=?0.013) genotypes polymorphisms were observed. Blood concentrations of Cd in NP patients (2.2 μg/L) were significantly higher than those of controls (0.5 μg/L). A significant interaction between ERCC3 (7122 A>G) polymorphism and blood-Cd levels (for the median of blood-Cd levels: OR?=?3.8, 95 % CI?=?1.3–10.8, p?=?0.014 and for the 75th percentiles of blood-Cd levels: OR?=?2.7, 95 % CI?=?1.1–7.2, p?=?0.041) was found in association with the risk of NP disease. In addition, when we stratified ERCC2, ERCC3 and XRCC1 polymorphism genotypes by the median and 75th percentiles of blood-Cd levels, we found also significant interactions between ERCC2 (Lys751Gln) and ERCC3 (7122 A>G) genotypes polymorphism and this metal in association with NP disease. However, no interaction was found between XRCC1 (Arg399Gln) polymorphism genotypes and Cd in association with NP disease.     

Delimiting a rice wide-compatibility gene <Emphasis Type="Italic">S</Emphasis><Stack><Subscript>5</Subscript><Superscript><Emphasis Type="Italic">n</Emphasis></Superscript></Stack> to a 50 kb region

Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S ₅ ⁿ gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S ₅ ⁿ gene. In the first-pass mapping, the S ₅ ⁿ gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S ₅ region. In the fine mapping, the S ₅ region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S ₅ ⁿ gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S ₅ region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S ₅ ⁿ gene. Qing Ji and Jufei Lu have contributed equally to this paper.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Overexpression of <Emphasis Type="Italic">ARO10</Emphasis> in <Emphasis Type="Italic">pdc5</Emphasis>Δmutant resulted in higher isobutanol titers in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate effects of different pyruvate decarboxylases on isobutanol titers in Saccharomyces cerevisiae, single-gene deletion of the three PDCs genes encoding pyruvate decarboxylases were constructed in this study. In addition, we over-expressed Ilv2, which catalyzed the first step in the valine synthetic pathway, and Bat2, which was the cytoplasmic branched-chain amino-acid aminotransferase that catalyzed L-valine to 2-ketoisovalerate, to increase isobutanol production in the genetically modified strains. Our results showed that knockout of PDC5 were one of the main factors among the three PDC genes for improving isobutanol titers in S. cerevisiae. Additionally, we found that deletion of PDC5 in strain carrying overexpressed ILV2 and ARO10 resulted in 8-fold higher isobutanol productivity as compared to the control strain in micro-aerobic fermentations. Our results also suggested that engineered strain pdc5ΔpILV2 pARO10 generated lower ethanol titers and higher acetate acid titers than the control strain, while the growth rate and glucose consumption rate of engineered strain pdc5ΔpILV2 pARO10 were slightly lower than that of the control strain. Meanwhile, the biomass concentration of pdc5ΔpILV2 pARO10 decreased dramatically than that of the control strain.