Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

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.

     

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.     

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.     

Efficient de novo synthesis of resveratrol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Resveratrol has been the subject of numerous scientific investigations due to its health-promoting activities against a variety of diseases. However, developing feasible and efficient microbial processes remains challenging owing to the requirement of supplementing expensive phenylpropanoic precursors. Here, various metabolic engineering strategies were developed for efficient de novo biosynthesis of resveratrol. A recombinant malonate assimilation pathway from Rhizobium trifolii was introduced to increase the supply of the key precursor malonyl-CoA and simultaneously, the clustered regularly interspaced short palindromic repeats interference system was explored to down-regulate fatty acid biosynthesis pathway to inactivate the malonyl-CoA consumption pathway. Down-regulation of fabD, fabH, fabB, fabF, fabI increased resveratrol production by 80.2, 195.6, 170.3, 216.5 and 123.7%, respectively. Furthermore, the combined effect of these genetic perturbations was investigated, which increased the resveratrol titer to 188.1 mg/L. Moreover, the efficiency of this synthetic pathway was improved by optimizing the expression level of the rate-limiting enzyme TAL based on reducing mRNA structure of 5′ region. This further increased the final resveratrol titer to 304.5 mg/L. The study described here paves the way to the development of a simple and economical process for microbial production of resveratrol.     

<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.     

Proteome wide identification of iron binding proteins of <Emphasis Type="Italic">Xanthomonas translucens</Emphasis> pv. <Emphasis Type="Italic">undulosa</Emphasis>: focus on secretory virulent proteins

Xanthomonas translucens pv. undulosa (Xtu) causes Bacterial Leaf Streak disease in the staple food crops such as wheat and barley. The survival strategies of pathogen and host are determined by the complex interactions occurring between the host plants and the pathogenic microbes. Iron binding proteins are important in the plant–microbe interactions as they are indulged in enzyme catalysis, virulence, metabolic and transport activities. In the presented study, we have identified that ~9.8% of Xtu proteome possess iron binding sequence motifs. Further, the analysis of Xtu proteome for secretory iron binding virulent proteins (IBVPs) revealed the fact that iron co-regulate the function of secretory proteins in virulence. We have found 26 secretory IBVPs and observed that these proteins are diverse in their biological functions ranging from transport to antimicrobial resistance, Reactive oxygen species detoxification and carbohydrate catabolism. The inferences may instigate to design the new strategies to combat and control the microbial diseases of staple food crops.     

Improving methionine and ATP availability by <Emphasis Type="Italic">MET6</Emphasis> and <Emphasis Type="Italic">SAM2</Emphasis> co-expression combined with sodium citrate feeding enhanced SAM accumulation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

S-adenosyl-l-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.     

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.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

Genome-scale model-driven strain design for dicarboxylic acid production in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Background

Recently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application.

Results

In this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica.

Conclusion

In silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.

     

Illumina-based analysis of the rhizosphere microbial communities associated with healthy and wilted Lanzhou lily (<Emphasis Type="Italic">Lilium davidii</Emphasis> var. <Emphasis Type="Italic">unicolor</Emphasis>) plants grown in the field

Lanzhou lily (Liliumdavidii var. unicolor) is the best edible lily as well as a traditional medicinal plant in China. The microbes associated with plant roots play crucial roles in plant growth and health. However, little is known about the differences of rhizosphere microbes between healthy and wilted Lanzhou lily (Lilium davidii var. unicolor) plants. The objective of this study was to compare the rhizosphere microbial community and functional diversity of healthy and wilted plants, and to identify potential biocontrol agents with significant effect. Paired end Illumina Mi-Seq sequencing of 16S rRNA and ITS gene amplicons was employed to study the bacterial and fungal communities in the rhizosphere soil of Lanzhou lily plants. BIOLOG technology was adopted to investigate the microbial functional diversity. Our results indicated that there were major differences in the rhizosphere microbial composition and functional diversity of wilted samples compared with healthy samples. Healthy Lanzhou lily plants exhibited lower rhizosphere-associated bacterial diversity than diseased plants, whereas fungi exhibited the opposite trend. The dominant phyla in both the healthy and wilted samples were Proteobacteria and Ascomycota, i.e., 34.45 and 64.01 %, respectively. The microbial functional diversity was suppressed in wilted soil samples. Besides Fusarium, the higher relative abundances of Rhizoctonia, Verticillium, Penicillium, and Ilyonectria (Neonectria) in the wilted samples suggest they may pathogenetic root rot fungi. The high relative abundances of Bacillus in Firmicutes in healthy samples may have significant roles as biological control agents against soilborne pathogens. This is the first study to find evidence of major differences between the microbial communities in the rhizospheric soil of healthy and wilted Lanzhou lily, which may be linked to the health status of plants.     

Improved production of 1-deoxynojirymicin in <Emphasis Type="Italic">Escherichia coli</Emphasis> through metabolic engineering

Azasugars, such as 1-deoxynojirymicin (1-DNJ), are associated with diverse pharmaceutical applications, such as antidiabetic, anti-obesity, anti-HIV, and antitumor properties. Different azasugars have been isolated from diverse microbial and plant sources though complicated purification steps, or generated by costly chemical synthesis processes. But the biosynthesis of such potent molecules using Escherichia coli as a heterologous host provides a broader opportunity to access these molecules, particularly by utilizing synthetic biological, metabolic engineering, and process optimization approaches. This work used an integrated approach of synthetic biology, enzyme engineering, and pathway optimization for rational metabolic engineering, leading to the improved production of 1-DNJ. The production of 1-DNJ in recombinant E. coli culture broth was confirmed by enzymatic assays and mass spectrometric analysis. Specifically, the pathway engineering for its key precursor, fructose-6-phosphate, along with optimized media condition, results in the highest production levels. When combined, 1-DNJ production was extended to ~?273 mg/L, which is the highest titer of production of 1-DNJ reported using E. coli.     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Comparative study on intestinal bacterial communities of <Emphasis Type="Italic">Boleophthalmus pectinirostris</Emphasis> and <Emphasis Type="Italic">Periophthalmus magnuspinnatus</Emphasis> with different sexes and feeding strategies

Intertidal mudflats are unique, highly productive ecosystems. Boleophthalmus pectinirostris and Periophthalmus magnuspinnatus are common fish species that are distributed in the intertidal mudflats of the Yangtze Estuary in China. They perform important ecological functions and have different feeding strategies. Herein, we studied the intestinal microbial diversity and structure of wild B. pectinirostris and P. magnuspinnatus with different sexes and feeding strategies during their breeding season. Gut samples of B. pectinirostris and P. magnuspinnatus individuals (female:male ratio?=?1:1) were collected and subjected to high-throughput DNA sequencing. The results showed Proteobacteria was the most dominant phylum in all the four sample groups: 73.5% in the males and 52.6% in the females of B. pectinirostris and 40.2% in the males and 40.9% in the females of P. magnuspinnatus. Aeromonas, Shewanella, Halomonas, and Acinetobacter of the phylum Proteobacteria were dominant genera in all the sample groups and accounted for 62.13% of the ten dominant genera. The diversity of the intestinal microflora in the omnivorous P. magnuspinnatus was significantly higher (P?<?0.05) than that in the herbivorous B. pectinirostris. Beta diversity, including PCoA and UPGMA of unweighted UniFrac distances, showed that B. pectinirostris samples were clustered together, and P. magnuspinnatus samples were clustered together, implying the effect of the feeding habits on the microbial community structure is more considerable than that of sex.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.     

Reprogramming <Emphasis Type="Italic">Halomonas</Emphasis> for industrial production of chemicals

Halomonas spp. are able to grow under a high salt concentration at alkali pH, they are able to resist contamination by other microbes. Development of Halomonas spp. as platform production strains for the next-generation industrial biotechnology (NGIB) is intensively studied. Among Halomonas spp., Halomonas bluephagenesis is the best studied one with available engineering tools and methods to reprogram it for production of various polyhydroxyalkanoates, proteins, and chemicals. Due to its contamination resistance, H. bluephagenesis can be grown under open and continuous processes not just in the labs but also in at least 1000 L fermentor scale. It is expected that NGIB based on Halomonas spp. be able to engineer for production of increasing number of products in a competitive manner.