Absent Genes Research Articles

Production of an Oncolytic Adeno-Associated Virus Containing the Pro-Apoptotic TRAIL Gene Can Be Improved by shRNA Interference.

Recombinant Adeno-associated virus (rAAV) is a popular vector for treating genetic diseases caused by absent or defective genes. rAAVs can be produced that contain a therapeutic transgene, i.e., a correct copy of the affected gene, which is then delivered into target cells. A further application of rAAV is to deliver pro-apoptotic genes such as TNF-related apoptosis-inducing ligand (TRAIL) into cancer cells, leading to tumor regression. However, rAAV production is expensive and insufficient yields may hinder wide-spread adoption especially in systemic conditions. During rAAV production, the therapeutic transgene may be expressed in the producer cell line, and in the case of an oncolytic gene, this would likely lead to cell death thus reducing rAAV yields. Here we demonstrate that expression of TRAIL during rAAV production in HEK293F cells negatively impacts rAAV yield. A shRNA-based strategy was developed to suppress the expression of TRAIL in rAAV-producing cells specifically during the production process. Incorporating a TRAIL-targeting shRNA expression cassette within the backbone of the rAAV genome-encoding plasmid during triple-transfection of HEK293F cells reduced transgene expression and led to a 60% increase in the yield of rAAV-TRAIL compared to controls.

Liquorilactobacillus: A Context of the Evolutionary History and Metabolic Adaptation of a Bacterial Genus from Fermentation Liquid Environments.

In the present work, we carried out a comparative genomic analysis to trace the evolutionary trajectory of the bacterial species that make up the Liquorilactobacillus genus, from the identification of genes and speciation/adaptation mechanisms in their unique characteristics to the identification of the pattern grouping these species. We present phylogenetic relationships between Liquorilactobacillus and related taxa such as Bacillus, basal lactobacilli and Ligilactobacillus, highlighting evolutionary divergences and lifestyle transitions across different taxa. The species of this genus share a core genome of 1023 genes, distributed in all COGs, which made it possible to characterize it as Liquorilactobacillus sensu lato: few amino acid auxotrophy, low genes number for resistance to antibiotics and general and specific cellular reprogramming mechanisms for environmental responses. These species were divided into four clades, with diversity being enhanced mainly by the diversity of genes involved in sugar metabolism. Clade 1 presented lower (< 70%) average amino acid identity with the other clades, with exclusive or absent genes, and greater distance in the genome compared to clades 2, 3 and 4. The data pointed to an ancestor of clades 2, 3 and 4 as being the origin of the genus Ligilactobacillus, while the species of clade 1 being closer to the ancestral Bacillus. All these traits indicated that the species of clade 1 could be soon separated in a distinct genus.

RIBAP: a comprehensive bacterial core genome annotation pipeline for pangenome calculation beyond the species level

Microbial pangenome analysis identifies present or absent genes in prokaryotic genomes. However, current tools are limited when analyzing species with higher sequence diversity or higher taxonomic orders such as genera or families. The Roary ILP Bacterial core Annotation Pipeline (RIBAP) uses an integer linear programming approach to refine gene clusters predicted by Roary for identifying core genes. RIBAP successfully handles the complexity and diversity of Chlamydia, Klebsiella, Brucella, and Enterococcus genomes, outperforming other established and recent pangenome tools for identifying all-encompassing core genes at the genus level. RIBAP is a freely available Nextflow pipeline at github.com/hoelzer-lab/ribap and zenodo.org/doi/10.5281/zenodo.10890871.

Resequencing and characterization of the first Corynebacterium pseudotuberculosis genome isolated from camel.

Corynebacterium pseudotuberculosis is a zoonotic Gram-positive bacterial pathogen known to cause different diseases in many mammals, including lymph node abscesses in camels. Strains from biovars equi and ovis of C. pseudotuberculosis can infect camels. Comparative genomics could help to identify features related to host adaptation, and currently strain Cp162 from biovar equi is the only one from camel with a sequenced genome. In this work, we compared the quality of three genome assemblies of strain Cp162 that used data from the DNA sequencing platforms SOLiD v3 Plus, IonTorrent PGM, and Illumina HiSeq 2500 with an optical map and investigate the unique features of this strain. For this purpose, we applied comparative genomic analysis on the different Cp162 genome assembly versions and included other 129 genomes from the same species. Since the first version of the genome, there was an increase of 88 Kbp and 121 protein-coding sequences, a decrease of pseudogenes from 139 to 53, and two inversions and one rearrangement corrected. We identified 30 virulence genes, none associated to the camel host, and the genes rpob2 and rbpA predicted to confer resistance to rifampin. In comparison to 129 genomes of the same species, strain Cp162 has four genes exclusively present, two of them code transposases and two truncated proteins, and the three exclusively absent genes lysG, NUDIX domain protein, and Hypothetical protein. All 130 genomes had the rifampin resistance genes rpob2 and rbpA. Our results found no unique gene that could be associated with tropism to camel host, and further studies should include more genomes and genome-wide association studies testing for genes and SNPs.

Genomic Analysis of Yersinia pestis Strains from Brazil: Search for Virulence Factors and Association with Epidemiological Data.

Yersinia pestis, the etiological agent of the plague, is considered a genetically homogeneous species. Brazil is currently in a period of epidemiological silence but plague antibodies are still detected in sentinel animals, suggesting disease activity in the sylvatic cycle. The present study deployed an in silico approach to analyze virulence factors among 407 Brazilian genomes of Y. pestis belonging to the Fiocruz Collection (1966-1997). The pangenome analysis associated several known virulence factors of Y. pestis in clades according to the presence or absence of genes. Four main strain clades (C, E, G, and H) exhibited the absence of various virulence genes. Notably, clade G displayed the highest number of absent genes, while clade E showed a significant absence of genes related to the T6SS secretion system and clade H predominantly demonstrated the absence of plasmid-related genes. These results suggest attenuation of virulence in these strains over time. The cgMLST analysis associated genomic and epidemiological data highlighting evolutionary patterns related to the isolation years and outbreaks of Y. pestis in Brazil. Thus, the results contribute to the understanding of the genetic diversity and virulence within Y. pestis and the potential for utilizing genomic data in epidemiological investigations.

Intensification in Genetic Information and Acquisition of Resistant Genes in Genome of Acinetobacter baumannii: A Pan-Genomic Analysis.

Acinetobacter baumannii (A. baumannii) attributes 26% of the mortality rate in hospitalized patients, and the percentage can rise to 46 in patients admitted to ICU as it is a major cause of ventilator-associated pneumonia. It has been nominated as the critical priority organism by WHO for which new therapeutic drugs are urgently required. To understand the genomic identification of different strains, antimicrobial resistance patterns, and epidemiological typing of organisms, whole-genome sequencing (WGS) analysis provides insight to explore new epitopes to develop new drugs against the organism. Therefore, the study is aimed at investigating the whole genome sequence of A. baumannii strains to report the new intensifications in its genomic profile. The genome sequences were retrieved from the NCBI database system. Pan-genome BPGA (Bacterial Pan-genome Analysis Tool) was used to analyze the core, pan, and species-specific genome analysis. The pan and core genome curves were extrapolated using the empirical power law equation f(x) = a.xb and the exponential equation f1(x) = c.e (d.x). To identify the resistant genes with resistant mutations against antibiotics, ResFinder and Galaxy Community hub bioinformatics tools were used. According to pan-genome analysis, there were 2227 core genes present in each species of the A. baumannii genome. Furthermore, the number of accessory genes ranged from 1182 to 1460, and the unique genes in the genome were 931. There were 325 exclusively absent genes in the genome of Acinetobacter baumannii. The pan-genome analysis showed that there is a 5-fold increase in the genome of A. baumannii in 5 years, and the genome is still open. There is the addition of multiple unique genes; among them, genes participating in the function of information and processing are increased.

The complete organellar genomes of the entheogenic plant Psychotria viridis (Rubiaceae), a main component of the ayahuasca brew.

Psychotria viridis (Rubioideae: Rubiaceae), popularly known as chacrona, is commonly found as a shrub in the Amazon region and is well-known to produce psychoactive compounds, such as the N,N-dimethyltryptamine (DMT). Together with the liana Banisteropsis caapi, P. viridis is one of the main components of the Amerindian traditional, entheogenic beverage known as ayahuasca. In this work, we assembled and annotated the organellar genomes (ptDNA and mtDNA), presenting the first genomics resources for this species. The P. viridis ptDNA exhibits 154,106 bp, encoding all known ptDNA gene repertoire found in angiosperms. The Psychotria genus is a complex paraphyletic group, and according to phylogenomic analyses, P. viridis is nested in the Psychotrieae clade. Comparative ptDNA analyses indicate that most Rubiaceae plastomes present conserved ptDNA structures, often showing slight differences at the junction sites of the major four regions (LSC-IR-SSC). For the mitochondrion, assembly graph-based analysis supports a complex mtDNA organization, presenting at least two alternative and circular mitogenomes structures exhibiting two main repeats spanning 24 kb and 749 bp that may symmetrically isomerize the mitogenome into variable arrangements and isoforms. The circular mtDNA sequences (615,370 and 570,344 bp) encode almost all plant mitochondrial genes (except for the ccmC, rps7, rps10, rps14, rps19, rpl2 and rpl16 that appears as pseudogenes, and the absent genes sdh3, rps2, rsp4, rsp8, rps11, rpl6, and rpl10), showing slight variations related to exclusive regions, ptDNA integration, and relics of previous events of LTR-RT integration. The detection of two mitogenomes haplotypes is evidence of heteroplasmy as observed by the complex organization of the mitochondrial genome using graph-based analysis. Taken together, these results elicit the primary insights into the genome biology and evolutionary history of Psychotria viridis and may be used to aid strategies for conservation of this sacred, entheogenic species.

Pangenomic analysis of Chinese gastric cancer

Pangenomic study might improve the completeness of human reference genome (GRCh38) and promote precision medicine. Here, we use an automated pipeline of human pangenomic analysis to build gastric cancer pan-genome for 185 paired deep sequencing data (370 samples), and characterize the gene presence-absence variations (PAVs) at whole genome level. Genes ACOT1, GSTM1, SIGLEC14 and UGT2B17 are identified as highly absent genes in gastric cancer population. A set of genes from unaligned sequences with GRCh38 are predicted. We successfully locate one of predicted genes GC0643 on chromosome 9q34.2. Overexpression of GC0643 significantly inhibits cell growth, cell migration and invasion, cell cycle progression, and induces cell apoptosis in cancer cells. The tumor suppressor functions can be reversed by shGC0643 knockdown. The GC0643 is approved by NCBI database (GenBank: MW194843.1). Collectively, the robust pan-genome strategy provides a deeper understanding of the gene PAVs in the human cancer genome.

Genome Analysis of Enterobacter asburiae and Lelliottia spp. Proliferating in Oligotrophic Drinking Water Reservoirs and Lakes.

Surface waters are one of the main sources for drinking water production, and thus microbial contamination should be as minimal as possible. However, high concentrations of coliform bacteria were detected in reservoirs and lakes used for drinking water production during summer months due to autochthonous proliferation processes. Here, we present the genomic analyses of 17 strains of Enterobacter asburiae and Lelliottia spp. proliferating in reservoirs and lakes with special focus on the hygienic relevance, antibiotic resistance, and adaptations to the oligotrophic environments. The genomes contain neither genes for the type III secretion system nor cytotoxins or hemolysins, which are considered typical virulence factors. Examination of antibiotic resistance genes revealed mainly efflux pumps and β-lactamase class C (ampC) genes. Phenotypically, single isolates of Enterobacter asburiae showed resistance to fosfomycin and ceftazidime. The genome analyses further suggest adaptations to oligotrophic and changing environmental conditions in reservoirs and lakes, e.g., genes to cope with low nitrate and phosphate levels and the ability to utilize substances released by algae, like amino acids, chitin, alginate, rhamnose, and fucose. This leads to the hypothesis that the proliferation of the coliform bacteria could occur at the end of summer due to algae die-off. IMPORTANCE Certain strains of coliform bacteria have been shown to proliferate in the oligotrophic water of drinking water reservoirs and lakes, reaching values above 104 per 100 mL. Such high concentrations challenge drinking water treatment, and occasionally the respective coliform bacteria have been detected in the treated drinking water. Thus, the question of their hygienic relevance is of high importance for water suppliers and authorities. Our genomic analyses suggest that the strains are not hygienically relevant, as typical virulence factors are absent and antibiotic resistance genes in the genomes most likely are of natural origin. Furthermore, their presence in the water is not related to fecal contamination. The proliferation in reservoirs and lakes during stable summer stratification is an autochthonic process of certain E. asburiae and Lelliottia strains that are well adapted to the surrounding oligotrophic environment.

Clostridioides difficile from Brazilian hospitals: characterization of virulence genes by whole genome sequencing

Clostridioides difficile (CD) is the most frequent cause of healthcare related diarrhea and its severity has increased in the last decade by the spread of hypervirulent strains. Most important CD virulence factor is toxin production; however, not only toxins are responsible for Clostridioides virulence. We sequenced 38 strains and analyzed the presence and integrity of 24 virulence (including toxin) genes. We identified 28 toxigenic strains, six also presented the cdt genes. Only six strains didn't present all others genes searched. All absent genes were adhesion related. Understand others CD virulence factors can lead to a best understanding on this matter.

Endometrial Cancer: Genetic, Metabolic Characteristics, Therapeutic Strategies and Nanomedicine.

Endometrial cancer is the fourth most common malignancy in female population worldwide. It was estimated that 65,620 new cases and 12.590 subsequent deaths occurred in 2020 in the United States. Patients with type II and advanced endometrial cancer do not respond well to the current treatments. Therefore, endometrial cancer should be better understood in order to develop more effective treatments. To provide an overview of genetic, metabolic characteristics, therapeutic strategies and current application of nanotechnology surrounding endometrial cancer. Relevant articles were retrieved from Pubmed and were systematically reviewed. Hypoxia inducible factor-1 and Von Hippel-Lindau factor participated in oncogenesis and progression of endometrial cancer and Nrf2 was associated with oncogenesis. Various genetic alterations were found in endometrial cancer. Examining the abnormal X chromosome inactivation may help in the diagnosis of endometrial cancer and its precancerous lesions. Some absent tumor suppressor genes, activated oncogenes were revealed by the genetically modified mouse models. Disorders in glucose and lipid metabolism were found in endometrial cancer. Current therapeutic strategies focused on the HIF-1α pathway, the mTOR pathway as well as the immunotherapy. Nanotechnology showed great potential in endometrial cancer's early diagnosis, metastasis determination and treatment. Endometrial cancer has been understood in various aspects but the underlying mechanisms still remain relatively unknown, which might be the source of novel diagnostic, prognostic and therapeutic targets. Nanomedicine in endometrial cancer is poorly studied but the current researches showed great results in treating endometrial cancer. It needs further researching.

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells.

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

Genetic Features of the Tomato Marker Line Мо938

In cultivated tomato hybrids (Marglobe × Mo938), the anthocyanin-free gene shows linked inheritance with the d (dwarf) gene on chromosome 2, but with a recombination frequency approximately three times higher than that according to the genetic map and in other hybrids with the Marglobe line. Cytological analysis of the mother pollen cells of the hybrids (Marglobe × Mo938) revealed no abnormalities of meiotic division and segregation of chromosomes, as well as no decrease in fertility. By means of the functional allelism test, it was established that, unlike Mo500, Mo504, and Mo755 marker tomato lines, in Mo938 the anthocyanin-free trait is not determined by the aw (anthocyanin without) or aa (anthocyanin absent) genes of chromosome 2. Using the F2 progeny of interspecies hybrids (Mo938 × Solanum pimpinellifolium), independent inheritance of the anthocyanin-free gene relative to the wv (white virescent) and d marker genes, as well as to six SSR anchor markers distributed at different sites of chromosome 2, was established. Thus, the Mo938 tomato line carries the d and wv markers on chromosome 2, as well as the anthocyanin-free gene not belonging to chromosome 2.

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genesnot seen in any other multicellular eukaryotes. Inparticular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of therespiratory complexes and mitochondrial functions.Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I.

Understanding Mucor circinelloides pathogenesis by comparative genomics and phenotypical studies

ABSTRACT The increasing number of infections by species of Mucorales and their high mortality constitute an important concern for public health. This study aims to decipher the genetic basis of Mucor circinelloides pathogenicity, which displays virulence in a strain dependent manner. Assuming that genetic differences between strains may be linked to different pathotypes, we have conducted a study to explore genes responsible for virulence in M. circinelloides by whole genome sequencing of the avirulent strain NRRL3631 and comparison with the virulent strain CBS277.49. This genome analysis revealed 773 truncated, discontiguous and absent genes in the NRRL3631 strain. We also examined phenotypic traits resulting in reduced heat stress tolerance, chitosan content and lower susceptibility to toxic compounds (calcofluor white and sodium dodecyl sulphate) in the virulent strain, suggesting the influence of cell wall on pathogenesis. Based on these results, we focused on studying extracellular protein-coding genes by gene deletion and further pathotype characterization of mutants in murine models of pulmonary and systemic infection. Deletion of gene ID112092, which codes for a hypothetical extracellular protein of unknown function, resulted in significant reduction of virulence. Although pathogenesis is a multifactorial process, these findings highlight the crucial role of surface and secreted proteins in M. circinelloides virulence and should promote further studies of other differential genes.

Complete genome sequence of the sesame pathogen Ralstonia solanacearum strain SEPPX 05.

Ralstonia solanacearum is a soil-borne phytopathogen associated with bacterial wilt disease of sesame. R. solanacearum is the predominant agent causing damping-off from tropical to temperate regions. Because bacterial wilt has decreased the sesame industry yield, we sequenced the SEPPX05 genome using PacBio and Illumina HiSeq 2500 systems and revealed that R. solanacearum strain SEPPX05 carries a bipartite genome consisting of a 3,930,849bp chromosome and a 2,066,085bp megaplasmid with 66.84% G+C content that harbors 5,427 coding sequences. Based on the whole genome, phylogenetic analysis showed that strain SEPPX05 is grouped with two phylotype I strains (EP1 and GMI1000). Pan-genomic analysis shows that R. solanacearum is a complex species with high biological diversity and was able to colonize various environments during evolution. Despite deletions, insertions, and inversions, most genes of strain SEPPX05 have relatively high levels of synteny compared with strain GMI1000. We identified 104 genes involved in virulence-related factors in the SEPPX05 genome and eight absent genes encoding T3Es of GMI1000. Comparing SEPPX05 with other species, we found highly conserved secretion systems central to modulating interactions of host bacteria. These data may provide important clues for understanding underlying pathogenic mechanisms of R. solanacearum and help in the control of sesame bacterial wilt.

Transfection by cationic gemini lipids and surfactants.

Diseases that are linked to defective genes or mutations can in principle be cured by gene therapy, in which damaged or absent genes are either repaired or replaced by new DNA in the nucleus of the cell. Related to this, disorders associated with elevated protein expression levels can be treated by RNA interference via the delivery of siRNA to the cytoplasm of cells. Polynucleotides can be brought into cells by viruses, but this is not without risk for the patient. Alternatively, DNA and RNA can be delivered by transfection, i.e. by non-viral vector systems such as cationic surfactants, which are also referred to as cationic lipids. In this review, recent progress on cationic lipids as transfection vectors will be discussed, with special emphasis on geminis, surfactants with 2 head groups and 2 tails connected by a spacer.

Phylogenetic Distribution of CMP-Neu5Ac Hydroxylase (CMAH), the Enzyme Synthetizing the Proinflammatory Human Xenoantigen Neu5Gc.

The enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid present on the cell surface proteins of most deuterostomes. The CMAH gene is thought to be present in most deuterostomes, but it has been inactivated in a number of lineages, including humans. The inability of humans to synthesize Neu5Gc has had several evolutionary and biomedical implications. Remarkably, Neu5Gc is a xenoantigen for humans, and consumption of Neu5Gc-containing foods, such as red meats, may promote inflammation, arthritis, and cancer. Likewise, xenotransplantation of organs producing Neu5Gc can result in inflammation and organ rejection. Therefore, knowing what animal species contain a functional CMAH gene, and are thus capable of endogenous Neu5Gc synthesis, has potentially far-reaching implications. In addition to humans, other lineages are known, or suspected, to have lost CMAH; however, to date reports of absent and pseudogenic CMAH genes are restricted to a handful of species. Here, we analyze all available genomic data for nondeuterostomes, and 322 deuterostome genomes, to ascertain the phylogenetic distribution of CMAH. Among nondeuterostomes, we found CMAH homologs in two green algae and a few prokaryotes. Within deuterostomes, putatively functional CMAH homologs are present in 184 of the studied genomes, and a total of 31 independent gene losses/pseudogenization events were inferred. Our work produces a list of animals inferred to be free from endogenous Neu5Gc based on the absence of CMAH homologs and are thus potential candidates for human consumption, xenotransplantation research, and model organisms for investigation of human diseases.

Genomic Characterization of Urethritis-Associated Neisseria meningitidis Shows that a Wide Range of N. meningitidis Strains Can Cause Urethritis.

Neisseria meningitidis, typically a resident of the oro- or nasopharynx and the causative agent of meningococcal meningitis and meningococcemia, is capable of invading and colonizing the urogenital tract. This can result in urethritis, akin to the syndrome caused by its sister species, N. gonorrhoeae, the etiologic agent of gonorrhea. Recently, meningococcal strains associated with outbreaks of urethritis were reported to share genetic characteristics with the gonococcus, raising the question of the extent to which these strains contain features that promote adaptation to the genitourinary niche, making them gonococcus-like and distinguishing them from other N. meningitidis strains. Here, we analyzed the genomes of 39 diverse N. meningitidis isolates associated with urethritis, collected independently over a decade and across three continents. In particular, we characterized the diversity of the nitrite reductase gene (aniA), the factor H-binding protein gene (fHbp), and the capsule biosynthetic locus, all of which are loci previously suggested to be associated with urogenital colonization. We observed notable diversity, including frameshift variants, in aniA and fHbp and the presence of intact, disrupted, and absent capsule biosynthetic genes, indicating that urogenital colonization and urethritis caused by N. meningitidis are possible across a range of meningococcal genotypes. Previously identified allelic patterns in urethritis-associated N. meningitidis strains may reflect genetic diversity in the underlying meningococcal population rather than novel adaptation to the urogenital tract.

Reconstruction of amino acid biosynthetic pathways increases the productivity of 2-keto-L-gulonic acid in Ketogulonicigenium vulgare-Bacillus endophyticus consortium via genes screening.

Defect in the amino acid biosynthetic pathways of Ketogulonicigenium vulgare, the producing strain for 2-keto-L-gulonic acid (2-KGA), is the key reason for its poor growth and low productivity. In this study, five different strains were firstly reconstructed by expressing absent genes in threonine, proline and histidine biosynthetic pathways for better 2-KGA productivity. When mono-cultured in the shake flasks, the strain SyBE_Kv02080002 expressing hsk from Gluconobacter oxydans in threonine biosynthetic pathway achieved the highest biomass and the titer increased by 25.13%. When co-cultured with Bacillus endophyticus, the fermentation cycle decreased by 28.57% than that of the original consortium in 5-L fermenter. Furthermore, reconstruction of threonine biosynthetic pathway resulted in up-regulation of genes encoding sorbosone dehydrogenase and idonate-dehydrogenase, which increased the 2-KGA productivity in SyBE_Kv02080002. This study shows that reconstruction of absent biosynthetic pathways in bacteria is an effective way to enhance the productivity of target products.