Citrus is a large genus that includes several major cultivated species, including C. sinensis (sweet orange), Citrus reticulata (tangerine and mandarin), Citrus limon (lemon), Citrus grandis (pummelo) and Citrus paradisi (grapefruit). In 2009, the global citrus acreage was 9 million hectares and citrus production was 122.3 million tons (FAO statistics, see URLs), which is the top ranked among all the fruit crops. Among the 10.9 million tons (valued at $9.3 billion) of citrus products traded in 2009, sweet orange accounted for approximately 60% of citrus production for both fresh fruit and processed juice consumption (FAO statistics, see URLs). Moreover, citrus fruits and juice are the prime human source of vitamin C, an important component of human nutrition.Citrus fruits also have some unique botanical features, such as nucellar embryony (nucellus cells can develop into apomictic embryos that are genetically identical to mother plant). Consequently, somatic embryos grow much more vigorously than the zygotic embryos in seeds such that seedlings are essentially clones of the maternal parent. Such citrus-unique characteristics have hindered the study of citrus genetics and breeding improvement 1,2 . Complete genome sequences would provide valuable genetic resources for improving citrus crops.Citrus is believed to be native to southeast Asia 3-5 , and cultivation of fruit crops occurred at least 4,000 years ago 3,6 . The genetic origin of the sweet orange is not clear, although there are some speculations that sweet orange might be derived from interspecific hybridization of some primitive citrus species 7,8 . Citrus is also in the order Sapindales, a sister order to the Brassicales in the Malvidae, making it valuable for comparative genomics studies with the model plant Arabidopsis.We aimed to sequence the genome of Valencia sweet orange (C. sinensis cv. Valencia), one of the most important sweet orange varieties cultivated worldwide and grown primarily for orange juice production. Normal sweet oranges are diploids, with nine pairs of chromosomes and an estimated genome size of ~367 Mb 9 . To reduce the complexity of the sequenced genome, we obtained a doublehaploid (dihaploid) line derived from the anther culture of Valencia sweet orange 10 . We first generated whole-genome shotgun pairedend-tag sequence reads from the dihaploid genomic DNA and built a de novo assembly as the citrus reference genome; we then produced shotgun sequencing reads from the parental diploid DNA and mapped the sequences to the haploid reference genome to obtain the complete genome information for Valencia sweet orange. In addition, we conducted comprehensive transcriptome sequencing analyses for four representative tissues using shotgun RNA sequencing (RNA-Seq) to capture all transcribed sequences and paired-end-tag RNA sequencing (RNA-PET) to demarcate the 5′ and 3′ ends of all transcripts. On the basis of the DNA and RNA sequencing data, we characterized the orange genome for its gene content, heterozygosity and evolutionary features. ...

The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity.

This study showed that modification of the lipid A moiety of C. sakazakii with PEA increased resistance to CAMP and recognition of the immune response although signalling of TLR4/MD2 cascade, suggesting that the organism could not successfully evade the host innate immune system without the transference of PEA to its lipid A moiety.

Background: In citrus, genetic improvement via biotechnology is hindered by the obstacle of in vitro regeneration via somatic embryogenesis (SE). Although a few B3 transcription factors are reported to regulate embryogenesis, little is known about the B3 superfamily in citrus, and which members might be involved in SE. Results: Genome-wide sequence analysis identified 72 (CsB3) and 69 (CgB3) putative B3 superfamily members in the genomes of sweet orange (Citrus sinensis, polyembryonic) and pummelo (C. grandis, monoembryonic), respectively. Genome duplication analysis indicated that segmental and tandem duplication events contributed to the expansion of the B3 superfamily in citrus, and that the B3 superfamily evolved under the effect of purifying selection. Phylogenetic relationships were well supported by conserved gene structure and motifs outside the B3 domain, which allowed possible functions to be inferred by comparison with homologous genes from Arabidopsis. Expression analysis identified 23 B3 superfamily members that were expressed during SE in citrus and 17 that may play functional roles at late SE stages. Eight B3 genes were identified that were specific to the genome of polyembryonic sweet orange compared to monoembryonic pummelo. Of these eight B3 genes, CsARF19 was found to be specifically expressed at higher levels in embryogenic callus (EC), implying its possible involvement in EC initiation. Conclusions: This study provides a genome-wide analysis of the citrus B3 superfamily, including its genome organization, evolutionary features and expression profiles, and identifies specific family members that may be associated with SE.

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Lipid A, the hydrophobic anchor of lipopolysaccharide, is an essential component in the outer membrane of most Gram-negative bacteria. Food-borne pathogen Cronobacter sakazakii synthesizes two lipid A species, differing by the length of the secondary acyl chain. In this work, we identified three genes ESA02293, ESA02951 and ESA01386 encoding for the late acyltransferases of lipid A biosynthesis pathway in C. sakazakii. Based on the sequence alignment, proteins YP_001438378.1 encoded by ESA02293, YP_001439016.1 encoded by ESA02951, and YP_001437482.1 encoded by ESA01386 are homologous to E. coli LpxL, LpxP and LpxM, respectively. Functions of the three acyltransferases were confirmed by overexpressing the genes in E. coli, isolating lipid As and analyzing their structures using an ESI/MS. C. sakazakii LpxL and LpxM transfer a C14:0 secondary acyl chain to the 2′- and 3′-position of lipid A, respectively. C. sakazakii LpxP can transfer either a C16:1 or a C14:0 secondary acyl chains to the 2′-position of lipid A.

The direct fermentative production of L-serine from renewable biomass using Corynebacterium glutamicum is attracting increasing attention. In this study, wild-type C. glutamicum SYPS-062 produced up to 6.65±0.23 g/L Lserine; to further improve L-serine production, the serA gene was cloned, and the C-terminal domain of 3-phosphoglycerate dehydrogenase (PGDH) from this strain was truncated. When expressed in Escherichia coli, the resultant mutein SerAΔ197 showed a specific PGDH activity of 1.092±0.05 U/mg protein, representing a decrease of 25.87 % from that encoded by serA, and was no longer sensitive to high concentrations of L-serine. When serA Δ591 was overexpressed in C. glutamicum SYPS-062, the activity of PGDH in C. glutamicum pJC1-tac-serA Δ591 increased by 47.72 %, and the resultant strain C. glutamicum pJC1-tac-serA Δ591 could accumulate 7.69± 0.22 g/L L-serine. Furthermore, when serA Δ591 was overexpressed in C. glutamicum SYPS-062ΔsdaA, the resultant strain could accumulate 8.84 ±0.23 g/L L-serine at 102 h, and the yield of L-serine on cells (Y p/x) improved by 60 % when compared with that noted in the control. These results demonstrate that L-serine production in C. glutamicum SYPS-062 could be improved by overexpressing a C-terminal truncation of PGDH in combination with other genetic modifications.

A comparative genomic analysis was performed to study the genetic variations between the L-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced L-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as L-alanine and L-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the L-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher L-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest L-serine production to date.