BackgroundSecretory expression of valuable proteins by B. subtilis and its related species has attracted intensive work over the past three decades. Although very high yields can be achieved with homologous proteins, production of heterologous proteins by B. subtilis is unfortunately not the straight forward. The Sec pathway is the major route for protein secretion in B. subtilis. Therefore, the aim of this work was to identify the bottlenecks of the Sec pathway and improve the secretion of heterologous proteins by molecular genetic techniques.ResultsTwo α-amylases (AmyL and AmyS) both under the control of the PHpaII promoter and equipped with their native signal peptides SPamyl and SPamyS were successfully secreted with significantly different expression levels. To improve the secretion efficiency, 23 main genes or gene operons involved in or closely related to the Sec pathway were overexpressed singly by increasing an additional copy on the chromosome, and the overexpression of prsA enhanced the production of α-amylases (AmyL and AmyS) by 3.2- and 5.5-fold, respectively. With the induction by xylose of different concentrations, prsA overexpression level was optimized and the secretion efficiency of α-amylase was further improved. Moreover, combinatorial overexpression of prsA and nine screened genes or gene operons, respectively, was performed, and the overexpression of prsA combined with partial dnaK operon improved the α-amylase activity of AmyL and AmyS by 160 and 173%, respectively, compared with the overexpression of prsA singly. Finally, the performance of the recombinant B. subtilis 1A237 was evaluated with the fed-batch fermentation in 7.5 L fermentor, and the level of secreted AmyL and AmyS reached 1,352 and 2,300 U/mL with the productivity of 16.1 U/mL h and 27.4 U/mL h, respectively.ConclusionsOur systematic gene overexpression approach was designed to investigate the bottleneck of Sec pathway in B. subtilis. The deficiency of PrsA lipoprotein and chaperones of DnaK series was main rate-limiting factors for heterologous proteins secretion. Systematic and deep insight into how components of Sec pathway interact with each other may be the key to improving the yield of heterologous proteins thoroughly.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0282-9) contains supplementary material, which is available to authorized users.

NACs (NAM, ATAF1/2, and CUC2) are plant-specific transcription factors that play diverse roles in various plant developmental processes. In this study, we identified the NAC gene family in birch (Betula pendula) and further analyzed the function of BpNACs. Phylogenetic analysis reveals that the 114 BpNACs can be divided into seven subfamilies. We investigated the expression levels of these BpNACs in different tissues of birch including roots, xylem, leaves, and flowers, and the results showed that the BpNACs seem to be expressed higher in xylem and roots than leaves and flowers. In addition to tissue-specific expression analysis, we investigated the expression of BpNACs under low-temperature stress. A total of 21 BpNACs were differentially expressed under low-temperature stress, of which 17 were up-regulated, and four were down-regulated. Using the gene expression data, we reconstructed the gene co-expression network for the 21 low-temperature-responsive BpNACs. In conclusion, our results provide insight into the evolution of NAC genes in the B. pendula genome, and provide a basis for understanding the molecular mechanism for BpNAC-mediated cold responses in birch.

Despite early domestication around 3000 BC, the evolutionary history of the ancient allotetraploid species Brassica juncea (L.) Czern & Coss remains uncertain. Here, we report a chromosome-scale de novo assembly of a yellow-seeded B. juncea genome by integrating long-read and short-read sequencing, optical mapping and Hi-C technologies. Nuclear and organelle phylogenies of 480 accessions worldwide supported that B. juncea is most likely a single origin in West Asia, 8,000–14,000 years ago, via natural interspecific hybridization. Subsequently, new crop types evolved through spontaneous gene mutations and introgressions along three independent routes of eastward expansion. Selective sweeps, genome-wide trait associations and tissue-specific RNA-sequencing analysis shed light on the domestication history of flowering time and seed weight, and on human selection for morphological diversification in this versatile species. Our data provide a comprehensive insight into the origin and domestication and a foundation for genomics-based breeding of B. juncea.

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