Abstract In this study, high-throughput sequencing (HTS) was used to compare and analyze the microbial diversity and variation law during the brewing process of xiaoqu Baijiu . The results showed that 34 phyla, 378 genera of bacteria and 4 phyla, 32 genera of fungi were detected. At the phylum level, Firmicutes, Proteobacteria, Bacteroidetes, Ascomycota and Bacteroidetes were the dominant groups. During the brewing process of xiaoqu Baijiu , the dominant bacteria were Weissella and unidentified Rickettsiales 2 days before brewing and Lactobacillus 3 days after brewing until the end of brewing. The dominant fungi were Rhizopus , Saccharomyces and Issatchenkia . The relative abundance of Rhizopus decreased with the extension of brewing time, while the relative abundance of Saccharomyces increased and became the dominant bacteria after the second day of brewing. This study revealed the diversity and variation of microbial community in the brewing process of xiaoqu Baijiu , and provide theoretical support and lay the foundation for future study on the contribution of microbial metabolism during brewing of xiaoqu Baijiu , thereby promote the development of xiaoqu baijiu industry.
O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.
Wild-type Escherichia coli usually does not accumulate l-threonine, but E. coli strain TWF001 could produce 30.35 g/L l-threonine after 23-H fed-batch fermentation. To understand the mechanism for the high yield of l-threonine production in TWF001, transcriptomic analyses of the TWF001 cell samples collected at the logarithmic and stationary phases were performed, using the wild-type E. coli strain W3110 as the control. Compared with W3110, 1739 and 2361 genes were differentially transcribed in the logarithmic and stationary phases, respectively. Most genes related to the biosynthesis of l-threonine were significantly upregulated. Some key genes related to the NAD(P)H regeneration were upregulated. Many genes relevant to glycolysis and TCA cycle were downregulated. The key genes involved in the l-threonine degradation were downregulated. The gene rhtA encoding the l-threonine exporter was upregulated, whereas the genes sstT and tdcC encoding the l-threonine importer were downregulated. The upregulated genes in the glutamate pathway might form an amino-providing loop, which is beneficial for the high yield of l-threonine production. Many genes encoding the 30S and 50S subunits of ribosomes were also upregulated. The findings are useful for gene engineering to increase l-threonine production in E. coli.
In order to better understand the transformation and distribution of different Se forms in wild peach. Se2−, Se0, Se4+ and Se6+ (from selenomethionine, Se powder, sodium selenite, and sodium selenate, respectively) were added to 4.1-L plastic pots with 15-cm high wild peach seedlings, for a final soil Se concentration of 1.0 mg/kg soil. Growth parameters, Se transaction factors (TFS) and bioconcentration factor, as well as the leaf sugar content and antioxidant ability were determined. We found that all four forms of Se increased shoot length, stem diameter, and dry weight, whereby treatment with Se4+ and Se6+ especially promoted the dry weight. The wild peach seedlings showed different Se enrichment ability, whereby the Se bioconcentration factor (BCF) and total Se uptake per plant were in the same order of Se2– > Se6+> Se4+> Se0 > Control. However, the transaction factors (TFS) in the control and Se6+ treatment groups were significantly higher than in the groups treated with other Se forms. The control and the Se forms Se0, Se4+ and Se2− showed the same trend of Se distribution, with the largest Se concentration in the roots, followed by the leaves and stems. No significant differences were observed in leaf pigment concentration of the seedlings treated with all Se forms except for Se0. Treatment with different Se forms significantly reduced the leaf malondialdehyde (MDA) concentration, obviously increased the reducing power, insignificantly increased the content of reducing sugars, and decreased the content of total sugars. Overall, different Se forms effected the growth and physiological parameters of wild peach seedlings to different extent, whereby Se6+ and Se2– led to the highest Se accumulation, which may be beneficial for the production of Se-enriched fruits.
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