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Proteus vulgaris is an important foodborne opportunistic pathogen, both environmentally and clinically. The use of appropriate antibiotics has significant therapeutic effects, but has led to the emergence and spread of drug-resistant strains. In this study, a P. vulgaris strain, designated "P3M," was isolated from Penaeus vannamei in Tianjin, China. The whole genome of P3M was sequenced, generating detailed information, including the key genes involved in important metabolic pathways and their physiological functions. A total of 218 antibiotic resistance genes (ARGs) were predicted in the genome. The determination of various minimum inhibitory concentrations indicated that P3M is a multidrug-resistant (MDR) bacterium, with significant resistance to 16 antibiotics in seven categories. Determination of fractional inhibitory concentration index showed that the combination of ciprofloxacin plus tetracycline exhibited synergistic antimicrobial activity. Bioinformatics and phylogenetic analyses detected the presence of two two-component systems that mediate multidrug resistance and several mobile genetic elements involved in the horizontal transfer of ARGs in P3M. P. vulgaris strains represent a serious challenge to clinicians and infection control teams for its ubiquity worldwide and close relevance with human life. To the best of our knowledge, we report the first isolation and characterization of an important foodborne MDR P. vulgaris strain, and this study will provide necessary theoretical basis for the selection and clinical use of the appropriate antibiotics.

The use of particulate adjuvants offers an interesting method for enhancing and modulating the immune responses elicited by vaccines. Aluminum salt (Alum) is one of the most important immune adjuvants approved by the Food and Drug Administration for use in humans because of its safety and efficacy, but it lacks the capacity to induce strong cellular and mucosal immune responses. In this study, we designed an antigen delivery system that combines aluminum salts with β-glucan particles. The β-glucan-aluminum particles (GP-Al) exhibited a highly uniform size of 2-4 μm and could highly specifically target antigen-presenting cells (APCs) and strongly induce dendritic cell (DC) maturation and cytokine secretion.

In this study, we applied a series of genetic modifications to wild-type S. cerevisiae strain BY4741 to address the bottlenecks in the l-tyrosine pathway. A tyrosine ammonia-lyase (TAL) gene from Rhodobacter capsulatus, which can catalyze conversion of l-tyrosine into p-coumaric acid, was overexpressed to facilitate the analysis of l-tyrosine and test the strain’s capability to synthesize heterologous derivatives. First, we enhanced the supply of precursors by overexpressing transaldolase gene TAL1, enolase II gene ENO2, and pentafunctional enzyme gene ARO1 resulting in a 1.55-fold increase in p-coumaric acid production. Second, feedback inhibition of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase was relieved by overexpressing the mutated feedback-resistant ARO4(K229L) and ARO7(G141S), and a 3.61-fold improvement of p-coumaric acid production was obtained. Finally, formation of byproducts was decreased by deleting pyruvate decarboxylase gene PDC5 and phenylpyruvate decarboxylase gene ARO10, and p-coumaric acid production was increased 2.52-fold. The best producer—when TAL1, ENO2, ARO1, ARO4(K229L), ARO7(G141S), and TAL were overexpressed, and PDC5 and ARO10 were deleted—increased p-coumaric acid production by 14.08-fold (from 1.4 to 19.71 mg L(−1)). Our study provided a valuable insight into the optimization of l-tyrosine metabolic pathway. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02223-3) contains supplementary material, which is available to authorized users.

To determine the effect of large genomic region deletion in a Saccharomyces cerevisiae strain on tyrosine yield and to identify new genetic modification targets through transcriptome analysis. TAL was used to produce p-coumaric acid (p-CA) from tyrosine to quantity tyrosine yield. S. cerevisiae mutant strain NK14 with deletion of a 23.8 kb genomic region was identified to have p-CA production of 10.3 mg L− 1, while the wild-type strain BY4741 had p-CA production of 1.06 mg L− 1. Analysis of growth patterns and stress tolerance showed that the deletion did not affect the growth phenotype of NK14. Transcriptome analysis suggested that, compared to BY4741, genes related to glycolysis (ENO2, TKL1) and the tyrosine pathway (ARO1, ARO2, ARO4, ARO7, TYR1) were upregulated in NK14 at different levels. Besides genes related to the tyrosine biosynthetic pathway, amino acid transporters (AVT6, VBA5, THI72) and transcription factor (ARO80) also showed changes in transcription levels. We developed a strain with improved tyrosine yield and identified new genetic modification candidates for tyrosine production.

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives. 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. Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.