Glycine-rich flexible peptide linkers have been widely adopted in fusion protein engineering; however, they can hardly be cleaved for the separation of fusion partners unless specific protease recognition sites are introduced. Herein, we report the use of the peptidoglycan-targeting staphylolytic enzyme lysostaphin to directly digest the glycine-rich flexible linkers of various lengths including oligoglycine linkers and (G4S)x linkers, without the incorporation of extra amino acids. Using His-MBP-linker-LbCpf1 as a model substrate, we show that both types of linkers could be digested by lysostaphin, and the digestion efficiency improved with increasing linker length. The enzyme LbCpf1 retained full activity after tag removal. We further demonstrated that the proteolytic activity of lysostaphin could be well maintained under different environmental conditions and in the presence of a series of chemical reagents at various concentrations that are frequently used in protein purification and stabilization. In addition, such a digestion strategy could also be applied to remove the SUMO domain linked to LwCas13a via an octaglycine linker. This study extends the applications of lysostaphin beyond an antimicrobial reagent and demonstrates its potential as a novel, efficient, and robust protease for protein engineering.

Background Eriodictyol is a bioactive flavonoid compound that shows potential applications in medicine development and food processing. Microbial synthesis of eriodictyol has been attracting increasing attention due to several benefits. In this study, we employed a GRAS strain Corynebacterium glutamicum as the host to produce eriodictyol directly from tyrosine. Results We firstly optimized the biosynthetic module of naringenin, the upstream intermediate for eriodictyol production, through screening of different gene orthologues. Next, to improve the level of the precursor malonyl-CoA necessary for naringenin production, we introduced matB and matC from Rhizobium trifolii into C. glutamicum to convert extracellular malonate to intracellular malonyl-CoA. This combinatorial engineering resulted in around 35-fold increase in naringenin production from tyrosine compared to the initial recombinant C. glutamicum. Subsequently, the hpaBC genes from E. coli encoding 4-hydroxyphenylacetate 3-hydroxylase were expressed in C. glutamicum to synthesize eriodictyol from naringenin. Further optimization of the biotransformation process parameters led to the production of 14.10 mg/L eriodictyol. Conclusions The biosynthesis of the ortho-hydroxylated flavonoid eriodictyol in C. glutamicum was achieved for the first time via functional expression of E. coli hpaBC, providing a baseline strain for biosynthesis of other complex flavonoids. Our study demonstrates the potential application of C. glutamicum as a host microbe for the biosynthesis of value-added natural compounds from tyrosine.

金黄色葡萄球菌是常见致病菌，易产生耐药性。溶葡球菌酶可高效杀灭金黄色葡萄球菌的普通菌株及耐药菌株，具有良好的临床应用前景。然而，在丰富营养环境中，代谢旺盛的金黄色葡萄球菌耐受溶葡球菌酶，作用机制不明。现有研究发现，金黄色葡萄球菌的耐药性与壁磷壁酸密切相关。在此基础上，本项目以壁磷壁酸的具体结构为切入点，具体研究壁磷壁酸对溶葡球菌酶的效用以及菌体耐受溶葡球菌酶的影响。首先通过CRISPR-dCas9的方法，针对壁磷壁酸合成途径各基因，构建了基因抑制突变菌株。通过体外酶活动力学测试发现，壁磷壁酸与溶葡球菌酶之间无明显结合作用，但壁磷壁酸的存在可影响溶葡球菌酶的功能。通过流式细胞实验分析发现，壁磷壁酸可阻止溶葡球菌酶与菌体表面的肽聚糖进行结合。对于代谢旺盛的菌体，壁磷壁酸的存在可显著增强菌体对溶葡球菌酶的耐受性，且壁磷壁酸的丰度、链长、胞内外分布对这一耐受过程的贡献程度相当，而壁磷壁酸的糖基化修饰对菌体耐受溶葡球菌酶的过程影响有限。抑制这些关键基因的表达，可显著提高代谢旺盛的菌体对溶葡球菌酶的敏感性。该研究可为新型抗金黄色葡萄球菌药物的开发提供新靶点和新思路，为其它革兰氏阳性致病菌的耐药机制研究提供参考。

Lysostaphin is a potent bacteriolytic enzyme with endopeptidase activity against the common pathogen Staphylococcus aureus. By digesting the pentaglycine crossbridge in the cell wall peptidoglycan of S. aureus including the methicillin-resistant strains, lysostaphin initiates rapid lysis of planktonic and sessile cells (biofilms) and has great potential for use in agriculture, food industries, and pharmaceutical industries. In the past few decades, there have been tremendous efforts in potentiating lysostaphin for better applications in these fields, including engineering of the enzyme for higher potency and lower immunogenicity with longer-lasting effects, formulation and immobilization of the enzyme for higher stability and better durability, and recombinant expression for low-cost industrial production and in situ biocontrol. These achievements are extensively reviewed in this article focusing on applications in disease control, food preservation, surface decontamination, and pathogen detection. In addition, some basic properties of lysostaphin that have been controversial and only elucidated recently are summarized, including the substrate-binding properties, the number of zinc-binding sites, the substrate range, and the cleavage site in the pentaglycine crossbridge. Resistance to lysostaphin is also highlighted with a focus on various mechanisms. This article is concluded with a discussion on the limitations and future perspectives for the actual applications of lysostaphin.

Microbial cell surface layers, which mainly include the cell membrane, cell wall, periplasmic space, outer membrane, capsules, S-layers, pili, and flagella, control material exchange between the cell and the extracellular environment, and have great impact on production titers and yields of various bio-products synthesized by microbes. Recent research work has made exciting achievements in metabolic engineering using microbial cell surface components as novel regulation targets without direct modifications of the metabolic pathways of the desired products. This review article will summarize the accomplishments obtained in this emerging field, and will describe various engineering strategies that have been adopted in bacteria and yeasts for the enhancement of mass transfer across the cell surface, improvement of protein expression and folding, modulation of cell size and shape, and re-direction of cellular resources, all of which contribute to the construction of more efficient microbial cell factories toward the synthesis of a variety of bio-products. The existing problems and possible future directions will also be discussed.

Wall teichoic acids (WTAs) are charged glycopolymers containing phosphodiester-linked polyol units and represent one of the major components of Gram-positive cell envelope. WTAs have important physiological functions in cell division, gene transfer, surface adhesion, drug resistance and biofilm formation, and are critical virulence factors and vital determinants in mediating cell interaction with and tolerance to environmental factors. Here, we first briefly introduce WTA structure, biosynthesis and its regulation, and then summarize in detail four major physiological roles played by WTAs, i.e. WTA-mediated resistance to antimicrobials, virulence to mammalian cells, interaction with bacteriolytic enzymes and regulation of cell metabolism. We also review the applications of WTAs in these fields that are closely related to the human society, including antibacterial drug discovery targeting WTA biosynthesis, development of vaccines and antibodies regarding WTA-mediated pathogenicity, specific and sensitive detection of pathogens in food using WTAs as a surface epitope and regulation of WTA-related pathways for efficient microbial production of useful compounds. We also point out major problems remaining in these fields, and discuss some possible directions in the future exploration of WTA physiology and applications.

Wall teichoic acids (WTAs) are charged glycopolymers containing phosphodiester-linked polyol units and represent one of the major components of Gram-positive cell envelope. WTAs have important physiological functions in cell division, gene transfer, surface adhesion, drug resistance and biofilm formation, and are critical virulence factors and vital determinants in mediating cell interaction with and tolerance to environmental factors. Here, we first briefly introduce WTA structure, biosynthesis and its regulation, and then summarize in detail four major physiological roles played by WTAs, i.e. WTA-mediated resistance to antimicrobials, virulence to mammalian cells, interaction with bacteriolytic enzymes and regulation of cell metabolism. We also review the applications of WTAs in these fields that are closely related to the human society, including antibacterial drug discovery targeting WTA biosynthesis, development of vaccines and antibodies regarding WTA-mediated pathogenicity, specific and sensitive detection of pathogens in food using WTAs as a surface epitope and regulation of WTA-related pathways for efficient microbial production of useful compounds. We also point out major problems remaining in these fields, and discuss some possible directions in the future exploration of WTA physiology and applications.