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Keto acid decarboxylase (Kdc) is a key enzyme in producing keto acid derived higher alcohols, like isobutanol. The most active Kdc's are found in mesophiles; the only reported Kdc activity in thermophiles is 2 orders of magnitude less active. Therefore, the thermostability of mesophilic Kdc limits isobutanol production temperature. Here, we report development of a thermostable 2-ketoisovalerate decarboxylase (Kivd) with 10.5-fold increased residual activity after 1h preincubation at 60 °C. Starting with mesophilic Lactococcus lactis Kivd, a library was generated using random mutagenesis and approximately 8,000 independent variants were screened. The top single-mutation variants were recombined. To further improve thermostability, 16 designs built using Rosetta Comparative Modeling were screened and the most active was recombined to form our best variant, LLM4. Compared to wild-type Kivd, a 13 °C increase in melting temperature and over 4-fold increase in half-life at 60 °C were observed. LLM4 will be useful for keto acid derived alcohol production in lignocellulosic thermophiles.

The current unsustainable usage of antibiotics for clinical and agricultural purposes has resulted in a global spread of antibiotic-resistant bacterial strains. This rapid reduction in antimicrobial potency is directly caused by the strong evolutionary selection pressure of classical antibiotics. One promising strategy to avoid this problem is the development of antivirulence compounds, which seeks to alleviate the aforementioned selection pressure by specifically mitigating pathogenic phenotypes, for example, toxin secretion or biofilm formation, but without killing the bacteria. In this work, we propose a new target for this emerging antivirulence approach and identify through molecular design followed by chemical screening that bacterial osmoprotectant analogues can be used as antivirulence metabolites against biofilm formation. Among the 19 compounds tested, ethylcholine, a biosynthetic precursor of ethyl glycine betaine and previously reported to induce glycine betaine catabolism, was found to most effectively inhibit biofilm establishment without detectable biological toxicity. The potential complementary usage of this molecule with traditional antibiotic compounds and its other impacts on bacterial physiology were also herein explored and discussed.

The first green chemistry-oriented antispore strategy is herein reported. The tricomponent system of low pH, anionic surfactants, and ethanol kills bacterial spores under relatively mild conditions by sequentially targeting spore resistance and viability, contrasting the current prevailing spore chemical sterilization protocols that invariably require the use of strong oxidizing agents. The sporicidal potency of this system was demonstrated with three spore species both in liquid suspensions as well as on surfaces. Furthermore, the chemical simplicity of this strategy allows it to be easily recreated using common household products.

Bacterial endospores can withstand common disinfection procedures and extreme environmental adversity. This tenacity for survival, coupled with pathogenicity, makes spores a major threat for the food and medical industries as well as national security. Though unsuitable for practical usage due to their high environmental toxicity, primary ammonium surfactants, dodecylamine (DDA) in particular, are the most potent antispore molecules known. However, over half a century after the initial discovery, the mechanism of DDA spore killing remains largely elusive and antispore compounds with practical utility are still greatly needed. Herein, we propose and provide evidence that DDA bioactivity may lie in its capacity to form hydrophobically stabilized salt bridges with carboxylate anions of the spore cortex, a structure critical in maintaining a low water content in the spore core. More importantly, the proposed mechanism of action was experimentally shown to be useful in guiding the design of potential antispore agents.

Global climate change linked to the accumulation of greenhouse gases has caused concerns regarding the use of fossil fuels as the major energy source. To mitigate climate change while keeping energy supply sustainable, one solution is to rely on the ability of microorganisms to use renewable resources for biofuel synthesis. In this Review, we discuss how microorganisms can be explored for the production of next-generation biofuels, based on the ability of bacteria and fungi to use lignocellulose; through direct CO2 conversion by microalgae; using lithoautotrophs driven by solar electricity; or through the capacity of microorganisms to use methane generated from landfill. Furthermore, we discuss how to direct these substrates to the biosynthetic pathways of various fuel compounds and how to optimize biofuel production by engineering fuel pathways and central metabolism.

Multifunctional magnetic-plasmonic Fe(3)O(4)-Au core-shell nanoparticles (Au-MNPs) were prepared for simultaneous fast concentration of bacterial cells by applying an external point magnetic field, and sensitive detection and identification of bacteria using surface-enhanced Raman spectroscopy (SERS). We demonstrated that a spread of a 10 μL drop of a mixture of 10(5) cfu/mL bacteria and 3 μg/mL Au-MNPs on a silicon surface can be effectively condensed into a highly compact dot within 5 min by applying an external point magnetic field, resulting in 60 times more concentrated bacteria in the dot area than on the spread area without concentration. Surrounded by dense uniformly packed Au-MNPs, bacteria can be sensitively and reproducibly detected directly using SERS. The principle component analysis (PCA) showed that three different Gram-negative bacterial strains can be clearly differentiated. We also demonstrated that the condensed multifunctional Au-MNPs dot can be used as a highly sensitive SERS-active substrate and a limit of detection better than 0.1 ppb was obtained in detection of small molecules such as 4-mercaptopyrine. This novel platform significantly simplifies the concentration and detection process, which holds great promise for applications in food safety, environmental monitoring, medical diagnoses, and chemical and biological threat detections.

Attack or defend! A smart polymer surface has two reversibly switchable equilibrium states, a cationic N,N-dimethyl-2-morpholinone (CB-Ring) and a zwitterionic carboxy betaine (CB-OH). CB-Ring will kill bacteria upon contact under dry conditions, whereas CB-OH will release the previously attached and dead bacteria and further resist adhesion of bacteria under wet conditions.

In this work, we report a study of the interactions between bacteria and zwitterionic polymers using a long-range surface plasmon resonance (LR-SPR) sensor. The LR-SPR with an extended probing field allows one to perform in-situ monitoring of bacterial adhesion and to investigate bacteria–surface interactions under various conditions. Alginate-producing and -deficient Pseudomonas aeruginosa were used in order to study the role of capsular exopolysaccharide in bacterial adhesion onto a surface. Polycarboxybetaine polymers with one and two carbons in spaces between two oppositely charged moieties on polymer side chains (pCBAA-1 and pCBAA-2) were prepared in order to unveil the effects of material properties on surface resistance to bacterial adhesion. In addition, environmental factors (e.g., divalent cations) were investigated. This work demonstrates the role of polysaccharides beyond proteins in bacteria adhesion and the effective resistance of pCBAA-1 against bacterial adhesion.