The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granuleassociated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymerproducing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli. Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals.IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E. coli. These findings open the road for the use of these proteins for the manipulation of bacterial strains to optimize the synthesis of diverse bioproducts from renewable carbon sources.KEYWORDS Escherichia coli, ethanol, butanol, 1,3-propanediol, chaperone, GroEL, PhaP, metabolic engineering F ossil oil, or petroleum, has been utilized by humankind for many centuries, but since the mid-18th century the number and variety of applications for this product have sharply increased, so that nowadays derivatives of this nonrenewable substrate are present in almost every aspect of modern life (1). We use petroleum derivatives as our main source of energy but also as a starting point for the synthesis of many different

Manipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary in different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra and Rob using single deletion mutants of the well characterized and completely sequenced strain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds and synthesis of native and non-native bioproducts in different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants, and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3- propanediol (1,3-PDO). The mutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the mutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semi-defined medium using glycerol, resulting in 24 g.L of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g.Lh Although the effects of many global regulators, especially ArcA and Cra, have been studied in , the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, essential for the design of metabolic engineering strategies. The genome of BW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion eliminating possible strain-dependent results. As a proof of concept, and mutants were subjected to further manipulations to obtain high amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts.

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Staphylococcus aureusandPseudomonas aeruginosaare two bacterial species that cause a wide variety of infections and coinfections. The interaction between these species is complex and involves the production of different metabolites and metabolic changes. The impact of elevated body temperature, such as fever, on the physiology and interaction of these pathogens remains poorly understood. Therefore, the aim of this work was to analyze the effect of moderate fever-like temperatures (39°C) onS. aureusUSA300 andP. aeruginosaPAO1 mono- and co-cultures in comparison with 37°C, by using RNAseq and physiological assays in microaerobiosis. Both bacterial species showed metabolic alterations in response to temperature but also in response to the competitor. Organic acid production and nitrite content in the supernatant were altered both by the presence of the competitor and the incubation temperature. Interaction ANOVA showed that, inS. aureus, gene expression presented interaction between the temperature and the presence of the competitor. Among these genes, the most relevant were theagroperon and three of its direct target genespsmβ,psmβ2andhld. In the A549 epithelial lung cell line, fever-like temperatures impacted in vitro virulence, antibiotic resistance, cell invasion, and cytokine production. In agreement with thein vitroassays, the survival of mice that had been intranasally inoculated withS. aureusmonocultures pre-incubated at 39°C showed reduced survival after 10 days. An even higher mortality of around 30% was observed in mice that had been inoculated with co-cultures pre-incubated at 39°C. The bacterial burden in the lungs, kidney, and liver was higher for both species when the mice were infected with co-cultures previously incubated at 39°C. Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

HIGHLIGHTS ➢Comparative genome analysis of Thermoanaerobacterium thermosaccharolyticum GSU5 was performed. ➢Ethanol and butanol production from different carbon sources without acetone was achieved. ➢ Solventogenic and acidogenic pathways in Thermoanaerobacterium were elucidated.