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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