A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

Abstract

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

Enhancing butanol tolerance of Escherichia coli reveals hydrophobic interaction of multi-tasking chaperone SecB

“…However, only GroEL and ClpB from E. coli [7], GroESL from C. acetobutylicum [43], Cupriavidus necator [44] and Lactococcus lactis [24], and PhaP from Azotobacter sp. strain FA8 with chaperone-like properties [10] have been proved to be effective in enhancing the host tolerance against butanol. As a result, genome mining for more molecular chaperones in improving the butanol tolerance of E. coli was of special interest.…”

“…Specific growth rate ( μ ) of E. coli strain was calculated according to Eq. 1 [10]. Tolerance ( T ) to solvents was determined based on the growth of engineered E. coli in the presence and absence of solvent as shown in Eq.…”

“…Penetration and accumulation of organic solvents in cell membrane and cytoplasm might lead to denaturation of functional proteins, oxidative stress and disorganization of cellular structure, resulting in the loss of ions and changes of intracellular pH and membrane fluidity, the leakage of cytoplasm, and eventually cell death [8]. Engineering host strain for enhanced product tolerance is often envisaged as one of the effective approaches to improve biofuel production [9, 10].…”

Understanding and engineering alcohol-tolerant bacteria using OMICS technology

“…Overexpressing the phasin polyhydroxyalkanoate granule-associated protein (PhaP) from Azotobacter sp. strain FA8 (Mezzina et al 2017 ) in E. coli also enhances alcohol tolerance PhaP from Azotobacter has chaperone activity and exerts a stress-alleviating effect in recombinant E. coli cells (de Almeida et al 2011 ; Mezzina et al 2015 ). In these approaches, beneficial genes associated with alcohol tolerance (e.g., GroESL or PhaP) were found in known alcohol-tolerant species such as C. acetobutylicum or Azotobacter spp.…”

Quantitative proteomic analysis ofRhodococcus ruberresponsive to organic solvents

“…Organic solvents are a variety of widely used organic compounds such as ethanol, toluene, chloroform, phenol, etc., which play an important role in the modern society [1]. For instance, organic solvents are often introduced in a process catalysed by whole cells.…”