Introduction

Transport processes are essential to living organisms and the control of transport across lipid membranes was one of the milestones to the generation of life on Earth (Lancet, Zidovetzki, & Markovitch, 2018; Mansy, 2010). Transport proteins play a big role in cell physiology, such as nutrient uptake and export of toxic compounds, and are predicted to make up 13.7% and 5.8% of the Escherichia coli andSaccharomyces cerevisiae proteome, respectively (Claus, Jezierska, & Bogaert, 2019).
However, research on transport processes has always be limited due to technical difficulties in comparison with enzymes. Most transport proteins are membrane integrated or membrane associated proteins. The membrane environment necessary for the correct folding of membrane proteins complicates their purification and the following in vitro studies or crystallizations for structural studies. This results in an underrepresentation of structures of membrane proteins in databases, as they represent less than 3% of protein structures in the Protein Data Bank while they comprise around 25% of all known proteins (Newport, Sansom, & Stansfeld, 2019). Therefore, when studying membrane proteins, it is necessary to rely on other techniques, such as homology modelling or mutagenesis (Futagi, Kobayashi, Narumi, Furugen, & Iseki, 2019). Besides this, heterologous expression or overexpression of membrane proteins can easily lead to membrane stress in cells, requiring careful strategies to ensure proper expression and activity (Kang & Tullman-Ercek, 2018). Another disadvantage related to transport processes is the lack of chemical change in their activity, which renders transport activity measurement more laborious and less straight-forward than enzymatic activity (Brouwer et al., 2013).
Despite all these limitations, transport proteins have been studied since the last century, when a lactate transporter was first identified in E. coli (Jones & Kennedy, 1969). Since then, many transporters have been identified for a wide diversity of molecular structures. However, the transport of hydrophobic substances has been traditionally associated with passive diffusion. During the last years, extensive evidence of protein assisted transport for hydrophobic molecules has led to a paradigm shift in the scientific community (Claus et al., 2019). Although the transport of hydrophobic compounds requires different mechanisms than soluble compounds, the employment of transport proteins allows cells to regulate this process and it opens opportunities for engineering and improving these transport processes.
Among the different hydrophobic cellular compounds, the most prevalent ones in metabolism are fatty acids, serving both as energy storage and membrane constituents. Transport of fatty acids in animals is tightly regulated, both intra and extracellularly. Fatty acids are transported across different organs and cellular compartments via soluble fatty acid binding proteins as well as membrane bound transporters (Glatz, 2015; Glatz, Luiken, & Bonen, 2010). Perturbation in the transport of fatty acids and lipids give place to pathologies, such as adrenoleukodystrophy, which is associated to mutations in the peroxisomal fatty acid transporter (Tarling, Vallim, & Edwards, 2013). This shows the importance of these mechanisms for correct physiological functions. Also in plants, lipid transport plays indispensable roles in processes such as seed development and cutin synthesis. In plants, fatty acids are synthesized in plastids, modified in the endoplasmic reticulum, stored in cytosolic lipid bodies and degraded in peroxisomes. This high compartmentalization of fatty acid metabolism in plants needs the coordinated participation of membrane transporters and carrier proteins for the intracellular trafficking of fatty acids. The transport of lipids and fatty acids in plants has been reviewed elsewhere (N. Li, Xu, Li-Beisson, & Philippar, 2016).
Fatty acid metabolism has been extensively studied in microbial model organisms such as E. coli and S. cerevisiae , but also in oleaginous bacteria, yeasts and microalgae (Hu, Zhu, Nielsen, & Siewers, 2019; Magnuson, Jackowski, Rock, & Cronan, 1993). Oleaginous organisms, such as Rhodococcus jostii or Yarrowia lipolytica , are those able to store lipids as 20% or more of their dry cell weight, reaching 80% in some cases (Alvarez et al., 2019). While microbial fatty acid metabolism has been highly studied and engineered, fatty acid transport in microorganisms still present knowledge gaps. The study of fatty acids in microorganisms is relevant because the scientific evidence acquired from the model organisms E. coli andS. cerevisiae lays the biochemical basis for research in higher organisms. In addition, their easy manipulation allows a more thorough and flexible study of the transport systems and their components. Moreover, microbial production of fatty acids is gaining attention due to their wide range of applications, mainly as biofuels and nutrition supplements, but also in animal feed, cosmetics and lubricants (Vasconcelos, Teixeira, Dragone, & Teixeira, 2019).
Increasing export rates for fatty acids can be highly beneficial for microbial cell factories for two reasons. On the one hand, overproduction of a metabolite causes feedback inhibition as well as toxic effects in the cell that severely affect productivity. The removal of product from the cellular space allows for a continuous production and a higher yield per cell mass (Kell, Swainston, Pir, & Oliver, 2015). On the other hand, the downstream processing of fermentation processes has a significant contribution to the final cost-effectiveness of the production process, due to the need to break open cells to extract the desired product, as well as the subsequent separation process from all cell constituents (Jezierska & Van Bogaert, 2017). The export of fatty acids into the extracellular space allows for a simpler downstream processing, where cells are removed, and the product can be directly obtained from a media that contains much less by-products (Borodina, 2019).
Despite the limitations associated with the study of membrane proteins and transport, several fatty acid import and export systems have been identified in microorganisms. This review presents the current scientific knowledge on fatty acid transport across biological membranes, as well as examples of engineering fatty acid transport to improve microbial cell factories.