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.