Biosynthesis and biotechnological application of the glycine-glucolipid from the marine bacterium <em>Alcanivorax borkumensis</em>

Abstract

This study investigated the production, biosynthesis pathway, and physiological function of the glycine-glucolipid produced by the marine bacterium <em>Alcanivorax borkumensis</em>. An absolute quantification method using HPLC was developed, which involves converting the glycine-glucolipid to phenacyl esters, followed by measurement with HPLC with UV detection. Different molecular species were separated by HPLC and identified via mass spectrometry (MS) as glucosyl-tetra(3-hydroxy-acyl)-glycine, containing varying numbers of 3-hydroxy-decanoic acid or 3-hydroxy-octanoic acid groups. HPLC and MS results show that the glycine-glucolipid accumulates in cells, it is not secreted into the medium, and its production is correlated with the alkane degradation process, rather than a response to phosphate deprivation. <br /> The deletion mutations in <em>A. borkumensis</em> and expression studies in <em>E. coli</em> demonstrate that the glycine-glucolipid is produced in <em>A. borkumensis</em> via a non-ribosomal peptide synthetase (NRPS)-dependent pathway involving the enzymes ABO_1784 (GglsA, NRPS), ABO_1783 (GglsB, glycosyltransferase), and ABO_1782 (GglsC, phosphopantetheinyl transferase, PPTase). GglsC activates GglsA through phosphopantetheinylation on the Ser1039 of the thiolation (T) domain. The adenylation (A) domain of GglsA activates and transfers a glycine onto the phosphopantetheine arm tethered to the T domain, consuming ATP in this process. The condensation (C) domain of GglsA catalyzes the condensation reaction, transferring a 3-D-hydroxydecanoyl moiety from 3-D-hydroxydecanoyl-CoA, forming an amide bond between glycine's amino group and the acyl group of the 3-D-hydroxydecanoyl moiety. GglsA subsequently attaches three additional 3-D-hydroxydecanoyl moieties onto the hydroxy groups of the intermediate on the T domain. Finally, the T domain shuttles the aglycone from the C/T surface to the T/TE globular core, where the aglycone is hydrolyzed from GglsA by the thioesterase (TE) domain at catalytic site Ser1177. The aglycone released from GglsA is then glycosylated by GglsB using UDP-glucose as a substrate. <br /> Physiological experiments, including the bacterial adhesion to hydrocarbons (BATH) assay, hexadecane emulsification, and observation via confocal microscopy, were performed with <em>ΔgglsB</em> and <em>ΔgglsA</em> mutants grown in pyruvate or hexadecane-containing medium. The hexadecane-grown mutant cells lacking the glycine-glucolipid and pyruvate-grown cells showed reduced adhesion to hexadecane and poorer emulsification efficiency compared to hexadecane-grown wild-type cells. The absence of the glycine-glucolipid not only impacted the attachment to hexadecane, but also reduced growth on hexadecane medium, decreased storage lipid accumulation, and resulted in smaller cell size. <br /> Expression of GglsA in <em>E. coli</em> severely limits growth and glycine-glucolipid production. Thus, <em>A. borkumensis</em> is required for glycine-glucolipid production. However, <em>A. borkumensis</em> can only be grown with expensive carbon sources. Alternatively, co-cultivation with hydrocarbon-producing cyanobacteria could offer a more sustainable approach for cultivating <em>A. borkumensis</em>. These cyanobacteria produce hydrocarbons through photosynthesis using CO2 and sunlight, and the insoluble carbon produced could stimulate glycine-glucolipid production in <em>A. borkumensis</em>. The glycine-glucolipid can potentially be employed as a biosurfactant in agriculture, replacing ecotoxic chemical surfactants like Triton X-100. However, its high cost currently limits widespread agricultural application despite its environmental benefits.