Erucic acid utilization by Lactobacillus johnsonii N6.2

Abstract

A multivariate nutritional analysis indicated that the consumption of erucic acid-rich food, a fatty acid (FA) found primarily in rapeseed and mustard oil, was positively correlated with higher counts of lactic acid bacteria (LAB). Furthermore, we showed Lactobacillus johnsonii N6.2, as well as other species of LAB tested from the former Lactobacillus genus, were able to efficiently use erucic acid (EA) as the source of FA. In this work, we identified significant changes induced in the FA profiles of L. johnsonii cultured with EA as the source of FA. We performed global transcriptomics to identify genes and pathways involved in EA utilization. It was found that L. johnsonii incorporates external fatty acids via a FakA/FakB and the plsX/plsY/plsC pathway for phosphatidic acid synthesis. It was found that cells grown in MRS with EA (MRS-E) significantly upregulated fakB2 and fakB4 when compared to cells grown in standard MRS with tween 80 as the source of FA. Additionally, in MRS-E, L. johnsonii N6.2 induced the expression of plsY2, plsC2 and plsC4 while the expression of pslX was constitutive during short term EA exposure. LC-MS analyses revealed that L. johnsonii N6.2 rapidly incorporates EA and synthesizes a variety of long chain fatty acids, including the health-relevant omega-9 monounsaturated fatty acids such as nervonic and gondoic acids.

Keywords: Lactobacillus johnsonii; erucic acid; long chain fatty acid; nervonic acid; probiotic.

Figure 1

Relative gene expression of fatty acid incorporation genes in L. johnsonii N6.2. Cells were grown in MRS-TD to exponential phase, centrifuged and washed pellets were resuspended in either MRS-E (E), MRS-O (O), or MRS-TD (T) for 5, 15, 30, and 60 min. Gene expression is expressed as log(2) fold change relative to MRS-TD 5 min. Genes involved in incorporation of fatty acids included (A) fakB1, (B) fakB2, (C) fakB3, and (D) fakB4. Gene expression was normalized to rpoD. Statistical analyses were performed by two-way ANOVA and Tukey’s post-hoc test. Different letters denote significant difference of p-adj < 0.05.

Figure 2

Relative gene expression of phosphatidic acid biosynthesis genes in L. johnsonii N6.2. Cells were grown in MRS-TD to exponential phase, centrifuged and washed pellets were resuspended in either MRS-E (E), MRS-O (O), or MRS-TD (T) for 5, 15, 30, and 60 min. Gene expression is expressed as log(2) fold change relative to MRS-TD 5 min. Phosphatidic biosynthesis genes included (A) plsX, (B) plsY1, (C) plsY2, (D) plsC1, (E) plsC2, (F) plsC3, and (G) plsC4. Gene expression was normalized to rpoD. Statistical analyses were performed by two-way ANOVA and Tukey’s post-hoc test. Different letters denote significant difference of p-adj < 0.05.

Figure 3

Relative gene expression of lipid head group biosynthesis genes in L. johnsonii N6.2. Cells were grown in MRS-TD to exponential phase, centrifuged and washed pellets were resuspended in either MRS-E (E), MRS-O (O), or MRS-TD (T) for 5, 15, 30, and 60 min. Gene expression is expressed as log(2) fold change relative to MRS-TD 5 min. Lipid head group biosynthesis (A) psd1, (B) psd2, (C) pmtA, and (D) pgpA were evaluated. Gene expression was normalized to rpoD. Statistical analyses were performed by two-way ANOVA and Tukey’s post-hoc test. Different letters denote significant difference of p-adj < 0.05.

Figure 4

Model of the phosphatidic acid biosynthesis pathway in L. johnsonii N6.2. Exogenous fatty acids, saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) are bound by FakB and converted to acyl-phosphate by FakA. L. johnsonii N6.2 encodes four fakB genes: fakB1, fakB2, fakB3, and fakB4. fakB2 and fakB4 are both upregulated in MRS-E media. In L. johnsonii N6.2, plsX is constitutively expressed in the short term exposure experiments, and modification of fatty acids is likely mediated through alternate enzymes. Acyl-phosphate can be utilized by PlsY which catalyzes the formation of lysophosphatidic acid, or by PlsX to synthesize acyl-ACP. L. johnsonii N6.2 encodes two plsY genes. PlsY2 is upregulated in both MRS-E and MRS-O media. Finally, lysophosphatidic acid and acyl-ACP are converted to phosphatidic acid by PlsC. L. johnsonii N6.2 encodes four plsC genes. PlsC2 and plsC4 are both upregulated in MRS-E media. Upregulation of the genes by the addition of EA or OA is indicated with an upward arrow next to the FA. Figure created with Biorender.com.