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Front Microbiol
2022 Apr 14;13:855736. doi: 10.3389/fmicb.2022.855736.
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A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics.
Malla S
,
van der Helm E
,
Darbani B
,
Wieschalka S
,
Förster J
,
Borodina I
,
Sommer MOA
.
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Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titers during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel L-lysine transporter, belonging to a previously uncharacterized EamA superfamily, which is further in vivo characterized as L-lysine exporter using Xenopus oocyte expression system as well as Escherichia coli host. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced yield, titer, and the specific production of L-lysine in an industrial Corynebacterium glutamicum strain by 7.8%, 9.5%, and 12%, respectively. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titers and productivity of toxicity-limited bioprocesses.
FIGURE 3. Functional characterization of MglE transporter activity in Xenopus oocytes, E.coli and Corynebacterium glutamicum. (A) L-lysine export assay of MglE transporter in Xenopus oocytes in Kulori buffer (i) at pH 5 and (ii) at pH 7.4. The bars represent lysine contents in the oocytes or the buffer (means ± SD, 3–4 biological replicates each involving 20 oocytes). Significant changes are in comparison with the controls marked by asterisks (** p < 0.01; Fishers one-way ANOVA). (B) The extracellular L-lysine concentration of reference E. coli W3110 and its isogenic mutant strain DMLC along with the cell OD600nm values confirming the exporter activity of the MglE protein in (i) LB media and (ii) in M9 minimal media supplemented with yeast extract (* p < 0.0001, t-test). (C) Fold enhancement of specific production of L-lysine by MglE protein in the industrial C. glutamicum VL5 strain compared to the empty vector control. Error bars are s.e.m., * denotes p = 0.023, n = 4.
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