Function of L-Pipecolic Acid as Compatible Solute in Corynebacterium glutamicum as Basis for Its Production Under Hyperosmolar Conditions

Abstract

Pipecolic acid or L -PA is a cyclic amino acid derived from L -lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L -PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L -lysine biosynthetic pathway. L -PA is a six-membered ring homolog of the five-membered ring amino acid L -proline, which serves as compatible solute in C. glutamicum . Here, we show that de novo synthesized or externally added L -PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L -PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L -PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L -PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L -PA production. Under hyper-osmotic conditions, a Δ proP strain showed similar growth behavior than the parent strain when L -proline was added externally. By contrast, the growth impairment of the Δ proP strain under hyper-osmotic conditions could not be alleviated by addition of L -PA unless proP was expressed from a plasmid. This is commensurate with the view that L -proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L -PA uptake under hyper-osmotic stress conditions.

Exploring the Potential of Corynebacterium glutamicum to Produce the Compatible Solute Mannosylglycerate

“…C. glutamicum not only is a suitable host for MG production due to the vast set of established engineering tools and its genetic accessibility ( Becker and Wittmann, 2015 ; Pérez-García and Wendisch, 2018 ) but also does not take up and metabolize MG (as shown here) like other industrially relevant organisms, such as E. coli and Bacillus subtilis ( Sampaio et al, 2004 ). C. glutamicum has been engineered to produce the native compatibles solutes trehalose ( Carpinelli et al, 2006 ) and L-proline ( Jensen and Wendisch, 2013 ) and the nonnative compatible solutes α-D-glucosylglycerol ( Roenneke et al, 2018 ), L-pipecolic acid ( Pérez-García et al, 2019 ), ectoine/hydroxyectoine ( Gießelmann et al, 2019 ), and very recently also MG ( Tian et al, 2020 ). In the latter work, MG production has been reported from glucose and mannose plus glycerate and authors applied MG synthase from Rhodothermus marinus DSM 4252 and additionally overexpressed the genes nahK from Bifidobacterium longum , encoding N-acetylhexosamine 1-kinase, manB , encoding phosphomannomutase from E. coli , and manC , encoding mannose 1-phosphate guanylyl transferase from E. coli to convert glucose/mannose to MG ( Tian et al, 2020 ).…”

“…Similar techniques have been applied before for the production of compatible solutes in their native hosts, e.g., ectoine/hydroxyectoine with Halomonas elongata ( Sauer and Galinski, 1998 ; up to 4.1 g L −1 ) and MG with Thermus thermophilus RQ-1 ( Egorova et al, 2007 ; 4.6 g L −1 ). Recombinant C. glutamicum cells grown under elevated osmotic pressure released L-pipecolic acid (about 2.8 g L −1 ) after an osmotic down-shock ( Perez-Garcia et al, 2019 ), in principle also enabling bacterial milking with repeated cycles. However, the former two processes in contrast to the one that is described here required increased concentrations of sodium chloride of up to 30 g L −1 , which is well known to act corrosively on steel reactors and thus might hamper industrial feasibility ( Speidel, 1981 ; Chisti, 1992 ).…”

“…During the past decade, however, the range of products has been expanded drastically and comprises nowadays not only other proteinogenic amino acids, such as L-valine ( Blombach et al, 2007a ; Schwentner et al, 2018 ), L-arginine ( Park et al, 2014 ), L-tryptophan ( Zhang et al, 2015 ), or L-histidine ( Schwentner et al, 2019 ), but also organic acids ( Wieschalka et al, 2013 ), alcohols ( Blombach et al, 2011 ), carotenoids ( Henke and Wendisch, 2019 ), and proteins ( Bakkes et al, 2020 ; Hemmerich et al, 2020 ). Moreover, C. glutamicum has been engineered to overproduce native compatible solutes such as trehalose ( Carpinelli et al, 2006 ) and L-proline ( Jensen and Wendisch, 2013 ), as well as the nonnative α-D-glucosylglycerol ( Roenneke et al, 2018 ), L-pipecolic acid ( Pérez-García et al, 2019 ), and ectoine/hydroxyectoine ( Gießelmann et al, 2019 ), principally demonstrating the capability of C. glutamicum to serve as a host for production of compatible solutes. Very recently, C. glutamicum has been also engineered to produce MG from glucose plus glycerate and from mannose plus glycerate in an artificially designed starch-mannose-fermentation biotransformation process, respectively ( Tian et al, 2020 ).…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…Upon abolishment of the export of the precursor l-lysine and the improvement of expression of the pathway genes, l-pipecolic acid was produced to a titre of 14.4 g.l −1 [128]. The cell-protective properties of l-pipecolic acid as an osmo-protectant could also be demonstrated for the recombinant C. glutamicum strain [126].…”

“…l-Pipecolic acid (piperidine 2-carboxylic acid, PA) serves as chiral building block for therapeutic agents [125], and recently its value as a cell-protecting compatible solute has been revealed [126]. Because pipecolic acid is derived from l-lysine, l-lysine overproducers were engineered for l-pipecolic acid production by a synthetic pathway involving oxidative deamination, dehydration, and reduction by l-lysine 6-dehydrogenase (deaminating) from Silicibacter pomeroyi and endogenous pyrroline 5-carboxylate reductase [127] (Table 4).…”