Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector® micro-fermentation system. Results: Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl -D-thiogalactopyranoside) and IPTG (isopropyl -D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions: We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.(VLID)101641

Identification of Oxygen-Responsive Transcripts in the Silage Inoculant Lactobacillus buchneri CD034 by RNA Sequencing

Eikmeyer FG, Heinl S, Marx H, Pühler A, Grabherr R, Schlüter A. Identification of Oxygen-Responsive Transcripts in the Silage Inoculant Lactobacillus buchneri CD034 by RNA Sequencing. PLoS ONE. 2015;10(7): e0134149.The Lactobacillus buchneri CD034 strain, known to improve the ensiling process of green fodder and the quality of the silage itself was transcriptionally analyzed by sequencing of transcriptomes isolated under anaerobic vs. aerobic conditions. L. buchneri CD034 was first cultivated under anaerobic conditions and then shifted to aerobic conditions by aeration with 21% oxygen. Cultivations already showed that oxygen was consumed by L. buchneri CD034 after aeration of the culture while growth of L. buchneri CD034 was still observed. RNA sequencing data revealed that irrespective of the oxygen status of the culture, the most abundantly transcribed genes are required for basic cell functions such as protein biosynthesis, energy metabolism and lactic acid fermentation. Under aerobic conditions, 283 genes were found to be transcriptionally up-regulated while 198 genes were found to be down-regulated (p-value < 0.01). Up-regulated genes i. a. play a role in oxygen consumption via oxidation of pyruvate or lactate (pox, lctO). Additionally, genes encoding proteins required for decomposition of reactive oxygen species (ROS) such as glutathione reductase or NADH peroxidase were also found to be up-regulated. Genes related to pH homeostasis and redox potential balance were found to be down-regulated under aerobic conditions. Overall, genes required for lactic acid fermentation were hardly affected by the growth conditions applied. Genes identified to be differentially transcribed depending on the aeration status of the culture are suggested to specify the favorable performance of the strain in silage formation

Enhancement of solubility in <it>Escherichia coli </it>and purification of an aminotransferase from <it>Sphingopyxis </it>sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Abstract Background Fumonisin B1 is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B1, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B1. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity. Results When expressed from a T7 promoter at 30°C, the aminotransferase accumulated invariably in inclusion bodies in DE3 lysogens of the E. coli strains BL21, HMS174, Rosetta 2, Origami 2, or Rosetta-gami. Omission of the isopropyl-beta-D-thiogalactopyranoside (IPTG) used for induction caused a reduction of expression level, but no enhancement of solubility. Likewise, protein production but not solubility correlated with the IPTG concentration in E. coli Tuner(DE3). Addition of the solubility enhancers betaine and sorbitol or the co-enzyme pyridoxal phosphate showed no effect. Maltose-binding protein, used as an N-terminal fusion partner, promoted solubility at 30°C or less, but not at 37°C. Low enzyme activity and subsequent aggregation in the course of purification and cleavage indicated that the soluble fusion protein contained incorrectly folded aminotransferase. Expression in E. coli ArcticExpress(DE3), which co-expresses two cold-adapted chaperonins, at 11°C finally resulted in production of appreciable amounts of active enzyme. Since His tag-mediated affinity purification from this strain was hindered by co-elution of chaperonin, two steps of chromatography with optimized imidazole concentration in the binding buffer were performed to obtain 1.45 mg of apparently homogeneous aminotransferase per liter of expression culture. Conclusions We found that only reduction of temperature, but not reduction of expression level or fusion to maltose-binding protein helped to produce correctly folded, active aminotransferase FumI in E. coli. Our results may provide a starting point for soluble expression of related aminotransferases or other aggregation-prone proteins in E. coli.</p

The 25 most abundantly transcribed genes of <i>L</i>. <i>buchneri</i> CD034 cultivated under aerobic <i>vs</i>. anaerobic conditions.

<p>¹ Reads per kilobase of transcript per million mapped reads.</p><p>The 25 most abundantly transcribed genes of <i>L</i>. <i>buchneri</i> CD034 cultivated under aerobic <i>vs</i>. anaerobic conditions.</p

Fermentation of <i>L</i>. <i>buchneri</i> CD034 grown under aerobic and anaerobic conditions.

<p>The fermentation was separated in three phases: phase 1 represents aerobic, phase 2 anaerobic and phase 3 again aerobic conditions with respect to the oxygen concentration of the gas inlet (O_2,in). (A) Gas phase composition during fermentation as described by oxygen and carbon dioxide concentrations in the gas inlet (O_2,in, CO_2,in) and in the gas outlet (O_2,out, CO_2,out). (B) pH, dissolved oxygen concentration in the medium (dO₂) and optical density (OD). (C) Concentration of carbohydrates glucose, xylose and arabinose and (D) concentration of organic acids lactate and acetate. Fermentations were performed in duplicates. Arrows indicate sampling time points for RNA-Seq. Parameters shown originate from fermenter 1. For fermenter 2 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134149#pone.0134149.s001" target="_blank">S1 Fig</a>.</p

Volcano plot representing transcriptional levels for <i>L</i>. <i>buchneri</i> CD034 genes under aerobic <i>vs</i>. anaerobic conditions.

<p>For each protein encoding gene the −log₂(p-value) is plotted against its log₂(fold change). Genes up-regulated (p < 0.01) under aerobic conditions are colored blue while down-regulated genes are colored red. Genes found to be located between bacteriophagous attachment sites are colored in yellow (<i>ΦLbu-1</i>) and orange (<i>ΦLbu-2</i>) irrespective of their relative transcriptional levels. For genes with gene names that were found to be highly up- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134149#pone.0134149.t003" target="_blank">Table 3</a>) or down- (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134149#pone.0134149.t004" target="_blank">Table 4</a>) regulated the gene name is given. Abbreviations represent gene names: <i>pox2</i>, <i>-3</i>: pyruvate oxidase; <i>lctO</i>: lactate oxidase; <i>hsp1</i>, <i>-2</i>: molecular chaperones; <i>oppA3</i>: oligopeptide transport system, substrate binding protein; <i>arcC3</i>: carbamate kinase; <i>aguA1</i>: agmatine deiminase; <i>pctA</i>: putrescine carbamoyltransferase; <i>gntP</i>: H+/gluconate symporter; <i>gntR</i>: gluconate operon transcriptional regulator; <i>gntK1</i>: gluconokinase; <i>pyrG</i>: CTP synthetase; <i>rihB</i>: ribosylpyrimidine nucleosidase; <i>pgd</i>: 6-phosphogluconate dehydrogenase; <i>adhA</i>: alcohol dehydrogenase; <i>hisD</i>: histidinol dehydrogenase; <i>tagG</i>: teichoic acid permease; <i>tagB</i>, teichoic acid biosynthesis protein; <i>argH</i>: argininosuccinate lyase; <i>nupC</i>: pyrimidine specific nucleoside symporter. For genes lacking gene names the index numbers of their respective locus tags (LBUCD034_XXXX) are given.</p

Fermentation of <i>L</i>. <i>buchneri</i> CD034 grown under aerobic and anaerobic conditions.

<p>The fermentation was separated in three phases: phase 1 represents aerobic, phase 2 anaerobic and phase 3 again aerobic conditions with respect to the oxygen concentration of the gas inlet (O_2,in). (A) Gas phase composition during fermentation as described by oxygen and carbon dioxide concentrations in the gas inlet (O_2,in, CO_2,in) and in the gas outlet (O_2,out, CO_2,out). (B) pH, dissolved oxygen concentration in the medium (dO₂) and optical density (OD). (C) Concentration of carbohydrates glucose, xylose and arabinose and (D) concentration of organic acids lactate and acetate. Fermentations were performed in duplicates. Arrows indicate sampling time points for RNA-Seq. Parameters shown originate from fermenter 1. For fermenter 2 see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134149#pone.0134149.s001" target="_blank">S1 Fig</a>.</p