Engineering probiotics to produce short chain fatty acids for the management of metabolic syndrome

th January 2019 Chapter 2: ENGINEERING PROBIOTICS TO PRODUCE BUTYRATE 2. Introduction 2. Butyrate as a therapeutic metabolite against metabolic syndrome As discussed in chapter 1, one of the most critical roles played by gut microbes is to regulate a wide of array of host metabolic processes, which plays a significant role in the development of metabolic syndrome. The bacterial fermentation products, SCFA, are vital mediators, which are involved in the microbial modulation of host glucose and energy homeostasis. Kimura, 2014 #474) Butyrate, among other SCFA, attributes the most significant function in glucose balance by promoting GLP-1 secretion(Yadav, 2013 #42), intestinal gluconeogenesis(De Vadder, 2014 #391), and the pancreatic ß-cell health(Khan, 2014 #430). have demonstrated that the supplementation of butyrate in mice on a high-fat diet completely abolished weight gain, reduced food intake, and improved glucose tolerance. The authors have claimed that butyrate protects mice from diet-induced obesity by stimulating incretins, GLP-1 and GIP.

These incretins are secreted from the GI tract and upregulate insulin secretion from pancreas after meals to lower plasma glucose level. More importantly, butyrate has shown to be the most potent metabolite to stimulate the anorexigenic hormone GLP-1 compared to other SCFA. Also, butyrate promotes the pancreatic ß-cell health that further enhances its glucose-lowering effects. The butyrate supplement in mice has shown to upregulate the expression of thermogenesis related genes, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and UCP-1, in brown adipose tissue. PGC-1α regulates energy metabolism by interacting with crucial various transcription factors to control mitochondrial biogenesis and respiration. In brown fat, PGC-1α promotes the expression of UCP-1 that generates heat through thermogenic respiration. Gao et al. also have shown that the butyrate supplement in mice upregulates the expression of PGC-1α and the ratio of the type 1 fibers in skeletal muscles.

One direct method to increase luminal butyrate levels is to administer butyrate tablets orally. These dietary supplements contain an esterified form of butyrate that can be hydrolyzed upon pancreatic lipases. However, the butyrate tablet may not be degraded and release butyrate at its target location since there the luminal transit time, and pH varies among individuals. Thus, the limitations of the strategies above suggest that an alternative approach that can deliver the therapeutically appropriate amount of butyrate at target locations safely and effectively would be desirable. One alternative method to overcome these limitations could be the admiration of natural butyrate producers, which are known to colonize the human GI tract stably. Considering all the above desirable characteristics, a probiotic strain could be a promising probiotic candidate to secrete butyrate as a therapeutic molecule.

Nissle holds many desirable traits which would be discussed below. First, the biosafety aspect of Nissle has been extensively studied for its human consumption. Mutaflor® is a licensed microbial drug with Nissle as an active component that has been traditionally used to treat various human diseases since 1917. Thus, numerous investigations have been performed to underline the clinical safety of the Nissle treatment in human. coli for industrial applications, making a probiotic strain E. coli Nissle as an attractive probiotic candidate to express butyrate as therapeutic molecule against metabolic syndrome. Construction of a butyrate-producing pathway in Nissle Numerous studies have genetically engineered E. coli to produce butyrate for industrial applications using a synthetic biology approach. This strategy combines different butyrogenic genes from various natural butyrate producers to create optimum and robust butyrogenic pathways in E.

These genes include hbd (encoding 3-hydroxybutyryl-CoA dehydrogenase) and crt (encoding crotonase) arising from C. acetobutylicum, and ter (encoding trans-enoyl-CoA reductase) originating from T. denticola. The E. coli native genes atoB (encoding acetyl-CoA acetyltransferase) and testB (acyl-CoA thioesterase II) were introduced to complete the butyrate-producing pathway. Sonnenborn, 2009 #151) These probiotic strain colonies the distal part of the GI tract[116], especially in the colon, where butyrate can be produced to elicit enteroendocrine L-cells to secrete GLP-1[117]. Also, numerous well-established butyrogenic pathways and genetic engineering tools available in E. coli make Nissle a suitable chassis to be genetically modified as a robust butyrate producer for therapeutic applications. To construct a butyrogenic pathway, the synthetic butyrogenic pathway previously constructed in E. coli K-12 (Lim, 2013 #408) was adapted in this study to engineer a probiotic strain E. List of strains used Table 2.

List of plasmids used *rbs5 was used for all gene expressions unless stated otherwise *AmpR – ampicillin resistance ; CmRR- chloramphenicol ; chloramphenicol resistance ; KanR – kanamycin resistance. List of plasmids Table 2. List of plasmids used *rbs5 was used for all gene expressions unless stated otherwise *AmpR – ampicillin resistance ; CmRR- chloramphenicol ; chloramphenicol resistance ; KanR – kanamycin resistance. Methodology Bacterial culture All bacterial cells were maintained in Luria broth (LB) medium (Becton Dickinson) supplemented with appropriate antibiotics and/or supplements at 37 °C with shaking at 225rpm. Bacterial cloning Chemical and electric competent cells were prepared according to the lab protocols and used for bacterial cloning. ng of plasmids were used for transformation, and the transformed bacterial cultures were recovered in LB media at 37 °C with shaking at 225rpm for 1 hour. The recovered bacterial cultures were plated on LB agar plates with appropriate antibiotics and were incubated at 37 °C overnight.

Bacterial gene deletion The Red/ET Recombination system was used to delete bacterial genes as described previously. Bacterial gene deletion fragments consist of the kanamycin resistance gene flanked by the FRT sequences and the homologous sequences upstream and downstream of the targeted genes. using spectrophotometer Biophotometer Plus (Eppendorf). µl of bacterial pre-culture was added to each well of 96-well clear microplate (Corning). Bacterial cell densities were determined using Synergy H1 Multi-Mode plate reader (BioTek), and all growth assays were performed in triplicates. GC-MS quantification of butyrate Nissle cells were inoculated in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with D-alanine and chloramphenicol. After 24 hours of incubation at 37 °C with shaking at 225 rpm, Nissle cells were harvested by centrifuging at 10. was adapted to engineer Nissle to produce butyrate. This synthetic butyrogenic pathway consists of five butyrogenic genes originating from different natural butyrate-producers including Clostridium acetobutylicum and Treponema denticola.

Figure 2. A) (B) Figure 2. A) Overview of the butyrogenic pathway used in this study. B) was inserted downstream of the promoter J23100 in the vector pBbE8a-j23100-r5 digested with BamHI and Xhol. The positive construct was screened by colony PCR (Figure 2. C) and was sequence verified. This showed the successful construction of the vector harboring the Nissle atoB gene (pBbE8a-j23100-r5-atoB). The protein expression test for the positive colony was performed to validate that the expression of the Nissle atoB gene the promoter J23100. coli T10 harboring the plasmid pBbE8a-j23100-r5-atoB, 2: Whole cell extract of E. coli T10, M: Prestained protein molecular weight marker. Due to numerous restriction sites present in the plasmid pBASP, the Gibson assembly method was used to construct the butyrogenic plasmid pC4.

Figure 2. A) (revision)In brief, the previously constructed vector pBbE8a-j23100-r5-atoB was the used to clone the atoB gene, with its promoter j23100 attached, a fragment containing flanking regions of homology at its ends to the linearized plasmid pBASP. k bp) and the linearized vector pBASP (7k bp) for Gibson assembly. The PCR product of the linearized pBASP vector; 2: The PCR product of atoB; M: DNA ladder. C) Gel electrophoresis of colony PCR showing the successful construction of the butyrogenic plasmid pC4 through Gibson assembly. Two internal primers were used to verify the presence of atoB 1: pBASP without atoB insertion; 2: pBASP with atoB yielding the butyrogenic plasmid pC4; M: DNA ladder. D) Plasmid map of pC4 2. Sonnenborn, 2009 #151) To determine the levels of the butyrate produced, the areas under the peaks at the retention time of 5.

min of the GC-MS chromatograms were calculated. Based on this, Nissle produced less than 5 mg/L of butyrate. On the other hand, the engineered butyrogenic strain (Nissle pC4) produced about 60 mg/L of butyrate, which is approximately 0. mM. This suggested that the engineered butyrate-producing Nissle may not produce sufficient level of butyrate to trigger GLP-1 secretion. Thus, it was necessary to improve the butyrate production in Nissle to ensure that therapeutically sufficient amount of butyrate is to confer its therapeutic functions. A) (B) (C) Figure 2. A) GC-MS chromatogram of the engineered butyrogenic strain Nissle pC4 and wildtype control Nissle. B) Bar graph was representing butyrate production from the wildtype control Nissle and the engineered butyrogenic Nissle pC4. H. Lim et al. to compete with butyrate synthesis for a carbon source. The disruption of these pathways was suggested to redirect the metabolic flux towards butyrate synthesis and to enhance butyrate production.

Lim, 2013 #30) For the disruption of metabolically competing pathways in Nissle, an auxotrophic version of Nissle was used. These genes are involved in the acetate, ethanol, succinate, and lactate synthesizing pathways, which compete with butyrate synthesis for the carbon source. Nissle∆2 with these five genes deleted were named Nissle∆7. The Red recombination system was performed to delete these five genes, as described in a previous study. Datsenko & Wanner, 2000b) (Figure 2. A) In this system, the gene fragment containing the kanamycin resistance marker flanked by two FRT sites were amplified using primers with homologous sequences of the 5’ and 3’ ends of the target gene. Together, the five genes were deleted in the auxotrophic Nissle∆2, resulting in the strain Nissle∆7. The auxotrophic Nissle∆2 was used to ensure that the engineered probiotic strain is optimal for future in-vivo studies.

The five genes were deleted to redirect the metabolic flux towards butyrate synthesis. Also, the growth assay performed showed that the gene deletion did not cause growth defects in Nissle. The resultant strain Nissle∆7 was next tested for butyrate production. coli Nissle with seven metabolic pathways deleted. Butyrate production was determined using the previously constructed strain Nissle∆7 harboring the butyrogenic plasmid pC4 (Nissle∆7 pC4). The GC-MS chromatogram showed that Nissle∆7 pC4 were eluted with higher ion abundances at the retention time of 5. compared to that of Nissle pC4. Figure 2. It was found that Nissle∆2 pC4 produced approximately 60 mg/L of butyrate, similar to that of Nissle pC4. data not shown) This indicated that the deletion two genes to create the auxotrophic Nissle strain (Nissle∆2) did not affect the butyrate production. Overall, these results indicated that higher butyrate production (an 8-fold increase) was achieved after disrupting the metabolically competing pathways to redirect the metabolic flux towards butyrate synthesis.

Also, a growth assay of Nissle∆7 pC4 was performed to investigate if the expression of butyrogenic pathway causes growth defects in Nissle∆7. As previously mentioned, no growth defect was observed under the conditions tested for Nissle∆7. More importantly, the resultant strain (Nissle∆7 pC4) produced sufficient levels of butyrate that was shown to exhibit therapeutic functions based on a previous finding. Yadav, 2013 #42) Of note, Nissle∆7 pC4 was observed to have a growth defect. It may be possible that the gene deletion and the expression of the five exogenous butyrogenic enzymes caused metabolic burdens that slow down the cell growth. A) V (B) (C) Figure 2. A) GC-MS chromatogram of the engineered butyrogenic strains (Nissle∆7 pC4 and Nissle pC4) and controls (Nissle and Nissle∆7). mM) of butyrate. However, the Nissle ∆7 pC4 strain exhibited a growth defect.

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