Engineering probiotics to produce short chain fatty acids for the management of metabolic syndrome
_________________ 18th January 2019 Chapter 2: ENGINEERING PROBIOTICS TO PRODUCE BUTYRATE 2. 1 Introduction 2. 1 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. 4 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. [119-123] 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, especially in the colon, where butyrate can be produced to elicit enteroendocrine L-cells to secrete GLP-1. 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. coli Top10 - cloning Lab collection E. coli Nissle - probiotic chassis Mutaflor E. coli Nissle∆2 Nissle∆dadB∆alr probiotic chassis Lab collection Table 2. 1 List of strains used Table 2. 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. Agarose gel electrophoresis was performed using 1% agarose gel (1st Base) and SYBR safe DNA gel stain (ThermoFisher Scientific). DNA ligation and plasmid DNA extraction were performed using T4 DNA ligase (New England Biolabs) and QIAprep Spin Miniprep Kit (Qiagen), respectively. Biological construct sequence verifications were performed using Sanger sequencing (1st Base).
Bacterial cloning Chemical and electric competent cells were prepared according to the lab protocols and used for bacterial cloning. 10ng 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. Bacterial growth assay Bacterial pre-cultures were grown in LB media with appropriate antibiotics and supplements. The absorbance of bacterial cells at 600nm (OD600) was measured to determine cell density. Initial cell densities of bacterial pre-cultures at lag phase were adjusted at OD600 0. 2 using spectrophotometer Biophotometer Plus (Eppendorf). 200 µl of bacterial pre-culture was added to each well of 96-well clear microplate (Corning). 3 Results and Discussion 2. 1 Construction of the butyrogenic plasmid, pC4. The synthetic butyrate-producing pathway constructed by Lim et al.
2013 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. The restriction/ligation cloning strategy was used to clone the atoB gene into the vector pBbE8a-j23100-r5. Forward and reverse primers with flanking restriction sites, BglII and XhoI respectively were designed. The sub-cloned atoB gene (Figure 2. 4 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. 1: pBbE8a-j23100-r5 with atoB, 2: pBbE8a-j23100-r5 without atoB M: DNA ladder. (D) Coomassie Blue-stained, 10% SDS-PAGE, showing the expressed atoB protein (40 kDa). 1: Whole cell extract of E.
coli T10 harboring the plasmid pBbE8a-j23100-r5-atoB, 2: Whole cell extract of E. coli T10, M: Prestained protein molecular weight marker. 5 Construction of the butyrogenic plasmid pC4. (A) Overview of the Gibson assembly method used to construct the butyrogenic plasmid pC4. (B) Gel electrophoresis showing the PCR amplification of atoB (1. 5k bp) and the linearized vector pBASP (7k bp) for Gibson assembly. 1: The PCR product of the linearized pBASP vector; 2: The PCR product of atoB; M: DNA ladder. 24 min, the non-butyrogenic strain (Nissle) sample was eluted with comparable ion abundances to that of the baseline, medium only. (Figure 2. 6 A) Thus, this indicated that wildtype Nissle strain is not a native producer of butyrate, as previously described. (Sonnenborn, 2009 #151) To determine the levels of the butyrate produced, the areas under the peaks at the retention time of 5.
24 min of the GC-MS chromatograms were calculated. Based on a previous finding, at least 1mM of butyrate was required to promote GLP-1 secretion in a human enteroendocrine cell line. (Yadav, 2013 #41) However, the engineered butyrogenic Nissle produced approximately 0. 8 mM of butyrate. 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. (Lim, 2013 #408) Four competing pathways were disrupted to redirect metabolic flux towards butyrate synthesis. (Figure 2. 7 A) These were acetate, ethanol, succinate, and lactate synthesizing pathways, which were suggested by J. H. Lim et al. 7 (A) Schematic representation of native Nissle metabolic genes deleted in this study.
(B) List of innate metabolic genes deleted and, their encoding enzymes and functions. To disrupt four metabolically competing pathways in the auxotrophic Nissle strain (Nissle∆2), five genes were targeted. 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 growth assay showed there is no significant difference in the growth curves between Nissle and Nissle∆7. (Figure 2. 8 C) This indicated that the gene deletion (the two alanine racemase genes and the five genes involved in the metabolic competing pathways) did not cause growth defect. 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.
(C) Growth assay of deletion strain Nissle ∆7 (red), Nissle (black) included as a control. Nissle represents E. coli Nissle harboring empty vector; Nissle ∆7 represents E. 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). 9 B) This indicated that Nissle∆7 pC4 produced approximately eight times higher levels of butyrate compared to that of Nissle pC4. More importantly, this result indicated that Nissle∆7 pC4 produce sufficient amount of butyrate that was shown to confer therapeutic effects based on a previous finding. (Yadav, 2013 #42) To rule out the possibility that the deletion two genes (alr and dadX) to create the auxotrophic Nissle strain (Nissle∆2) affects butyrate production, we have determined the butyrate production of the Nissle∆2 strain harboring the butyrogenic plasmid pC4 (Nissle∆2 pC4).
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.  To summarise, the five genes involved in the metabolically competing pathways were deleted. The gene deletion aimed to redirect the metabolic flux towards butyrate synthesis and thus increase butyrate production. The redirection of the metabolic flux results in an 8-fold increase in the production. 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.
8 mM) of butyrate. To improve the butyrate production to achieve the therapeutically relevant levels of butyrate to induce GLP-1 based on previous findings, metabolically competing pathways were deleted, obtaining the strain Nissle ∆7 pC4. The deletion resulted in the upregulation of the butyrate level, where Nissle ∆7 pC4 produced approximately 500 mg/L (5. 5 mM) of butyrate. However, the Nissle ∆7 pC4 strain exhibited a growth defect. REFERENCES 1. Keating, L. , SCFAs and Signaling Pathways. Kobyliak, N. , O. , Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science, 2010. 328(5975): p. Alberti, K. G. , Health care utilization and costs by metabolic syndrome risk factors. Metab Syndr Relat Disord, 2009. 7(4): p. Grundy, S. M. and E. E. Blaak, Adipose tissue dysfunction and impaired metabolic health in human obesity: a matter of oxygen? Frontiers in endocrinology, 2015.
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