Euro Biotechnology Congress 2015 : Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli - Jingwen Zhou - Jiangnan University

Abstract

The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides, flavonoids and biofuels. Here, a Clustered Regularly Inter-Spaced Short Palindromic Repeats (CRISPR) interference system was constructed for fine-tuning the central metabolic pathways to efficiently channel carbon flux toward malonyl- CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these target genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene silencing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L which was 7.4-fold higher than the control strain (50.5 mg/L). The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for the production of other important products that require malonyl-CoA as a precursor such as flavonoids, polyketides and fatty acids. High‐strength polymers, for example, aramid strands, are significant materials in space innovation. To acquire these materials in remote areas, for example, Mars, organic creation is of intrigue. The fragrant polymer forerunner para ‐aminobenzoic corrosive (pABA ) can be gotten from the shikimate pathway through metabolic designing of Bacillus subtilis , a life form appropriate for space engineered science. Our building technique included fix of the faulty indole‐3‐glycerol phosphate synthase (trpC ), knockout of one chorismate mutase isozyme (aroH ) and overexpression of the aminodeoxychorismate synthase (pabAB ) and aminodeoxychorismate lyase (pabC ) from the microorganisms Corynebacterium callunae and Xenorhabdus bovienii separately. Further, a fusion‐protein chemical (pabABC ) was made for diverting of the carbon transition. Utilizing versatile advancement, freaks of the creation strain, ready to use xylose, were made, to investigate and think about pABA creation limit from various carbon sources. Instead of the effectiveness of the substrate or execution of the biochemical pathway, the item poisonousness, which was firmly subject to the pH , gave off an impression of being the general constraining component. The most noteworthy titre accomplished in shake flagons was 3.22 g l−1 with a carbon yield of 12.4% [C‐mol/C‐mol] from an amino sugar. This guarantees appropriateness of the framework for in situ asset use (ISRU ) in space biotechnology, where feedstocks that can be gotten from cyanobacterial cell lysate assume a job. Plastics and polymers are ubiquitous in our regular day to day existence as well as are possibly of much more noteworthy significance in space innovation. Biaxially situated polyethylene terephthalate (BoPET, exchange nameMylar®) is esteemed for its high elasticity, substance and dimensional dependability, hindrance properties and electrical protection; layers of metallized BoPET are, for instance, utilized in high‐altitude expands just as in spacesuits for warm protection and radiation obstruction. Aramids, similar to the texture and sheet material Kevlar®, highlight comparatively extraordinary properties, including high constancy and quality modulus, low flex exhaustion, just as superb compound security and warm soundness and furthermore radiation opposition. In this manner, they are perfect for a scope of forte applications, including ballistic insurance. That these materials are particularly appropriate for development of natural suits and residences in space innovation, shows their usage in inflatable shuttles like the ones of Bigelow Aerospace® (NASA, 2017). The feedstocks of fragrant polymers are normally petroleum product determined, which is neither feasible over the long haul on Earth, nor accessible in space or at goals, for example, Earth's moon or Mars. Metabolic Engineering may give the innovation to tackle this issue, by empowering creation of bioreplacement forerunners through in situ asset usage (ISRU). ISRU targets using manufactured science to recharge items on deep‐space investigation missions (Rothschild, 2016). Microbial metabolic pathways offer ascent to numerous intensifies that can possibly substitute as of now petroleum‐based synthetic concoctions with bio‐derived ones or supplant them with bio‐based choices. This incorporates a huge number of sweet-smelling and aromatic‐derived mixes (Averesch and Krömer, 2018). The shikimate pathway middle para ‐aminobenzoic corrosive (pABA) is one of these aromatics with adaptable pertinence – it is being utilized as cross‐linking operator for saps and colors, forerunner in the pharmaceutical business and as a remedial itself (for example as the medication POTABA®). pABA can likewise be changed over to terephthalic corrosive (Farlow and Krömer, 2016), as feedstock for creation of PET/Mylar®. It might likewise be conceivable to change over pABA to para ‐phenylenediamine (for example by means of Kochi‐ or Hunsdiecker response followed by nucleophilic replacement), which is (other than terephthalic corrosive) the second monomer of the aramid‐fibre Kevlar®. Further, pABA can likewise be polymerized with itself (Morgan, 1977), conceivably yielding a para ‐aramid with a sub-atomic structure undifferentiated from Kevlar®.The attainability of creating pABA microbiologically to be utilized as a mechanical forerunner was first indicated utilizing the yeast Saccharomyces cerevisiae (Krömer et al ., 2013), where a titre of 0.03 g l−1 (0.22 mM) was arrived at utilizing glucose as the sole carbon source. In a committed follow‐up study, the titre could be expanded to 0.22 g l−1 (1.57 mM) from glycerol/ethanol (Averesch et al ., 2016). Additionally, microscopic organisms have been used for the creation of pABA. In Escherichia coli, a centralization of 4.8 g l−1 (35 mM) was reached from glucose (Koma et al ., 2014), while the most noteworthy creation to date was cultivated with Corynebacterium glutamicum , arriving at 43.06 g l−1 (314 mM) from glucose (Kubota et al ., 2016). To use this innovation in space and eventually empower the union of aramid strands, it would be exceptionally attractive to create pABA in Bacillus subtilis , the creature generally fit to space engineered science. Bacillus subtilis structures endospores (Nicholson et al ., 2000; Horneck et al ., 2010), which are incredibly impervious to a few natural parameters, for example, dry spell, saltiness, pH and solvents and stay reasonable for a considerable length of time; insofar as shielded from UV radiation, they even bear the vacuum of room (Horneck, 1993). Biography Jingwen Zhou obtained his PhD degree in Fermentation Engineering in 2009. After that, he became Assistant Professor in 2009, Associate Professor in 2011 and Full Professor in 2014 in the School of Biotechnology, Jiangnan University. He finished his Postdoc training in Department of Chemistry and Chemical Biology in Harvard from 2012 to 2013. His current research works mainly focused on the metabolic engineering of microorganisms to produce organic acids and plant natural products, especially L-ascorbic acid and flavonoids. He published 52 peer reviewed papers in journals such as Metabolic Engineering, Applied and Environment Microbiology, and also several invited reviews on Current Opinion in Biotechnology and Biotechnology Advances. Several of the typical products he had been working on ares now produced by several manufactures on industrial scale. His achievements were awarded several times in China. He is now the Editorial Board Member of Scientific Reports (Nature Press) and Electronic Journal of Biotechnology (Elsevier Press).   zhoujw1982@jiangnan.edu.cn