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Mattheos KOFFAS 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
Sulfated glycosaminoglycans (sGAGs) are linear polysaccharides that have important applications in the medical and food industries. Engineering bacteria for microbial production of sGAGs will facilitate the one-step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. Advancing towards this goal, we engineered E. coli to accumulate 3’-phosphoadenosine-5’-phosphosulfate (PAPS), the universal sulfate donor, using traditional metabolic engineering approaches. PAPS is one of the least explored components required for biosynthesis of SGAGs. The resulting engineered E. coli strain showed a ~1000-fold increase in intracellular PAPS concentrations. In a similar fashion, we were able to identify using computational tools a number of chondroitin sulfotransferases with enhanced activities when provided unsulfated chondroitin and PAPS. These mutants were identified using PROS, a computational platform that allows the identification of point mutations that improve enzyme stability and solubility. By combining the PAPS produced in E.coli with the functionally expressed sulfotransferases, we were able to show for the first time the production of animal-free bioengineered CS in an E.coli recombinant strain. A similar approach was followed for the production of deuterated heparin, another sulfated GAG, also for the first time.
Pandey, R.P.,Parajuli, P.,Koffas, M.A.G.,Sohng, J.K. Pergamon Press ; Elsevier Science Ltd 2016 BIOTECHNOLOGY ADVANCES Vol.34 No.5
<P>In this review, we address recent advances made in pathway engineering, directed evolution, and systems/synthetic biology approaches employed in the production and modification of flavonoids from microbial cells. The review is divided into two major parts. In the first, various metabolic engineering and system/synthetic biology approaches used for production of flavonoids and derivatives are discussed broadly. All the manipulations/engineering accomplished on the microorganisms since 2000 are described in detail along with the biosynthetic pathway enzymes, their sources, structures of the compounds, and yield of each product. In the second part of the review, post-modifications of flavonoids by four major reactions, namely glycosylations, methylations, hydroxylations and prenylations using recombinant strains are described. (C) 2016 Elsevier Inc. All rights reserved.</P>
Roypim Thananusak,Kobkul Laoteng,Nachon Raethong,Mattheos Koffas,Wanwipa Vongsangnak 한국생물공학회 2023 Biotechnology and Bioprocess Engineering Vol.28 No.3
Dissecting the cellular metabolism of Cordyceps militaris is important for the efficient production of bioactive compounds of a target with medicinal and industrial applications. However, the metabolic functions during developmental stages in this fungus at a system level are still unexplored. In this study, we aimed to reveal the metabolic functions and regulation of C. militaris TBRC6039 relevant to its developmental stages, including mycelial growth (MY) and fruiting body (FB) stages through integrative transcriptome analysis. The transcriptome analysis showed that 9,256 genes of C. militaris were expressed in both stages. Of them, 1,877 genes, residing primarily in the cell division cycle and amino acid, carbohydrate, and lipid metabolisms, exhibited significant differences in transcript levels between the MY and FB stages. Through integration with genome-scale networks analysis, the unique reporter metabolites (e.g., α-D-glucose, β-D-glucose, D-galactose, triacylglycerol, and diacylglycerol) and key regulators (e.g., AtfA, Atf2, and Yap1 transcription factors) were identified in C. militaris when grown at FB stage, linking to up-regulation of the metabolic genes involved in galactose and polysaccharide metabolisms, as well as glycerolipid and glycerophospholipid biosynthesis. Moreover, the high cordycepin content is related to the upregulated genes in lipid metabolism during the FB stage. Our findings suggest that the transcriptional regulation of these metabolic pathways played a crucial role in specific developmental stages of C. militaris. This study serves for cultivation process improvement for overproduction of valuable metabolites in C. militaris through an emerging systems and synthetic biology approach.
Multi-level Rebalancing of the Naringenin Biosynthetic Pathway Using Synthetic RNA Biosensors
Hyun Gyu HWANG,Myung Hyun NOH,Mattheos A.G. KOFFAS,Sungho JANG,Gyoo Yeol JUNG 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.10
Recombinant microbes have emerged as promising alternatives to natural sources of naringenin—a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5′-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 ㎎/L naringenin, which is the highest concentration from glycerol and p-coumaric acid reported to date. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.