Production of Tagatose by Whole-cell Bioconversion from Fructose Using Corynebacterium glutamicum

다국어 초록 (Multilingual Abstract)

Tagatose is a rarely occurring sugar found in food and sweet fruits. It is a potential sweetener with low calories but a sweetness similar to that of sucrose. In this study, we developed a whole-cell biocatalyst using Corynebacterium glutamicum for direct bioconversion of fructose to tagatose. First, we constructed a biological conversion pathway for the conversion of fructose to tagatose by expressing tagatose 4-epimerase (TN) from Thermotoga neapolitana in C. glutamicum. Next, to increase the expression level of the enzyme, we engineered copy number of plasmid. Plasmid library was constructed by randomizing the cgrI antisense RNA region in the plasmid, and the plasmid with high-copy number was isolated using fluorescence-activated single cell sorting (FACS)-based high-throughput screening. The isolated plasmid, pHCP7, had 2-fold higher copy numbers than the original plasmid. A higher expression level and conversion yield could be achieved using pHCP7. Finally, we examined a novel tagatose 4-epimerase TN(KNF4E) isolated from Kosmotoga olearia. The gene expression level was further increased (33.9% of total fraction) through codon optimization and expression in pHCP7, and a conversion yield as high as 21.7% was achieved.

참고문헌 (Reference)

1 Jayamuthunagai, J., "d-Tagatose production by permeabilized and immobilized Lactobacillus plantarum using whey permeate" 235 : 250-255, 2017

2 Lin, B., "Whole-cell biocatalysts by design" 16 : 106-, 2017

3 Zhang, G., "Two-stage biosynthesis of D-tagatose from milk whey powder by an engineered Escherichia coli strain expressing L-arabinose isomerase from Lactobacillus plantarum" 305 : 123010-, 2020

4 Gruber, A. R., "The Vienna RNA websuite" 36 (36): W70-W74, 2008

5 Oh, D. K., "Tagatose: properties, applications, and biotechnological processes" 76 : 1-8, 2007

6 Espinosa, I., "Tagatose: from a sweetener to a new diabetic medication?" 19 : 285-294, 2010

7 Gilbert V. Levin, "Tagatose, the New GRAS Sweetener and Health Product" 한국식품영양과학회 5 (5): 23-36, 2002

8 Lu, Y., "Tagatose, a new antidiabetic and obesity control drug" 10 : 109-134, 2008

9 Roy, S., "Tagatose as a potential nutraceutical:production, properties, biological roles, and applications" 83 : 2699-2709, 2018

10 Zhao, H., "Regeneration of cofactors for use in biocatalysis" 14 : 583-589, 2003

11 BARITUGOKEIANNE ; 손진아 ; 손유정 ; 김희택 ; 주정찬 ; 최종일 ; 박시재, "Recent progress in metabolic engineering of Corynebacterium glutamicum for the production of C4, C5, and C6 chemicals" 한국화학공학회 38 (38): 1291-1307, 2021

12 Wachtmeister, J., "Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale" 42 : 169-177, 2016

13 Ceroni, F., "Quantifying cellular capacity identifies gene expression designs with reduced burden" 12 : 415-418, 2015

14 Son, J., "Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum" 20 : 145-, 2021

15 Son, J., "Production of cinnamaldehyde through whole-cell bioconversion from trans-cinnamic acid using engineered Corynebacterium glutamicum" 70 : 2656-2663, 2022

16 Yang, S. J., "Production method for tagatose"

17 Beadle, J. R., "Process for manufacturing tagatose"

18 Bentley, W. E., "Plasmid-encoded protein: the principal factor in the "metabolic burden" associated with recombinant bacteria" 35 : 668-681, 1990

19 He, L., "PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids" 9 : 1131-1142, 1993

20 Lee, Y. M., "Novel fructose-4-epimerase and tagatose production method using same"

21 Henao-Velásquez, A. F., "Lactose and milk urea nitrogen: fluctuations during lactation in Holstein cows" 43 : 479-484, 2014

22 Yim, S. S., "Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum" 110 : 2959-2969, 2013

23 Yong Jun Jeong ; Jae Woong Choi ; Min Soo Cho ; Ki Jun Jeong, "Isolation of Novel Exo-type β-Agarase from Gilvimarinus chinensis and High-level Secretory Production in Corynebacterium glutamicum" 한국생물공학회 24 (24): 250-257, 2019

24 Skulj, M., "Improved determination of plasmid copy number using quantitative real-time PCR for monitoring fermentation processes" 7 : 6-, 2008

25 Lee, S. H., "Highyield production of pure tagatose from fructose by a three-step enzymatic cascade reaction" 39 : 1141-1148, 2017

26 Qi, X., "Fructose, galactose and glucose - in health and disease" 33 : 18-28, 2019

27 Kerpedjiev, P., "Forna (force-directed RNA): simple and effective online RNA secondary structure diagrams" 31 : 3377-3379, 2015

28 Shin, K. -C., "Development of tagaturonate 3-epimerase into tagatose 4-epimerase with a biocatalytic route from fructose to tagatose" 10 : 12212-12222, 2020

29 Yim, S. S., "Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum" 113 : 163-172, 2016

30 Choi, J. W., "Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum" 102 : 873-883, 2018

31 Grigoriou, S., "Development of a Corynebacterium glutamicum bio-factory for self-sufficient transaminase reactions" 22 : 4128-4132, 2020

32 Venkova-Canova, T., "Control of rep gene expression in plasmid pGA1 from Corynebacterium glutamicum" 185 : 2402-2409, 2003

33 Kallscheuer, N., "Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones" 38 : 47-55, 2016

34 Yang, S. J., "Composition for producing tagatose and method of producing tagatose using the same"

35 Martinez, A., "Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli" 15 : 891-897, 1999

36 Okibe, N., "Antisense-RNA-mediated plasmid copy number control in pCG1-family plasmids, pCGR2 and pCG1, in Corynebacterium glutamicum" 156 : 3609-3623, 2010

37 Wolf, S., "Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being" 65 : 197-212, 2021

38 정성희 ; 권문혁 ; 김선원, "Advanced Whole-cell Conversion for D-allulose Production Using an Engineered Corynebacterium glutamicum" 한국생물공학회 27 (27): 276-285, 2022

39 Lee, C., "Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli" 123 : 273-280, 2006