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研究生：	陳盈淳 Chen, Ying-Chun
論文名稱：	以共表達CadA和CadB之重組大腸桿菌作為全細胞催化劑生產1,5-戊二胺 Production of cadaverine using recombinant Escherichia coli co-overexpressing CadA and CadB as the whole-cell biocatalyst
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2020
畢業學年度：	108
語文別：	英文
論文頁數：	96
中文關鍵詞：	1, 5-戊二胺、離氨酸脫羧酶(CadA) 、離氨酸-屍胺反轉運蛋(CadB) 、BCP比色測定法、磷酸吡哆醛(PLP) 、全細胞生物催化劑、通透性、可重複使用性
外文關鍵詞：	Cadaverine, lysine decarboxylase (CadA), lysine-cadaverine antiporter (CadB), BCP colorimetric assay, pyridoxal phosphate, whole-cell biocatalyst, permeability, reusability
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由於人們愈來愈關注日益嚴重的資源枯竭及環境惡化的問題，因此開始重視以綠色製程替代傳統的石油化學製程，生產日常生活用的化學品，以降低對環境的衝擊。譬如近年來對於生物基聚醯胺(也稱為生物基尼龍)進行了許多研究，試圖取代傳統的化學合成聚醯胺(尼龍6和66)，進而生產生物基尼龍，如尼龍54、56和510。而1, 5-戊二胺 (俗稱屍胺) 是生物基尼龍的單體，其可利用基因改造微生物進行生產，如在重組大腸桿菌中，可利用離氨酸脫羧酶(CadA)催化離氨酸合成1, 5-戊二胺，並以磷酸吡哆醛(PLP)作為離氨酸脫羧酶的輔因子，而CadB蛋白則是促進離氨酸和1, 5-戊二胺運輸的逆轉運蛋白。
本研究構建了在T7誘導系統中共表達CadA和CadB的重組大腸桿菌，命名為BL21-AB。為了測定CadA活性，建立了使用pH指示劑（溴甲酚紫，BCP）偵測反應中顏色變化的比色測定法，其一個單位活性定義為每分鐘可催化生成1 mmol 的1, 5-戊二胺，且定義1 a.u. CadA為可以催化1 mmolcadaverine /min (unit)之酵素量。接著進行BL21-AB表達CadA的條件優化。在調查誘導的基本條件之後，0.007 mM的IPTG誘導劑、溫度32°C、80 rpm攪拌速率被決定為誘導蛋白表達的優化條件。當添加不同濃度離氨酸以搖瓶醱酵生產1, 5-戊二胺時，添加0.27 M和0.38 M的離氨酸分別生產15.33 g/L和18.40 g/L的1, 5-戊二胺。其中0.38 M離氨酸有較佳的轉化率為53.11%。由於BL21-AB對高濃度基質和產物的耐受性有限，影響到未來的商業化潛力，因此以全細胞生物轉化高濃度離氨酸被認為是有前景高效率生產1, 5-戊二胺的方法。
此外，也針對CadA活性進行動力學性質之探討。在Michaelis-Menten酵素動力學研究中，比較作為PLP前體的吡哆醇(PN)和吡哆醛(PL)對轉化不同濃度離胺酸的影響，並比較其Vmax、kM、kcat值。與使用PL和PLP作為輔因子相比之下，使用PN作為輔因子可得到最小的kcat/KM值，說明了在酵素反應中使用PN作為輔因子有最佳的轉化效率。另一方面，在酵素反應中提供前體PN和ATP可達到與直接添加PLP相近的活性(Vmax)。此外，為大量生產全細胞生物催化劑並降低醱酵成本，M9培養基被使用來替代LB培養基，用於培養BL21-AB菌株。在T7啟動子系統中，葡萄糖的存在導致CadA表達水平降低，因此選擇甘油作為碳源。在提升碳源和氮源的含量後，比細胞生長速率(µ)在M9CN(Y) 培養基中達到0.25 h-1，比LB培養基 (0.24 h-1)略高。另外，使用M9CN(Y) 培養基並控制pH在適當的條件，與LB培養基相比可達到約1.61倍的總活性，而每單位活性所需之培養基成本則可減少約87%。
為確認CadB促進離氨酸和1, 5-戊二胺的轉運，比較BL21-A和BL21-AB轉化0.27 M離氨酸的結果。BL21-AB產生了27.45±2.87 g/L的戊二胺，高於BL21-A（11.13±4.26 g/L）。此結果說明CadB扮演著重要角色，藉由CadB攝取離氨酸同時分泌1, 5-戊二胺促進生物轉化效率，並減少1, 5-戊二胺在體內過度累積而產生毒性。當經由乙醇通透性處理後，不僅通透性BL21-A的產量增加至30.50±1.44 g/L，而且通透性BL21-AB的產量也提升至31.10±1.77 g/L，兩者經由滲透性處理後幾乎達到相同產量。為了進一步了解滲透性處理對生物轉化作用的影響，對未處理的、冷凍處理的和酒精處理的細胞作為生物催化劑進行了比較和討論。細胞在通透性處理後，細胞膜受損，小分子可以自由進入和離開細胞，因而提高了生物轉化效率。使用未經處理的細胞進行重複使用且循環轉化，細胞在第一次使用後，在第二次重複使用時轉化效率顯著提升，且其轉化效率達到與滲透性細胞相同。此實驗結果說明可能高濃度基質或產物對細胞產生毒性，因此其細胞膜受損，通透性增加，而提升生物轉化效率。也就是說，不僅滲透化的細胞，重複使用的細胞也被推測為死亡細胞。兩者皆因為細胞膜受損不具有調控功能，而小分子可以自由進入和離開細胞。另一方面，未處理的細胞在五次重複使用後，仍維持70.73±4.53%的生物轉化效率。
最後，為發展不直接添加昂貴的外源PLP的系統，嘗試將PN作為PLP前體應用於高濃度離氨酸生物轉化過程。結果顯示，在PLP補救合成途徑中，增強PN/PL/PM激酶(PdxK或PdxY)的表達是必要的未來工作。

Due to the increased concerns toward resource depletion and environmental issues, bio-based chemical production has been a hot research topic, aiming to reduce the environmental impacts arising from excessive uses of the conventional petroleum-based process for chemical production. For instance, bio-based polyamides (also known as nylon) have been receiving increasing research attention to replace traditional petroleum-based polyamides (nylon-6 and 66). Cadaverine, also known as 1, 5-diaminopentane, is the building block of bio-based nylons, such as nylon-54, 56, and 510. In the model bacteria Escherichia coli (E. coli), cadaverine is synthesized from lysine, catalyzed by the enzyme lysine decarboxylase (CadA) using pyridoxal phosphate (PLP) as the cofactor. On the other hand, CadB is an antiporter facilitating the excretion of lysine and cadaverine.
In this study, recombinant E. coli overexpressing CadA and CadB in a T7 inducible expression system was constructed and named as BL21-AB. To detect the CadA activity, a colorimetric assay based on the color change of pH indicator (bromocresol purple, BCP) was established. In this study, one unit (U) of CadA activity is defined as the production of one mmol cadaverine per min (mmolcadaverine/min); one a.u. CadA is defined as the quantity of enzyme required to produce one mmol cadaverine per min. After establishing the method for detecting CadA activity, the basic conditions for the induction of BL21-AB expressing CadA were optimized as follows: IPTG concentration, 0.007 mM; temperature, 32°C; agitation rate, 80 rpm. In the bio-production of cadaverine by flask fermentation, cadaverine titers of 15.33 g/L and 18.40 g/L were achieved from lysine concentration of 0.27 M and 0.38 M, respectively. The lysine conversion efficiency with 0.27 M lysine was 53.11%. The strain BL21-AB demonstrated limited tolerance to the product cadaverine and substrate lysine. Thus, whole-cell bioconversion was evaluated for the efficient production of cadaverine from high lysine concentrations.
Kinetics investigation of the recombinant CadA activity was also carried out. In the enzyme kinetics analysis using the Michaelis-Menten kinetic model, pyridoxine (PN) and pyridoxal (PL) were compared as precursors of PLP to convert different concentrations of lysine. Using PN as a cofactor exhibited a lower value of kcat/KM, while using PL and PLP as the cofactor gave the highest lysine conversion efficiency in the enzymatic reaction. Also, the activity (Vmax) obtained using precursor PN along with ATP in the enzymatic reaction was as fast as direct PLP supply. For enhancing BL21-AB biomass production to be used as biocatalysts, M9 medium was used to replace LB medium for culturing BL21-AB to reduce the fermentation cost. In the T7 promoter system, the presence of glucose seems to repress the expression level of CadA, so glycerol was used as the alternative carbon source. With increasing the content of carbon and nitrogen source, specific cell growth rate (µ) of the M9CN (Y) medium achieved 0.25 h-1 faster than LB medium (0.24 h-1). In addition, with the pH controlled at an appropriate level, total activity was 1.61 times higher than that of LB medium, and the price per unit of M9CN(Y) decreased by 87%.
To confirm CadB increasing the transport of lysine and cadaverine, conversion of 0.27 M lysine was investigated, and BL21-AB overexpressing CadB produced 27.45±2.87 g/L cadaverine, which was higher than BL21-A (11.13±4.26 g/L). Moreover, CadB seems to facilitate bioconversion of lysine uptake and cadaverine excretion and relieves the toxic accumulation of cadaverine in vivo. After permeability-treatment by ethanol, not only permeabilized BL21-A increased cadaverine titer to 30.50±1.44 g/L, while permeabilized BL21-AB also increased cadaverine titer to 31.10±1.77 g/L, which is nearly the same as permeabilized BL21-A. To further understand the influence of permeabilization on bioconversion efficiency, non-treated, freeze-treated, and ethanol-treated biocatalysts were prepared and compared. The cell wall/membrane of the permeabilized cells are damaged, and low-molecular-weight molecules can freely enter and leave the cell to increase bioconversion efficiency. After the first cycle of bioconversion, non-treated cells increased cadaverine production and lysine consumption during the second cycle of repeated use, whose conversion efficiency was the same as the permeabilized cells. This indicates that the cells were damaged by the toxic effect of high titer of substrate or product, so cell wall permeability increased to enhance biotransformation. Not only the permeabilized cells but also reused cells were seen as dead cells. On the other hand, non-treated cells were able to maintain a bioconversion efficiency of 70.73±4.53% after five cycles. Finally, PN as a PLP precursor was applied in the high-concentration lysine bioconversion process to develop a system without a direct supply of exogenous PLP. The results suggest that enhancing the expression levels of PN/PL/PM kinase (PdxK or PdxY) is an essential future work in the de novo pathway of PLP biosynthesis.

摘要	I
Abstract	IV
Acknowledgments	VII
Contents	VIII
List of Tables	XII
List of Figures	XIII
Chapter 1	Introduction	1
1	Background	1
2	Motivation and purpose	3
3	Research scheme of this study	4
Chapter 2	Literature review	6
1	Introduction to cadaverine	6
2	Advances in cadaverine bacterial production	8
3	Functions of CadB	13
4	Whole-cell biocatalysts and permeability issues	15
5	Salvage pathway for the pyridoxal-5’-phosphate (PLP) biosynthesis	17
6	Assay of measuring lysine decarboxylase activity	18
7	The option of medium in lactose induced system	19
7.1	Glucose effect on lac operon expression system	19
7.2	Influence of the minimal salts medium (M9) on E. coli	20
7.3	Glycerol utilized in lactose induced system	20
Chapter 3	Materials and methods	21
1	Chemicals and materials	21
2	Equipment	24
3	Microorganism strains and cultivation medium	26
3.1	Cloning and construct of cadA and cadB genes	26
3.2	Storage of bacterial strains	28
3.3	Pre-culture medium and cultivation condition	28
3.4	Main-culture medium and cultivation condition	29
3.5	Expression of recombinant CadA in E. coli BL21(DE3)	29
4	Experimental methods	30
4.1	Calibration of cadaverine concentration in CadA activity assay	30
4.2	Enzyme kinetics of lysine decarboxylase	30
4.3	In vivo bioproduction of cadaverine in flask fermentation	31
4.4	Flask cultivation of BL21-AB for whole-cell biocatalyst production	31
4.5	Optimization of cultivation medium	32
4.6	Whole-cell bioconversion of lysine to cadaverine	34
4.7	Preparation of ethanol-treated permeabilized cells	34
4.8	Reusability of whole-cell biocatalysts	34
5	Analytical methods	35
5.1	Determination of cell concentration	35
5.2	Total protein concentration analysis	35
5.3	Determination of lysine and cadaverine concentration	36
5.4	Bromocresol purple (BCP) assay for CadA activity	37
5.5	Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and sample preparation	38
5.6	Determination of the concentration of carbon sources and metabolites in the medium	40
5.7	Measurements of carbon, nitrogen, and phosphorus concentration in culture medium	41
5.8	Scanning electron microscopy (SEM) analysis	41
Chapter 4	Results and discussion	42
1	The construct BL21-AB strain overexpresses CadA and CadB protein	42
2	Establishment of a continual spectrophotometric assay for determining activity of lysine decarboxylase (CadA)	43
3	Optimization of basic conditions of induction	46
4	Time course of BL21-AB cultivation expressing recombinant protein	48
5	In vivo bioproduction of cadaverine in flask fermentation	50
6	Kinetic analysis of CadA enzyme extract	53
6.1	Influence of B6 vitamers on kinetic constants	53
6.2	Supplementation with PN and ATP to form PLP	56
7	Degradation of activity in the stored cells	58
8	Effect of cultivation medium on BL21-AB cultivation as biocatalysts	60
8.1	Composition of LB medium compared to M9 medium	60
8.2	Using modified minimal medium for flask cultivation	62
8.3	Using modified minimal medium for flask cultivation with ammonium hydroxide to maintain pH	64
8.4	Comparison of all medium in flask cultivation	66
9	Overexpression of CadB to improve the bioconversion of lysine to cadaverine	68
10	Permeabilization of biocatalysts	70
10.1	Optimization of ethanol concentration for permeabilized cells	70
10.2	Permeabilization to improve the bioconversion of lysine to cadaverine	72
10.3	Influence of permeabilization on BL21-AB as biocatalysts	74
10.4	Cycling and reusability of non-treated and permeabilized BL21-AB	78
10.5	BL21-AB biocatalysts stored in refrigerator	80
11	Effect of cofactor on bioproduction of cadaverine	82
Chapter 5	Conclusions	84
Chapter 6	Future work	88
Reference	89
Appendix	93
                                    

AQUALYTIC®. 2017. Photometer System AL450 Instruction manual.
Caron, G.N.v. 1998. Assessment of bacterial viability status by flow cytometry and single cell sorting. Journal of applied microbiology, 84(6), 988-998.
Chen, R.R. 2007. Permeability issues in whole-cell bioprocesses and cellular membrane engineering. Applied microbiology and biotechnology, 74(4), 730-738.
Chung, H., Yang, J.E., Ha, J.Y., Chae, T.U., Shin, J.H., Gustavsson, M., Lee, S.Y. 2015. Bio-based production of monomers and polymers by metabolically engineered microorganisms. Curr Opin Biotechnol, 36, 73-84.
Eisenthal, R., Danson, M.J., Hough, D.W. 2007. Catalytic efficiency and kcat/KM: a useful comparator? Trends in biotechnology, 25(6), 247-249.
Felix, H. 1982. Permeabilized cells. Analytical biochemistry, 120(2), 211-234.
Grossman, A. Use of glucose to control basal expression in the pET System.
Inada, T., Kimata, K., Aiba, H. 1996. Mechanism responsible for glucose–lactose diauxie in Escherichia coli: challenge to the cAMP model. Genes to Cells, 1(3), 293-301.
Kim, H.J., Kim, Y.H., Shin, J.-H., Bhatia, S.K., Sathiyanarayanan, G., Seo, H.-M., Choi, K.Y., Yang, Y.-H., Park, K. 2015a. Optimization of direct lysine decarboxylase biotransformation for cadaverine production with whole-cell biocatalysts at high lysine concentration. J. Microbiol. Biotechnol, 25(7), 1108-1113.
Kim, H.T., Baritugo, K.-A., Oh, Y.H., Hyun, S.M., Khang, T.U., Kang, K.H., Jung, S.H., Song, B.K., Park, K., Kim, I.-K., Lee, M.O., Kam, Y., Hwang, Y.T., Park, S.J., Joo, J.C. 2018. Metabolic Engineering of Corynebacterium glutamicum for the High-Level Production of Cadaverine That Can Be Used for the Synthesis of Biopolyamide 510. ACS Sustainable Chemistry & Engineering, 6(4), 5296-5305.
Kim, J.H., Kim, J., Kim, H.J., Sathiyanarayanan, G., Bhatia, S.K., Song, H.S., Choi, Y.K., Kim, Y.G., Park, K., Yang, Y.H. 2017. Biotransformation of pyridoxal 5'-phosphate from pyridoxal by pyridoxal kinase (pdxY) to support cadaverine production in Escherichia coli. Enzyme Microb Technol, 104, 9-15.
Kim, Y.H., Kim, H.J., Shin, J.-H., Bhatia, S.K., Seo, H.-M., Kim, Y.-G., Lee, Y.K., Yang, Y.-H., Park, K. 2015b. Application of diethyl ethoxymethylenemalonate (DEEMM) derivatization for monitoring of lysine decarboxylase activity. Journal of Molecular Catalysis B: Enzymatic, 115, 151-154.
Kim, Y.H., Sathiyanarayanan, G., Kim, H.J., Bhatia, S.K., Seo, H.-M., Kim, J.-H., Song, H.-S., Kim, Y.-G., Park, K., Yang, Y.-H. 2015c. A liquid-based colorimetric assay of lysine decarboxylase and its application to enzymatic assay. J. Microbiol. Biotechnol, 25(12), 2110-2115.
Kind, S., Neubauer, S., Becker, J., Yamamoto, M., Volkert, M., Abendroth, G., Zelder, O., Wittmann, C. 2014. From zero to hero - production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum. Metab Eng, 25, 113-23.
Kind, S., Wittmann, C. 2011. Bio-based production of the platform chemical 1,5-diaminopentane. Appl Microbiol Biotechnol, 91(5), 1287-96.
Kopp, J., Slouka, C., Ulonska, S., Kager, J., Fricke, J., Spadiut, O., Herwig, C. 2018. Impact of glycerol as carbon source onto specific sugar and inducer uptake rates and inclusion body productivity in E. coli BL21 (DE3). Bioengineering, 5(1), 1.
Lee, S.Y., Kim, H.U. 2015. Systems strategies for developing industrial microbial strains. Nature biotechnology, 33(10), 1061-1072.
Li, M., Li, D., Huang, Y., Liu, M., Wang, H., Tang, Q., Lu, F. 2014. Improving the secretion of cadaverine in Corynebacterium glutamicum by cadaverine–lysine antiporter. Journal of industrial microbiology & biotechnology, 41(4), 701-709.
Lin, B., Tao, Y. 2017. Whole-cell biocatalysts by design. Microbial Cell Factories, 16(1), 106.
Lu, W.W.-W., Mallette, M. 1970. Colorimetric assay for lysine decarboxylase in Escherichia coli. Appl. Environ. Microbiol., 19(2), 367-369.
Ma, W., Cao, W., Zhang, H., Chen, K., Li, Y., Ouyang, P. 2015. Enhanced cadaverine production from L-lysine using recombinant Escherichia coli co-overexpressing CadA and CadB. Biotechnol Lett, 37(4), 799-806.
Ma, W., Chen, K., Li, Y., Hao, N., Wang, X., Ouyang, P. 2017. Advances in Cadaverine Bacterial Production and Its Applications. Engineering, 3(3), 308-317.
Moon, Y.-M., Yang, S.Y., Choi, T.R., Jung, H.-R., Song, H.-S., hoon Han, Y., Park, H.Y., Bhatia, S.K., Gurav, R., Park, K. 2019a. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5′-phosphate and ATP. Enzyme and microbial technology, 127, 58-64.
Moon, Y.M., Yang, S.Y., Choi, T.R., Jung, H.R., Song, H.S., Han, Y.H., Park, H.Y., Bhatia, S.K., Gurav, R., Park, K., Kim, J.S., Yang, Y.H. 2019b. Enhanced production of cadaverine by the addition of hexadecyltrimethylammonium bromide to whole cell system with regeneration of pyridoxal-5'-phosphate and ATP. Enzyme Microb Technol, 127, 58-64.
Nishi, K., Endo, S., Mori, Y., Totsuka, K., Hirao, Y. 2007. Method for producing cadaverine dicarboxylate, Google Patents.
Oh, Y.H., Kang, K.H., Kwon, M.J., Choi, J.W., Joo, J.C., Lee, S.H., Yang, Y.H., Song, B.K., Kim, I.K., Yoon, K.H., Park, K., Park, S.J. 2015. Development of engineered Escherichia coli whole-cell biocatalysts for high-level conversion of L-lysine into cadaverine. J Ind Microbiol Biotechnol, 42(11), 1481-91.
Park, S.H., Soetyono, F., Kim, H.K. 2017. Cadaverine Production by Using Cross-Linked Enzyme Aggregate of Escherichia coli Lysine Decarboxylase. J Microbiol Biotechnol, 27(2), 289-296.
Phan, A., Ngo, T., Lenhoff, H. 1982. Spectrophotometric assay for lysine decar☐ ylase. Analytical biochemistry, 120(1), 193-197.
Qian, Z.G., Xia, X.X., Lee, S.Y. 2011. Metabolic engineering of Escherichia coli for the production of cadaverine: a five carbon diamine. Biotechnology and bioengineering, 108(1), 93-103.
Rhee, J.I., Bode, J., Diaz-Ricci, J.C., Poock, D., Weigel, B., Kretzmer, G., Schügerl, K. 1997. Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI:: SPA. Journal of biotechnology, 55(2), 69-83.
Rui, J., You, S., Zheng, Y., Wang, C., Gao, Y., Zhang, W., Qi, W., Su, R., He, Z. 2020. High-efficiency and low-cost production of cadaverine from a permeabilized-cell bioconversion by a Lysine-induced engineered Escherichia coli. Bioresour Technol, 302, 122844.
Samartzidou, H., Delcour, A.H. 1999. Excretion of endogenous cadaverine leads to a decrease in porin-mediated outer membrane permeability. Journal of bacteriology, 181(3), 791-798.
Scriven, F., Wlasichuk, K.B., Palcic, M.M. 1988. A continual spectrophotometric assay for amino acid decarboxylases. Analytical biochemistry, 170(2), 367-371.
Shin, J., Joo, J.C., Lee, E., Hyun, S.M., Kim, H.J., Park, S.J., Yang, Y.-H., Park, K. 2018a. Characterization of a whole-cell biotransformation using a constitutive lysine decarboxylase from Escherichia coli for the high-level production of cadaverine from industrial grade L-lysine. Applied biochemistry and biotechnology, 185(4), 909-924.
Shin, J., Joo, J.C., Lee, E., Hyun, S.M., Kim, H.J., Park, S.J., Yang, Y.H., Park, K. 2018b. Characterization of a Whole-Cell Biotransformation Using a Constitutive Lysine Decarboxylase from Escherichia coli for the High-Level Production of Cadaverine from Industrial Grade L-Lysine. Appl Biochem Biotechnol, 185(4), 909-924.
Soksawatmaekhin, W., Kuraishi, A., Sakata, K., Kashiwagi, K., Igarashi, K. 2004. Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli. Molecular microbiology, 51(5), 1401-1412.
Thakur, C.S., Brown, M.E., Sama, J.N., Jackson, M.E., Dayie, T.K. 2010. Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides. Applied microbiology and biotechnology, 88(3), 771-779.
Wachtmeister, J., Rother, D. 2016. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale. Current opinion in biotechnology, 42, 169-177.
Wang, J., Lu, X., Ying, H., Ma, W., Xu, S., Wang, X., Chen, K., Ouyang, P. 2018. A Novel Process for Cadaverine Bio-Production Using a Consortium of Two Engineered Escherichia coli. Front Microbiol, 9, 1312.
Xue, C., Hsu, K.-M., Ting, W.-W., Huang, S.-F., Lin, H.-Y., Li, S.-F., Chang, J.-S., Ng, I.S. 2020. Efficient biotransformation of l-lysine into cadaverine by strengthening pyridoxal 5’-phosphate-dependent proteins in Escherichia coli with cold shock treatment. Biochemical Engineering Journal, 161.
Yang, P., Li, X., Liu, H., Li, Z., Liu, J., Zhuang, W., Wu, J., Ying, H. 2019. Thermodynamics, crystal structure, and characterization of a bio-based nylon 54 monomer. CrystEngComm, 21(46), 7069-7077.
Yang, Y., Zhao, G., Winkler, M.E. 1996. Identification of the pdxK gene that encodes pyridoxine (vitamin B6) kinase in Escherichia coli K-12. FEMS Microbiology Letters, 141(1), 89-95.
Zhao, G., Winkler, M.E. 1995. Kinetic limitation and cellular amount of pyridoxine (pyridoxamine) 5'-phosphate oxidase of Escherichia coli K-12. Journal of Bacteriology, 177(4), 883-891.

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