L-Phenyllactic acid is a natural antibacterial substance, which has a spectral inhibitory effect on a variety of pathogenic microorganisms. It is expected to become a new type of biological preservative. A multi-enzyme cascade catalytic reaction system was constructed to improve the ability of L-phenyllactic acid synthesis by Escherichia coli whole cell. A new L-phenyllactic acid biosynthesis method was established by co-expressing L-amino acid deaminase and phenylpyruvate reductase coupled with glucose dehydrogenase for coenzyme regeneration. The genes were successfully expressed in E.coli. The whole cell transformation conditions were optimized. The optimal transformation conditions were reaction at 30 ℃, initial pH 8.0, substrate phenylalanine of 30 g/L, OD600 of 30, and auxiliary substrate glucose of one-time molar equivalent, conversion reaction time of 12 h. A total of 21.39 g/L of L-phenyllactic acid was produced, and the molar conversion rate was 71.33%. A simple and efficient biocatalysis method is provided, which lays the foundation for the realization of large-scale production.
SHAO Yu
,
ZHANG Xian
,
HU Mengkai
,
WEI Yuxia
,
YANG Taowei
,
XU Meijuan
,
SHAO Minglong
,
GAO Minjie
,
RAO Zhiming
. Synthesis of L-phenyllactic acid catalyzed by recombinant Escherichia coli whole cell biotransformation[J]. Food and Fermentation Industries, 2021
, 47(14)
: 1
-8
.
DOI: 10.13995/j.cnki.11-1802/ts.026819
[1] ARMAFORTE E, CARRI S, FERRI G, et al.High-performance liquid chromatography determination of phenyllactic acid in MRS broth[J].Journal of Chromatography A, 2006, 1 131(1-2):281-284.
[2] PENG Q, ZENG X F, ZHU J L, et al.Effects of dietary Lactobacillus plantarum B1 on growth performance, intestinal microbiota, and short chain fatty acid profiles in broiler chickens[J].Poultry Science, 2016, 95(4):893-900.
[3] SCHNRER J, MAGNUSSON J.Antifungal lactic acid bacteria as biopreservatives[J].Trends in Food Science & Technology, 2005, 16(1-3):70-78.
[4] HASHIMOTO Y, KOBAYASHI E, ENDO T, et al.Conversion of a cyanhydrin compound into S-(-)-3-phenyllactic acid by enantioselective hydrolytic activity of Pseudomonas sp.BC-18[J].Bioscience, Biotechnology, and Biochemistry, 1996, 60(8):1 279-1 283.
[5] BUSTOS A Y, VALDEZ G F, GEREZ C L.Optimization of phenyllactic acid production by Pediococcus acidilactici CRL 1753.application of the formulated bio-preserver culture in bread[J].Biological Control, 2018, 123(3):137-143.
[6] LEE H S, PARK J, YOO Y J, et al.A novel d-2-hydroxy acid dehydrogenase with high substrate preference for phenylpyruvate originating from lactic acid bacteria:Structural analysis on the substrate specificity[J].Enzyme and Microbial Technology, 2019, 125:37-44.
[7] LIU S P, ZHANG L, MAO J, et al.Metabolic engineering of Escherichia coli for the production of phenylpyruvate derivatives[J].Metabolic Engineering, 2015, 32(1):55-65.
[8] XU G C, ZHANG L L, NI Y.Enzymatic preparation of D-phenyllactic acid at high space-time yield with a novel phenylpyruvate reductase identified from Lactobacillus sp.CGMCC 9967[J].Journal of Biotechnology, 2016, 222(1):29-37.
[9] LUO X, ZHANG Y, YIN F, et al.Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis[J].3 Biotech, 2020, 10(3):101.
[10] COBAN H B, DEMIRCI A, PATTERSON P H, et al.Screening of phenylpyruvic acid producers and optimization of culture conditions in bench scale bioreactors[J].Bioprocess and Biosystems Engineering, 2014, 37(11):2 343-2 352.
[11] ZHANG L, LIU Q, PAN H, et al.Metabolic engineering of Escherichia coli to high efficient synthesis phenylacetic acid from phenylalanine[J].AMB Express, 2017, 7(1):105.
[12] HOU Y, HOSSAIN G S, LI J H, et al.Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst[J].Applied Microbiology and Biotechnology, 2016, 100(5):2 183-2 189.
[13] LI J F, LI X Q, LIU Y, et al.Directed modification of l-LcLDH1, an l-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid91[J].Journal of Biotechnology, 2018, 281(1):193-198.
[14] HOU Y, GAO B, CUI J, et al.Combination of multi-enzyme expression fine-tuning and co-substrates addition improves phenyllactic acid production with an Escherichia coli whole-cell biocatalyst[J].Bioresource Technology, 2019, 287(2):121 423.
[15] 段培峰, 尤甲甲, 徐美娟, 等.重组枯草芽孢杆菌全细胞催化合成 2-O-α-D-甘油葡糖苷展[J].生物工程学报, 2020, 36(8):1-11.
DUAN P F, YOU J J, XU M J, et al.Whole-cell biosynthesis of 2-O-α-D-glu-copyranosyl-sn-glycerol by recombinant Bacillus subtilis[J].Chinese Journal of Biotechnology, 2020, 36(8):1-11.
[16] 刘巧利, 杨套伟, 周俊平, 等.酶法高效转化苯乙酮酸合成L-苯甘氨酸[J].应用与环境生物学报.2019, 25 (2):451-156.
LIU Q L, YANG T W, ZHOU J P, et al.Efficient enzymatic synthesis of L-phenylglycine from benzoylformic acid[J].Chinese Journal of Applied & Environmental Biology, 2019, 25 (2):451-456.
[17] KAWAGUCHI H, MIYAGAWA H, NAKAMURA T S, et al.Enhanced phenyllactic acid production in Escherichia coli via oxygen limitation and shikimate pathway gene expression[J].Biotechnology Journal, 2019, 14(6):1 800 478.
[18] ZHAO W, DING H, LYU C J, et al.Two-step biocatalytic reaction using recombinant Escherichia coli cells for efficient production of phenyllactic acid from L-phenylalanine[J].Process Biochemistry, 2018, 64(2):7-31.
[19] 黄国昌, 顾斌涛, 黄朝, 等.苯乳酸生产菌的筛选及鉴定[J].食品与发酵工业, 2018, 44(3):97-100.
HUANG G C,GU B T, HUANG Z, et al.Screening and identification of phenyllactic acid-producing bacteria[J].Food and Fermentation Industries,2018, 44(3):97-100.
[20] WU S, CHEN Y, XU Y, et al.Enantioselective trans-dihydroxylation of aryl olefins by cascade biocatalysis with recombinant Escherichia coli coexpressing monooxygenase and epoxide hydrolase[J].ACS Catalysis, 2014, 4(2):409-420.
[21] DAO Y, ZHANG K, LU X F, et al.Role of glucose and 2-oxoglutarate/malate translocator(OMT1) in the production of phenyllactic acid and p-hydroxyphenyllactic acid, two food-borne pathogen inhibitors[J].Journal of Agricultural and Food Chemistry, 2019, 67(20):5 820-5 826.
