L-亮氨酸是一种重要的支链氨基酸,在医药、食品、饲料、化妆品等多领域中具有重要价值。目前,工业上用于生产L-亮氨酸的菌株存在转化率低、副产物多、结晶后产率大幅下降等问题。为获得一株性能优越的L-亮氨酸生产菌株,该研究对L-亮氨酸生产菌株谷氨酸棒状杆菌CP进行系统代谢工程改造。首先,敲除竞争途径并引入外源磷酸酮醇酶(由fxpk编码)加强前体物(丙酮酸和乙酰辅酶A)供应;其次,加强主代谢流(亮氨酸途径和支链氨基酸共同途径)提高L-亮氨酸产量;随后,加强胞内NADPH的供应;最后,敲除L-亮氨酸内转运蛋白BrnQ,最终获得的可高效生产L-亮氨酸的工程菌株CP06-1在5 L生物反应器中经50 h发酵后L-亮氨酸产量达65.3 g/L,糖酸转化率25.1%。此外,为解决L-亮氨酸结晶后产生的泡沫影响菌体吸收营养和溶氧的问题,该研究采取了半连续发酵的方式在L-亮氨酸结晶前通过放液-补液来延长菌株产酸高峰期,最终5 L生物反应器发酵50 h共生产L-亮氨酸228.8 g,糖酸转化率达27.9%,较分批补料提高了11.2%。该研究为L-亮氨酸生产菌株的改造和应用提供了一定的指导价值。
L-Leucine is an important branched-chain amino acid with significant value in various fields such as pharmaceuticals, food, feed, and cosmetics.Currently, the industrial production of L-leucine using strains faces challenges including low conversion rates, high levels of by-products, and substantial decreases in yield after crystallization.To obtain a superior strain for L-leucine production, this study conducted systematic metabolic engineering on the L-leucine-producing strain Corynebacterium glutamicum CP.Firstly, competitive pathways were knocked out, and exogenous phosphoenolpyruvate carboxykinase (encoded by fxpk) was introduced to enhance the supply of precursors (pyruvate and acetyl-CoA).Secondly, the main metabolic flux (including the pathways for L-leucine and branched-chain amino acids) was reinforced to increase L-leucine production.Subsequently, intracellular NADPH supply was enhanced.Finally, the L-leucine intracellular transport protein BrnQ was knocked out.The engineered strain CP06-1 obtained could efficiently produce L-leucine, with a yield of 65.3 g/L after 50 hours of fermentation in a 5 L bioreactor, and a yield of 0.251 g/g glucose.Moreover, to address the issue of foam generation affecting nutrient absorption and oxygen solubility after L-leucine crystallization, semi-continuous fermentation was adopted.Before L-leucine crystallization, the effluent was released and supplemented, extending the acid production peak of the strain.Ultimately, in the 50-hour fermentation in a 5 L bioreactor, a total of 228.8 g of L-leucine was produced, with a yield of 0.279 g/g glucose, representing an 11.2% improvement over batch feeding.This study provides valuable guidance for the modification and utilization of L-leucine-producing strains.
[1] VAN LOON L J C.Leucine as a pharmaconutrient in health and disease[J].Current Opinion in Clinical Nutrition and Metabolic Care, 2012, 15(1):71-77.
[2] LIU T, ZUO B, WANG W, et al.Dietary supplementation of leucine in premating diet improves the within-litter birth weight uniformity, antioxidative capability, and immune function of primiparous SD rats[J].BioMed Research International, 2018, 2018:1523147.
[3] YANG Y, WU Z L, MEININGER C J, et al.L-leucine and NO-mediated cardiovascular function[J].Amino Acids, 2015, 47(3):435-447.
[4] ZHANG S H, ZENG X F, REN M, et al.Novel metabolic and physiological functions of branched chain amino acids:A review[J].Journal of Animal Science and Biotechnology, 2017, 8:10.
[5] WU G Y.Amino acids:Metabolism, functions, and nutrition[J].Amino Acids, 2009, 37(1):1-17.
[6] WANG Y Y, XU J Z, ZHANG W G.Metabolic engineering of L-leucine production in Escherichia coli and Corynebacterium glutamicum:A review[J].Critical Reviews in Biotechnology, 2019, 39(5):633-647.
[7] CHENG H B, ZHU X, ZHU C, et al.Hydrolysis technology of biomass waste to produce amino acids in sub-critical water[J].Bioresource Technology, 2008, 99(9):3337-3341.
[8] IVANOV K, STOIMENOVA A, OBRESHKOVA D, et al.Biotechnology in the production of pharmaceutical industry ingredients:Amino acids[J].Biotechnology & Biotechnological Equipment, 2013, 27(2):3620-3626.
[9] MARTIN J, EISOLDT L, SKERRA A.Fixation of gaseous CO2 by reversing a decarboxylase for the biocatalytic synthesis of the essential amino acid l-methionine[J].Nature Catalysis, 2018, 1:555-561.
[10] WANG Y Y, ZHANG F, XU J Z, et al.Improvement of L-leucine production in Corynebacterium glutamicum by altering the redox flux[J].International Journal of Molecular Sciences, 2019, 20(8):2020.
[11] WANG Y Y, XU J Z, JIN Z Y, et al.Improvement of acetyl-CoA supply and glucose utilization increases L-leucine production in Corynebacterium glutamicum[J].Biotechnology Journal, 2022, 17(8):e2100349.
[12] WANG Y Y, SHI K, CHEN P D, et al.Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production[J].Journal of Industrial Microbiology & Biotechnology, 2020, 47(6-7):485-495.
[13] CHEN S L, LIU T S, ZHANG W G, et al.An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain[J].International Microbiology, 2023, 26(1):11-24.
[14] GUI Y L, MA Y C, XU Q Y, et al.Complete genome sequence of Corynebacterium glutamicum CP, a Chinese L-leucine producing strain[J].Journal of Biotechnology, 2016, 220:64-65.
[15] 王颖妤. 代谢工程改造谷氨酸棒杆菌发酵生产L-亮氨酸[D].无锡:江南大学, 2020.
WANG Y Y.Metabolic engineering of Corynebacterium glutamicum for L-leucine fermentation[D].Wuxi:Jiangnan University, 2020.
[16] 张玉富. L-亮氨酸生产菌的代谢工程改造及发酵工艺优化[D].天津:天津科技大学, 2021.
ZHANG Y F.Metabolic engineering and fermentation process optimization of L-leucine producing strain[D].Tianjin:Tianjin University of Science of Technology, 2021.
[17] BOGORAD I W, LIN T S, LIAO J C.Synthetic non-oxidative glycolysis enables complete carbon conservation[J].Nature, 2013, 502(7473):693-697.
[18] DING X H, YANG W J, DU X B, et al.High-level and-yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering[J].Metabolic Engineering, 2023, 78:128-136.
[19] GONG Y, WANG R Q, MA L, et al.Optimization of trans-4-hydroxyproline synthesis pathway by rearrangement center carbon metabolism in Escherichia coli[J].Microbial Cell Factories, 2023, 22(1):240.
[20] BRUNE I, JOCHMANN N, BRINKROLF K, et al.The IclR-type transcriptional repressor LtbR regulates the expression of leucine and tryptophan biosynthesis genes in the amino acid producer Corynebacterium glutamicum[J].Journal of Bacteriology, 2007, 189(7):2720-2733.
[21] MA Y C, CHEN Q X, CUI Y, et al.Comparative genomic and genetic functional analysis of industrial L-leucine- and L-valine-producing Corynebacterium glutamicum strains[J].Journal of Microbiology and Biotechnology, 2018, 28(11):1916-1927.
[22] YIN L H, HU X Q, XU D Q, et al.Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase L-isoleucine production in Corynebacterium glutamicum[J].Metabolic Engineering, 2012, 14(5):542-550.
[23] 杨汉昆, 徐建中, 张伟国.阻断辅因子NADPH合成对谷氨酸棒杆菌生长及产物合成的影响[J].食品与发酵工业, 2019, 45(10):1-9.
YANG H K, XU J Z, ZHANG W G.Blocking NADPH biosynthesis affected growth and metabolites formation of Corynebacterium glutamicum[J].Food and Fermentation Industries, 2019, 45(10):1-9.
[24] SON H F, PARK W, KIM S, et al.Structure-based functional analysis of a novel NADPH-producing glyceraldehyde-3-phosphate dehydrogenase from Corynebacterium glutamicum[J].International Journal of Biological Macromolecules, 2024, 255:128103.
[25] LANGE C, MUSTAFI N, FRUNZKE J, et al.Lrp of Corynebacterium glutamicum controls expression of the brnFE operon encoding the export system for L-methionine and branched-chain amino acids[J].Journal of Biotechnology, 2012, 158(4):231-241.
