張?chǎng)?,陳?guó)強(qiáng)
清華大學(xué)生命科學(xué)學(xué)院,北京 100084
4-羥基丁酸 (4-hydroxybutyrate,4HB) 作為神經(jīng)遞質(zhì)是 γ-氨基丁酸的代謝物,以很低的含量存在于哺乳動(dòng)物大腦中,在醫(yī)學(xué)、藥學(xué)領(lǐng)域里被認(rèn)為是一種神經(jīng)遞質(zhì),對(duì)于自我平衡調(diào)整和產(chǎn)生有規(guī)律的睡眠等方面起著非常重要的作用[1]。4-羥基丁酸可經(jīng)氧化轉(zhuǎn)變?yōu)殓晁岚肴㈢晁?,最終進(jìn)入三羧酸循環(huán)而被代謝降解[2-3]。琥珀酸半醛脫氫酶缺陷的患者會(huì)在體液中積累很高濃度的 4-羥基丁酸,并表現(xiàn)出遲鈍、癲癇等癥狀[4]。4-羥基丁酸具有與鹽酸氯胺酮類似的麻醉效果,且能刺激體內(nèi)荷爾蒙素的分泌,因而被歸為管制藥品[5-6]。
4-羥基丁酸的另一常見用途是作為生物塑料聚3-羥基丁酸酯 4-羥基丁酸酯 (P3HB4HB) 的合成前體,它在共聚物中的比例直接影響著最終P3HB4HB的材料學(xué)性質(zhì)和降解性[7-11]。盡管 4-羥基丁酸可在厭氧菌克氏梭菌Clostridium kluyveri對(duì)乙醇、乙酸發(fā)酵過程中產(chǎn)生[12],也存在于古細(xì)菌勤奮金屬球菌Metallosphaera sedula等的 3-羥基丁酸/4-羥基丁酸固碳途徑中[13],卻不能在羅氏真養(yǎng)菌 Ralstonia eutropha、大腸桿菌Escherichia coli等常用的PHA合成菌中形成。因此需要添加結(jié)構(gòu)相關(guān)碳源,如 4-羥基丁酸、γ-丁內(nèi)酯或者1,4-丁二醇作為4-羥基丁酸的來源[14-17],這些添加物主要以石化產(chǎn)品為原料經(jīng)化學(xué)轉(zhuǎn)化制備[18-20]。
張磊等利用來源于惡臭假單胞菌 Pseudomonas putida KT2442的醇脫氫酶 (DhaT) 和醛脫氫酶(AldD) 催化完成 1,4-丁二醇向 4-羥基丁酸的轉(zhuǎn)化[21]。這里DhaT和AldD的催化作用需要輔酶因子NAD的參與 (圖1)。
對(duì)輔酶因子的調(diào)節(jié)是代謝工程改造中的一個(gè)重要手段,提高胞內(nèi)NADH水平已成功用于促進(jìn)合成還原性產(chǎn)物如1,3-丙二醇[22]、琥珀酸[23]、丁醇[24]等。同理,提高胞內(nèi)NAD含量可能促進(jìn)1,4-丁二醇向4-羥基丁酸的轉(zhuǎn)化。煙酸磷酸核糖轉(zhuǎn)移酶 (Nicotinic Acid Phosphoribosyltransferase,PncB) 和NAD合成酶 (NAD Synthetase,NadE) 是大腸桿菌NAD合成途徑的兩個(gè)關(guān)鍵酶,單獨(dú)表達(dá)時(shí)均使胞內(nèi)NAD (H)總量提高約 2.5倍,共表達(dá)時(shí)能夠提高近 7倍,且NAD增加量要多于 NADH增量[25]。因此,本研究通過在 E. coli S17-1 (pZL-dhaT-aldD) 中共表達(dá)pncB-nadE基因,提高1,4-丁二醇向4-羥基丁酸的轉(zhuǎn)化效率。
菌種Escherichia coli S17-1為清華大學(xué)微生物實(shí)驗(yàn)室保存。質(zhì)粒 pZL-dhaT-aldD由本實(shí)驗(yàn)室前期構(gòu)建[21]。本文所用菌種、質(zhì)粒及引物序列見表1。
fastpfu DNA聚合酶以及用于基因克隆的E. coli DH5α為北京全式金生物技術(shù)有限公司產(chǎn)品。各種限制性內(nèi)切酶為Fermentas產(chǎn)品。質(zhì)粒提取、膠回收試劑盒為博邁德公司產(chǎn)品。
質(zhì)粒構(gòu)建過程以及搖瓶種子液均采用 LB培養(yǎng)基。搖瓶發(fā)酵培養(yǎng)基為另外添加了10 g/L 1,4-丁二醇的 LB培養(yǎng)基。根據(jù)重組菌攜帶質(zhì)粒的抗性向滅菌冷卻后的培養(yǎng)基中加入抗生素,其中氨芐青霉素濃度為100 mg/L,卡那霉素濃度為50 mg/L。
培養(yǎng)條件為37 ℃,200 r/min。除搖瓶發(fā)酵培養(yǎng)時(shí)間為48 h外,其余情況均培養(yǎng)12 h。
圖1 1,4-丁二醇向4-羥基丁酸的轉(zhuǎn)化反應(yīng)Fig. 1 Conversion of 1,4-BD to 4HB.
表1 本研究中所使用的菌種、質(zhì)粒和引物Table 1 Strains, plasmids and primers used in this study
按照已報(bào)道方法提取胞內(nèi)NAD和NADH[22],略有修改。將3 mL搖瓶發(fā)酵菌液于4 ℃離心收集細(xì)胞,棄上清。將細(xì)胞用冰冷的去離子水洗滌2次后充分重懸于0.2 mL去離子水中。加入50 μL 0.4 mol/L HCl (提取NAD) 或者0.4 mol/L KOH (提取NADH),充分混勻后在冰上放置10 min。NAD提取液在50 ℃水浴,NADH提取液在30 ℃水浴。10 min后迅速移到冰上加入50 μL 0.4 mol/L KOH (提取NAD) 或者0.4 mol/L HCl (提取NADH) 進(jìn)行中和。4 ℃、12 000 r/min離心10 min。上清液即為NAD (H) 的提取液,將其移至新的離心管中,立即檢測(cè)。
含量測(cè)定采用酶循環(huán)法,反應(yīng)體系為 (1 mL):450 μL HEPES 緩沖液 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,0.15 mol/L,pH 7.4);200 μL 5-乙基吩嗪硫酸鹽 (PES,4 mg/mL);200 μL噻唑藍(lán) (MTT,5 g/L);50 μL乙醇;50 μL的600 U/mL乙醇脫氫酶 (ADH)。上述反應(yīng)液混勻后37 ℃溫育5 min,加入50 μL的NAD (H) 的提取液起始反應(yīng)。測(cè)定5 min內(nèi)OD570的變化。吸光度-時(shí)間曲線的斜率反應(yīng)出提取液中NAD (H) 的濃度。
1.4.1 細(xì)胞干重的測(cè)定
培養(yǎng)結(jié)束后,12 000 r/min、10 min離心收集菌體,然后用去離子水洗滌、離心,去掉菌體表面粘附的培養(yǎng)基成分。將得到的細(xì)胞在抽真空條件下冰凍干燥至恒重,測(cè)定細(xì)胞干重 (Dry cell weight,DCW)。
1.4.2 產(chǎn)物濃度的測(cè)定
4-羥基丁酸的分析方法參考文獻(xiàn)[21],取菌液離心后上清進(jìn)行冰干、酯化和氣相色譜分析。
以E. coli MG1655基因組DNA為模板,表1中相應(yīng)引物用 fastpfu DNA聚合酶對(duì)基因 pncB和nadE進(jìn)行PCR擴(kuò)增。反應(yīng)程序:95 ℃預(yù)變性2 min;95 ℃變性20 s,58 ℃復(fù)性20 s,72 ℃延伸45/30 s,30個(gè)循環(huán);最后72 ℃延伸8 min。
將pncB/nadE基因的PCR回收片段和表達(dá)載體pEn分別進(jìn)行 Sac Ⅰ/Xba Ⅰ酶切,連接后化學(xué)法轉(zhuǎn)入E. coli DH5α感受態(tài)中,陽性克隆經(jīng)EcoR Ⅰ/Xho Ⅰ雙酶切,得到目的基因表達(dá)單元和載體骨架兩條帶 (圖2),證明質(zhì)粒pEnpncB和pEnnadE構(gòu)建成功。再用Avr Ⅱ/Xho Ⅰ酶切pEnnadE,回收nadE表達(dá)單元一段,將其插入經(jīng)Nhe Ⅰ/Xho Ⅰ酶切的pEnpncB中,因?yàn)锳vr Ⅱ和Nhe Ⅰ是同尾酶,因此兩片段可相連得到質(zhì)粒pEnpncBnadE。經(jīng)Xba Ⅰ酶切驗(yàn)證正確后與質(zhì)粒 pZL-dhaT-aldD一起經(jīng)電擊法轉(zhuǎn)入E. coli S17-1感受態(tài),獲得重組菌株。已有報(bào)道,基于 pBBR1MCS2所構(gòu)建的質(zhì)粒能夠與pMD19-T Simple的衍生質(zhì)粒共存[26],因此,這里的pZL-dhaT-aldD和 pEnpncBnadE能夠協(xié)同作用。進(jìn)一步質(zhì)粒提取證實(shí)了兩質(zhì)粒的相容性。
以E. coli S17-1 (pZL-dhaT-aldD) 為參照,驗(yàn)證PncB-NadE表達(dá)對(duì)1,4-丁二醇轉(zhuǎn)化的影響 (表2)。
野生型大腸桿菌也能由 1,4-丁二醇合成少量4-羥基丁酸,卻不能進(jìn)一步代謝利用 4-羥基丁酸[16,21,27]。另外,基因dhaT和 (或) aldD在嗜水氣假單胞菌 Aeromonas hydrophila 4AK4與 E. coli S17-1中顯示出相似的作用效果,而 A. hydrophila 4AK4 (pZL-dhaT-aldD) 的55 h發(fā)酵結(jié)果顯示4-羥基丁酸積累量不斷上升[21]??赏茢郋. coli S17-1重組菌的 4-羥基丁酸積累量以及 1,4-丁二醇的消耗量在發(fā)酵過程中應(yīng)是逐漸增大的,因此,這里以48 h發(fā)酵后的結(jié)果作為討論依據(jù)。
對(duì)照組E. coli S17-1 (pZL-dhaT-aldD) 由10 g/L的 1,4-丁二醇合成 4.31 g/L 4-羥基丁酸,轉(zhuǎn)化率(mol/mol%) 為36.44%。而同條件下,E. coli S17-1 (pZL-dhaT-aldD,pEnpncBnadE) 則得到4.87 g/L 4-羥基丁酸,1,4-丁二醇的轉(zhuǎn)化率為 41.18%,比對(duì)照組提高13.03%。計(jì)算單位細(xì)胞量的4-羥基丁酸產(chǎn)率能夠更直接說明PncB-NadE的作用。對(duì)照組單位細(xì)胞產(chǎn)率為1.32 g/g,實(shí)驗(yàn)組相應(yīng)值為1.86 g/g。綜上結(jié)果,PncB-NadE的共表達(dá)明顯促進(jìn)4-羥基丁酸的合成。
盡管如此,仍有近60%的1,4-丁二醇未被利用,這是因?yàn)榇济摎涿?(DhaT) 對(duì)1,4-丁二醇活性不高,且發(fā)酵后期會(huì)進(jìn)一步減弱[21],所以,需要篩選出更有效的酶來催化反應(yīng)以充分利用1,4-丁二醇。
此外,PncB-NadE的表達(dá)同時(shí)導(dǎo)致細(xì)胞干重下降??赡苁且?yàn)榘麅?nèi)NAD的增加導(dǎo)致氧化還原態(tài)失衡,影響了細(xì)胞的正常代謝。細(xì)胞總量減少是轉(zhuǎn)化率不能充分提高的另一原因,可通過添加葡萄糖等有利碳源來刺激細(xì)胞生長(zhǎng),進(jìn)而增加 1,4-丁二醇利用率。
圖2 質(zhì)粒pEnpncB、pEnnadE、pEnpncBnadE的酶切圖譜Fig. 2 Characterization of pEnpncB, pEnnadE, pEnpncBnadE. M: 1 kb DNA marker; p: pEnpncB digested with EcoR I/Xho I, two brands were 1 532 bp and 2 706 bp; n: pEnnadE digested with EcoR I/Xho I, two brands were 1 173 bp and 2 706 bp; pn: pEnpncBnadE digested with Xba I, two brands were 1 155 bp and 4 238 bp.
表2 重組菌搖瓶發(fā)酵生產(chǎn)4-羥基丁酸Table 2 Production of 4-hydroxybutyric acid by E. coli S17-1 (pZL-dhaT-aldD, pEnpncBnadE) and E. coli S17-1 (pZL-dhaT-aldD)
酶循環(huán)反應(yīng)中吸光度-時(shí)間曲線的斜率正比于提取液中 NAD (H) 的濃度。結(jié)果證明 PncB-NadE的表達(dá)確實(shí)能提高胞內(nèi)NAD (H) 濃度 (圖3)。結(jié)合表2計(jì)算單位質(zhì)量細(xì)胞內(nèi)NAD和NADH含量變化,E. coli S17-1 (pZL-dhaT-aldD,pEnpncBnadE) 比E. coli S17-1 (pZL-dhaT-aldD) 中NAD和NADH含量分別提高2.04倍和2.74倍。
圖3 重組菌NAD (H) 含量測(cè)定Fig. 3 Relative concentration of NAD (H) in different recombinants. 1: NAD (E. coli S17-1 (pZL-dhaT-aldD, pEnpncBnadE)); 2: NAD (E. coli S17-1 (pZL-dhaT-aldD)); 3: NADH (E. coli S17-1 (pZL-dhaT-aldD, pEnpncBnadE)); 4: NADH (E. coli S17-1 (pZL-dhaT-aldD)).
REFERENCES
[1] Ye F, Dong FW. Review of analysis methods of γ-hydroxybutyrate and its derivatives. Merch Qual, 2011(S3): 156?157.葉峰, 董鳳偉. γ-羥基丁酸及其相關(guān)物分析研究綜述.商品與質(zhì)量, 2011(S3): 156?157.
[2] Kaufman EE, Nelson T. An overview of γ-hydroxybutyrate catabolism: the role of the cytosolic NADP+-dependent oxidoreductase EC 1.1.1.19 and of a mitochondrial hydroxyacid-oxoacid transhydrogenase in the initial, rate-limiting step in this pathway. Neurochem Res, 1991, 16(9): 965?974.
[3] Wong CGT, Chan KFY, Gibson KM, et al. Gamma-hydroxybutyric acid: neurobiology and toxicology of a recreational drug. Toxicol Rev, 2004, 23(1): 3?20.
[4] Gibson KM, Hoffmann GF, Hodson AK, et al. 4-Hydroxybutyric acid and the clinical phenotype of succinic semialdehyde dehydrogenase deficiency, an inborn error of GABA metabolism. Neuropediatrics, 1998, 29(1): 14?22.
[5] Drasbek KR, Christensen J, Jensen K. Gamma-hydroxybutyrate-a drug of abuse. Acta Neurol Scand, 2006, 114(3):145?156.
[6] Efe C, Straathof AIJ, van der Wielen LAM. Options for biochemical production of 4-hydroxybutyrate and its lactone as a substitute for petrochemical production. Biotechnol Bioeng, 2008, 99(6): 1392?1406.
本次計(jì)算中取泊松比為υ=0.35,Ib=0.7,rm=12.5。室內(nèi)試驗(yàn)測(cè)得的主固結(jié)完成后的剪切模量為G=0.664 MPa。
[7] Saito Y, Doi Y. Microbial synthesis and properties of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans. Int J Biol Macromol, 1994, 16(2): 99?104.
[8] Saito Y, Nakamura S, Hiramitsu M, et al. Microbial synthesis and properties of poly(3-hydroxybutyrateco-4-hydroxybutyrate). Polym Int, 1996, 39(3): 169?174.
[9] Ishida K, Wang Y, Inoue Y. Comonomer unit composition and thermal properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s biosynthesized by Ralstonia eutropha. Biomacromolecules, 2001, 2(4): 1285?1293.
[10] Doi Y, Kanesawa Y, Kunioka M, et al. Biodegradation of microbial copolyesters: poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Macromolecules, 1990, 23(1): 26?31.
[11] Mukai K, Doi Y, Sema Y, et al. Substrate specificities in hydrolysis of polyhydroxyalkanoates by microbial esterases. Biotechnol Lett, 1993, 15(6): 601?604.
[12] S?hling B, Gottschalk G. Molecular analysis of the anaerobic succinate degradation pathway in Clostridium kluyveri. J Bacteriol, 1996, 178(3): 871?880.
[13] Berg IA, Kockelkorn D, Buckel W, et al. A 3-hydroxypropionate/4-hydroxybutyrate autotrophic carbon dioxide assimilation pathway in Archaea. Science, 2007, 318(5857): 1782?1786.
[14] Hiramitsu M, Koyama N, Doi Y. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Alcaligenes latus. Biotechnol Lett, 1993, 15(5): 461?464.
[15] Kunioka M, Kawaguchi Y, Doi Y. Production of biodegradable copolyesters of 3-hydroxybutyrate and 4-hydroxybutyrate by Alcaligenes eutrophus. Appl Microbiol Biotechnol, 1989, 30(6): 569?573.
[16] Hein S, S?hling B, Gottschalk G, et al. Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli. FEMS Microbiol Lett, 1997, 153(2): 411?418.
[17] Liu SJ, Steinbüchel A. A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli. Appl Environ Microbiol, 2000, 66(2): 739?743.
[18] Efe C, Straathof AJJ, van der Wielen LAM. Options for biochemical production of 4-hydroxybutyrate and its lactone as a substitute for petrochemical production. Biotechnol Bioeng, 2008, 99(6): 1392?1406.
[19] Qiu YN. Overview of the production method and application of γ-butyrolactone (γ-BL). Sci-Tech Info Dev Econ, 2008, 18(34): 83?84.邱婭男. γ-丁內(nèi)酯的生產(chǎn)方法及其應(yīng)用綜述. 科技情報(bào)開發(fā)與經(jīng)濟(jì), 2008, 18(34): 83?84.
[20] Jing S, Guan LJ. Introduction about properties of 1,4-butanediol and processing technique. Chem Eng Equi, 2011(2): 137?138.景森, 關(guān)麗娟. 1,4-丁二醇性能及各種生產(chǎn)工藝介紹. 化學(xué)工程與裝備, 2011(2): 137?138.
[21] Zhang L, Shi ZY, Wu Q, et al. Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms. Appl Microbiol Biotechnol, 2009, 84(5): 909?916.
[22] Zhang YP, Huang ZH, Du CY, et al. Introduction of an NADH regeneration system into Klebsiella oxytoca leads to an enhanced oxidative and reductive metabolism of glycerol. Metab Eng, 2009, 11(2): 101?106.
[23] Sánchez AM, Bennett GN, San KY. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metab Eng, 2005, 7(3): 229?239.
[24] Nielsen DR, Leonard E, Yoon SH, et al. Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng, 2009, 11(4/5):262?273.
[25] Heuser F, Schroer K, Lütz S, et al. Enhancement of the NAD(P)(H) pool in Escherichia coli for biotransformation. Eng Life Sci, 2007, 7(4): 343?353.
[26] Li ZJ, Shi ZY, Jian J, et al. Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli. Metab Eng, 2010, 12(4): 352?359.
[27] Yim H, Haselbeck R, Niu W, et al. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol, 2011, [Epub ahead of print].