偶联基于酮还原酶的NADH再生系统一锅法酶催化合成L-2-氨基丁酸

以廉价易得的L-苏氨酸为原料,利用在大肠杆菌中重组表达的苏氨酸脱氨酶和亮氨酸脱氢酶,并偶联基于酮还原酶的NADH再生系统一锅法制备L-2-氨基丁酸。以L-2-氨基丁酸的产率为指标,考察了一锅法酶催化制备L-2-氨基丁酸的最适p H、L-苏氨酸浓度及异丙醇浓度。在最适p H 7.5~8.0,L-苏氨酸浓度50g/L,添加5%的异丙醇及0.5g/L NAD+,分别加入0.6g/L苏氨酸脱氨酶、2g/L亮氨酸脱氢酶及2g/L酮还原酶,反应20h,可实现L-2-氨基丁酸的摩尔产率为99%,产量为43g/L。该结果为L-2-氨基丁酸的制备提供了一种新的思路。     

三酶级联催化L-苏氨酸生产L-2-氨基丁酸

L-2-氨基丁酸(L-ABA)是一种重要的化工原材料和手性医药中间体,为了实现L-ABA的高效生产,本研究在大肠杆菌EscherichiacoliBL21(DE3)中分别表达大肠杆菌来源的苏氨酸脱氨酶(Threonine deaminase,TD)、苏云金芽孢杆菌来源的亮氨酸脱氢酶(Leucine dehydrogenase,LDH)和博伊丁假丝酵母来源的甲酸脱氢酶(Formatedehydrogenase,FDH),构建体外级联酶催化反应实现L-苏氨酸向L-ABA的转化,体系中TD、LDH和FDH添加最适比例为1∶1∶0.2。为了简化生产工艺,将3种酶在一株菌E. coli 3FT+L中共表达并实现上述配比,在30 L发酵罐中用E. coli 3FT+L全细胞转化12 h,L-ABA的产量为68.5 g/L,底物L-苏氨酸的摩尔转化率达到99.0%。该工艺路线绿色高效,为未来大规模生产L-ABA提供借鉴。     

亮氨酸脱氢酶偶联NADH再生体系合成L-2-氨基丁酸

文中以大肠杆菌BL21(DE3)为宿主,构建两株分别共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/甲酸脱氢酶(FDH,来源水生弯杆菌)和亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/醇脱氢酶(ADH,来源红球菌)的重组大肠杆菌。通过偶联两种不同NADH再生体系,以L-苏氨酸为起始原料,利用苏氨酸脱氨酶(L-TD)与LDH-FDH或LDH-ADH一锅法合成L-2-氨基丁酸,并对LDH-FDH工艺和LDH-ADH工艺进行对比优化。LDH-FDH工艺的最适反应pH为7.5,最适反应温度为35℃,通过加入50 g/L甲酸铵、0.3 g/L NAD+、10%LDH-FDH粗酶液(V/V)和7 500 U/L的L-TD酶液,对L-苏氨酸进行分批补加,以便控制2-丁酮酸浓度小于15 g/L,反应28 h,实现了L-2-氨基丁酸的产量为161.8 g/L,产率97%。LDH-ADH工艺的最适pH为8.0,最适反应温度为35℃,通过加入0.3 g/L NAD+、10%LDH-ADH粗酶液(V/V)及7 500 U/L的L-TD酶液,分批补加L-苏氨酸及1.2倍摩尔量异丙醇,以便控制2-丁酮酸浓度小于15g/L,且每生成约40g/L的L-2-氨基丁酸,抽真空去除丙酮,反应24h,实现了L-2-氨基丁酸的产量为119.6 g/L,产率98%。文中所采用的工艺及结果可为L-2-氨基丁酸的工业化提供一定的参考依据。     

基于双酶级联协调表达策略高效催化合成D-甘露醇北大核心CSCD

D-甘露醇(D-mannitol)作为合成抗肿瘤药和免疫刺激剂的重要前体被广泛应用于制药和医疗等行业,酶法合成D-甘露醇反应成本昂贵无法满足工业化生产。本研究首先筛选关键酶获得较优性能的甘露醇脱氢酶Lp MDH和用于辅因子NADH再生的葡萄糖脱氢酶Ba GDH,在大肠杆菌(Escherichia coli)BL21(DE3)中共表达,实现了基于双酶级联反应催化底物D-果糖合成D-甘露醇,D-甘露醇的初步摩尔转化率为59.7%。针对双酶级联催化反应中辅酶再生用酶与催化用酶表达量不协调的问题,通过增加Bagdh拷贝量来提高辅因子循环能力,获得了双酶催化速率平衡的重组大肠杆菌E.coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh。进一步对重组菌的全细胞转化条件进行优化,确定了最适转化条件为反应温度30℃,初始pH值6.5,菌体量OD600=30,底物D-果糖100.0 g/L,辅底物葡萄糖与底物1︰1摩尔当量。于最优转化条件下5 L发酵罐转化24 h,D-甘露醇的最高产量为81.9g/L,摩尔转化率为81.9%。本研究提供了一种绿色、高效生物催化生产D-甘露醇的方法,为实现其规模化生产奠定了基础,同时也对其他相关稀有糖醇的研究具有指导意义。     

一株重组大肠杆菌/pET-28a-lpgad的构建及其高效生产γ-氨基丁酸转化条件的优化

为实现微生物法高效率生产γ-氨基丁酸(GABA),从一株经多次诱变筛选的具有较高谷氨酸脱羧酶(GAD)活力植物乳杆菌GB 01-21全基因组DNA中PCR扩增获得GAD酶基因lpgad,构建重组质粒pET-28a-lpgad,在大肠杆菌E.coli BL21(DE3)中高效诱导表达。并采用Ni柱亲和层析纯化获得重组GAD,并对其酶学性质进行初步研究,为改良转化工艺提高GABA产量提供可靠理论依据。结果显示,重组大肠杆菌中GAD酶活显著提高,可达8.53 U/mg,是植物乳杆菌GB 01-21中GAD酶活的4.24倍。将该重组菌应用于转化L-谷氨酸生产GABA,5 L发酵罐水平转化24 h产量可达143.5 g/L,摩尔转化率为97.32%,是植物乳杆菌GB 01-21的2.19倍。纯化后酶学性质进行初步研究表明:其最适pH为4.8;最适温度为37℃;Ca2+、Mg2+对其有较强的激活作用,将上述实验结果用于转化条件的优化,最终5 L发酵罐上进行转化实验,批次添加底物L-谷氨酸共600 g,转化24 h,GABA累计浓度可达204.5 g/L,摩尔转化率为97.92%,与最初转化条件相比,GABA浓度提高了42.5%,为其工业化应用打下了良好的基础。     

基于多酶级联协调表达策略高效催化合成 (S)-2-羟基丁酸

2-羟基丁酸 (2-hydroxybutyric acid,2-HBA) 是合成生物可降解材料和各种药物的重要中间体,化学法合成的外消旋2-HBA需要去消旋才能获得光学纯对映异构体,应用于工业。文中通过在大肠杆菌Escherichia coli BL21(DE3) 中共表达苏氨酸脱氨酶 (Threonine deaminase,TD)、l-乳酸脱氢酶 (l-lactate dehydrogenase,LDH)和甲酸脱氢酶 (Formate dehydrogenase,FDH),构建 (S)-2-HBA的合成途径及其辅因子NADH的循环系统,实现了基于三酶级联反应催化底物l-苏氨酸合成 (S)-2-HBA。为了解决多酶级联催化反应中中间产物2-酮丁酸的生成速率和消耗率不匹配的问题,文中通过启动子工程策略来调控TD和FDH的表达水平,获得了多酶催化速率平衡的重组大肠杆菌P21285FDH-T7V7827。在5 L发酵罐水平,全细胞催化反应16 h,(S)-2-HBA的最高产量为143 g/L,摩尔转化率为97%,为迄今报道的最高产量的1.83倍,使其具有较强的工业化应用潜力。此外,结果表明,在单细胞中构建可调节的多酶协调表达系统对生物催化制备羟基酸类化合物具有重要意义。     

固定化天冬氨酸酶制备(R)-3-氨基丁酸

从芽孢杆菌Bacillus sp.YM55-1基因组中克隆得到天冬氨酸酶基因,以pET-28a(+)为载体构建天冬氨酸酶基因的表达载体pET-28a(+)-Asp,将天冬氨酸酶基因进行定点突变,在E.coli BL21(DE3)系统中实现了天冬氨酸酶的异源表达。利用重组的天冬氨酸酶,以氨水、(NH_4)_2SO_4为辅料,将底物巴豆酸转化为(R)-3-氨基丁酸。将天冬氨酸酶的工程菌制备成固定化细胞,通过反应条件的优化研究,提高底物的转化率。结果表明:天冬氨酸酶最适pH为9.0,最适反应温度为40℃。在此反应条件下,加入30 g/L固定化细胞,转化22 h,(R)-3-氨基丁酸质量浓度达到220 g/L,对映体过量值e.e._s≥99.95%,底物转化率达到98%,固定化细胞重复使用次数不低于24次。     

应用合成生物学策略构建全细胞生物催化剂合成(S)-乙偶姻

目的:在大肠杆菌宿主中过量表达丁二酮还原酶(DAR),同时构建辅酶NADH原位再生系统,利用全细胞高效催化丁二酮不对称还原合成(S)-乙偶姻。方法:PCR克隆多黏芽孢杆菌(Paenibacillus polymyxa) dar基因连到质粒pETDuet-1,转化至大肠杆菌(Escherichia coli) BL21(DE3),构建重组菌E. coli BL21(DE3)-DAR;通过Hi Trap TALON柱亲和层析纯化表达产物DAR酶蛋白,测定DAR的比酶活和分子动力学参数。在重组菌E. coli BL21(DE3)-DAR中构建辅酶NADH原位再生系统,协同表达枯草芽孢杆菌(Bacillus subtilis)的葡萄糖脱氢酶(GDH),构建重组菌E. coli BL21(DE3)-DAR/GDH,并以此重组菌为全细胞生物催化剂,优化催化条件,提高(S)-乙偶姻的产量和产率。结果:获得重组工程菌E. coli BL21(DE3)-DAR和E. coli BL21(DE3)-DAR/GDH。DAR以NADH为辅酶还原丁二酮的米氏常数Km、最大催化速率Vmax、催化常数Kcat分别为2. 59mmol/L、1. 64μmol/(L·min·mg)、12. 3/s,还原丁二酮生成(S)-乙偶姻光学的纯度为95. 86%,具有较好的催化效率和立体异构体选择性。构建辅酶NADH原位再生系统后,重组菌E. coli BL21(DE3)-DAR/GDH可高效催化丁二酮合成乙偶姻。在最优催化条件下分批补料,乙偶姻产量达51. 26g/L,转化率为81. 37%,生产速率为5. 13g/(L·h)。结论:使用非手性化合物原料丁二酮生产高附加值的手性化合物(S)-乙偶姻,以重组菌为全细胞生物催化剂合成(S)-乙偶姻,不需额外添加昂贵的辅酶,具有较高的生产应用价值。     

生物转化法制备L-天冬酰胺

探索生物转化法制备L-天冬酰胺的技术与工艺。通过分子生物学方法,克隆来源于大肠杆菌(Escherichia coli, E.coli)JM109的天冬酰胺合成酶A基因asnA,并于E. coli BL21(DE3)中表达,利用构建的E.coli基因工程菌E.coli BL21(DE3)/pET28a(+)-asnA全细胞高密度催化L-天冬氨酸生产L-天冬酰胺,以PITC柱前衍生-高效液相检测底物和产物。表达的蛋白质分子质量约为37kDa,与预期大小相符,比酶活力为1786.6U/g。L-天冬氨酸转化率为95.8%,L-天冬酰胺产量可达126.5g/L,生产速率为15.81g/(L·h)。结果表明,已成功构建高效表达天冬酰胺合成酶A基因工程菌株,并用于催化L-天冬氨酸转化生产L-天冬酰胺,解决了L-天冬酰胺生物转化生产工艺中ATP成本过高的难题,为L-天冬酰胺制备提供新的绿色途径。     

亮氨酸脱氢酶与葡萄糖脱氢酶高效共表达制备L-叔亮氨酸北大核心CSCD

【目的】通过不同双基因共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶基因在大肠杆菌中表达影响的研究,获得具有高辅酶再生效率的双酶共表达重组生物催化剂,实现L-叔亮氨酸"一锅法"高效不对称合成。【方法】以来自于蜡状芽孢杆菌(Bacillus cereus)的亮氨酸脱氢酶(LDH)和来自芽孢菌属(Bacillus sp.)的葡萄糖脱氢酶(GDH)为模板,考察单质粒共表达,双质粒共表达和融合表达等3种共表达策略对重组细胞中亮氨酸脱氢酶和葡萄糖脱氢酶活的影响,比较不同酶活比例和不同催化剂形式对三甲基丙酮酸不对称还原制备L-叔亮氨酸效率的影响。【结果】研究发现不同共表达策略对亮氨酸脱氢酶和葡萄糖脱氢酶的影响存在明显差异。亮氨酸脱氢酶在不同策略下均能够正常表达,而葡萄糖脱氢酶在融合表达时没有活力,当C端含有组氨酸标签时,表达蛋白活性低。通过表达优化,获得3株亮氨酸脱氢酶和葡萄糖脱氢酶高效表达且具有不同酶活比例的重组菌。比较粗酶液和全细胞形式下的催化效率,发现酶活比例及催化剂形式对不对称还原反应效率具有重要影响。确定单质粒串联表达C端不含His标签重组菌E.coli BL21/p ET28a-L-SD-AS-G为最佳催化剂,以粗酶液进行转化时,完全转化0.5 mol/L底物所需菌体量为15 g/L,辅酶量为0.1 mmol/L。【结论】采用单质粒共表达策略,成功构建出1株具有较高亮氨酸脱氢酶和葡萄糖脱氢酶活性的重组菌,实现高效催化TMP合成L-Tle。     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in Escherichia coli

1. Washed-cell suspensions of Escherichia coli, incubated at the optimum pH of 6.4 and with a saturating substrate concentration of approx. 10mm, convert dl-1-aminopropan-2-ol into aminoacetone at a rate of approx. 4.0mmumoles/mg. dry wt. of cells/min. at 30 degrees . 2. Mg(2+), Mn(2+), Co(2+), Zn(2+), Ca(2+), K(+) and NH(4) (+), as sulphates, and EDTA have no effect on this rate, although Cu(2+) inhibits and Fe(2+) activates to some extent. 3. Conditions of growth markedly affect the rate of aminoacetone production by cell suspensions. 4. Dialysed cell-free extracts of E. coli exhibit 1-aminopropan-2-ol-dehydrogenase activity, the enzyme having optimum activity at pH7.0, a requirement for NAD(+) and K(+), and a K(m) for the amino alcohol substrate of 0.8mm, calculated for a single enantiomorph. 5. Under optimum conditions 1-aminopropan-2-ol dehydrogenase forms aminoacetone at rate of approx. 3.0mmumoles/mg. of protein/min. at 37 degrees . The enzyme is only slightly inhibited by dl-3-hydroxybutyrate and dl-2-hydroxy-2-phenylethyl-amine. 6. l-Threonine-dehydrogenase activity is exhibited by both whole cells and cell-free extracts. Whole cells produce aminoacetone from l-threonine more slowly than they do from dl-1-aminopropan-2-ol, whereas the situation is reversed in cell-free extracts. Both kinetic evidence, and the fact that synthesis of 1-aminopropan-2-ol dehydrogenase, but not of threonine dehydrogenase, is repressed by compounds such as glucose and pyruvate, provide evidence that the amino alcohol is oxidized by a specific enyme. 7. The metabolic role of 1-aminopropan-2-ol dehydrogenase is discussed.     

天蓝色链霉菌海藻糖合酶的异源表达、活性分析及重组菌全细胞转化合成海藻糖的条件优化

从天蓝色链霉菌Streptomyces coelicolor克隆得到海藻糖合酶基因 (ScTreS),在大肠杆菌Escherichia coli BL21(DE3) 中进行了异源表达,通过 Ni-NTA 亲和柱对表达产物进行分离纯化得到纯酶,经 SDS-PAGE 测定其分子量约为62.3 kDa。研究其酶学性质发现该酶最适温度35 ℃;最适pH 7.0,对酸性条件比较敏感。通过同源建模和序列比对分析,对该基因进行定点突变。突变酶K246A比酶活比野生酶提高了1.43倍,突变酶A165T相对提高了1.39倍,海藻糖转化率分别提高了14%和10%。利用突变体重组菌K246A进行全细胞转化优化海藻糖的合成条件并放大进行5 L罐发酵,结果表明：在麦芽糖浓度300 g/L、初始反应温度和pH分别为35 ℃和7.0的条件下,转化率最高达到71.3%,产量为213.93 g/L;当底物浓度增加到700 g/L时,海藻糖产量仍可达到465.98 g/L。     

Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound

The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212bp nucleotide fragment (oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences. Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of oye was constructed. Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of C. macedoniensis. (6R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638 mM (98.2 mg ml(-1)), the a molar yield being 96.9%. The asymmetric reduction of KIP to (6R)-levodione with E. coli cells, which co-expressed both oye and the glucose dehydrogenase gene (gdh), as a catalyst was investigated. The (6R)-levodione formed amounted to 627 mM (96.6 mg ml(-1)), the a molar yield being 95.4%. Since the use of E. coli BL21 (DE3) cells co-expressing oye and gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6R)-levodione.     

Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.     

Construction and co-expression of plasmid encoding xylitol dehydrogenase and a cofactor regeneration enzyme for the production of xylitol from D-arabitol

The biotransformation of D-arabitol into xylitol was investigated with focus on the conversion of D-xylulose into xylitol. This critical conversion was accomplished using Escherichia coli to co-express a xylitol dehydrogenase gene from Gluconobacter oxydans and a cofactor regeneration enzyme gene which was a glucose dehydrogenase gene from Bacillus subtilis for system 1 and an alcohol dehydrogenase gene from G. oxydans for system 2. Both systems efficiently converted D-xylulose into xylitol without the addition of expensive NADH. Approximately 26.91 g/L xylitol was obtained from around 30 g/L D-xylulose within system 1 (E. coli Rosetta/Duet-xdh-gdh), with a 92% conversion yield, somewhat higher than that of system 2 (E. coli Rosetta/Duet-xdh-adh, 24.9 g/L, 85.2%). The xylitol yields for both systems were more than 3-fold higher compared to that of the G. oxydans NH-10 cells (7.32 g/L). The total turnover number (TTN), defined as the number of moles of xylitol formed per mole of NAD(+), was 32,100 for system 1 and 17,600 for system 2. Compared with that of G. oxydans NH-10, the TTN increased by 21-fold for system 1 and 11-fold for system 2, hence, the co-expression systems greatly enhanced the NADH supply for the conversion, benefiting the practical synthesis of xylitol.     

Catabolism of threonine in mammals by coupling of l-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase

There is doubt about the l-threonine 3-dehydrogenase (EC 1.1.1.103) and threonine aldolase (EC 2.1.2.1) catabolic pathways of l-threonine in mammals which are believed to produce aminoacetone and glycine plus acetaldehyde, respectively. l-Threonine 3-dehydrogenase in disrupted guinea-pig liver mitochondria was investigated in a reaction mixture containing l-threonine without and with CoA and oxaloacetate; l-[U-¹⁴C]threonine was included in four similar experiments for autoradiograms. Threonine aldolase was examined in similar mitochondria from liver and kidney. CoA reduced the aminoacetone formed from l-threonine to 10–14% and CoA plus oxaloacetate produced citrate (from CoASAc) in approximately equal amounts to the decrease in aminoacetone. Autoradiograms confirmed the decrease in aminoacetone with the simultaneous appearance of citrate and glycine. No evidence was obtained that threonine aldolase catabolised l-threonine at the concentration used to assay the dehydrogenase. It is concluded that 2-amino-3-oxobutyrate (precursor of aminoacetone), which is produced from l-threonine by l-threonine 3-dehydrogenase, undergoes CoA-dependent cleavage to glycine and CoASAc by 2-amino-3-oxobutyrate-CoA ligase. The results suggest that the coupling of these enzymes provides a new pathway for the catabolism of threonine in mammals.