Klatte Stephanie, Wendisch Volker F
Chair of Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitaetsstr. 25, 33615, Bielefeld, Germany.
Microb Cell Fact. 2015 Jan 23;14:9. doi: 10.1186/s12934-014-0189-x.
In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed.
The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst.
The replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.
在白色生物技术中，生物催化是实现非天然化合物化学功能化的关键技术。通过质粒过量表达乙醇脱氢酶、L-丙氨酸依赖性转氨酶和丙氨酸脱氢酶，可在全细胞生物转化中实现醇类的氧化还原自足胺化反应。本文优化了（《生物有机与药物化学》22:5578 - 5585，2014）中提出的全细胞生物催化剂的条件，并分析了L-丙氨酸在1,10 - 癸二醇高效胺功能化生成1,10 - 二氨基癸烷中的作用。
对醇类胺功能化反应级联中的酶进行了体外表征，以寻找高效反应的最佳条件。来自紫色色杆菌的转氨酶TaCv的催化效率比来自河流弧菌的转氨酶TaVf高3倍，且在37°C时产量有所提高。在42°C时，TaCv活性更高，这与热稳定的乙醇脱氢酶和丙氨酸脱氢酶相匹配，使1,10 - 二氨基癸烷的生成速率提高了4倍。为研究L-丙氨酸在全细胞生物转化中的作用，改变L-丙氨酸浓度，在10 mM 1,10 - 癸二醇和100 mM NH4Cl恒定的条件下测试1,10 - 二氨基癸烷的形成。未添加L-丙氨酸时，仅观察到5.6%的二胺产物。与醇等摩尔浓度的L-丙氨酸可实现94%的产物形成，但更高浓度的L-丙氨酸可实现100%的产物形成。L-丙氨酸被大肠杆菌生物催化剂消耗，可能是由于丙酮酸分解代谢，因为积累了高达16 mM的乙酸盐。使用无法分解丙酮酸的大肠杆菌菌株YYC202/pTrc99a - ald - adh - ta Cv进行生物转化，转化率为42 mol-%。仅缺少丙酮酸氧化酶PoxB的大肠杆菌菌株进行生物转化时，1,10 - 癸二醇的胺化反应也有类似程度的降低，表明在使用该全细胞生物催化剂进行醇类氧化还原自足胺化反应过程中，PoxB将丙酮酸氧化脱羧为乙酸盐是丙酮酸分解代谢的主要原因。
在人工操纵子ald - adh - ta中，用在42°C时活性更高的TaCv替代转氨酶TaVf，可改善全细胞生物转化中醇类的胺化反应。添加L-丙氨酸是100%产物形成所必需的，L-丙氨酸可能通过提供维持能量而被大肠杆菌通过丙酮酸分解代谢消耗。代谢工程研究表明，在所选生物转化条件下丙酮酸分解代谢主要通过PoxB将其氧化脱羧为乙酸盐来进行。
