Müller Christina A, Dennig Alexander, Welters Tim, Winkler Till, Ruff Anna Joelle, Hummel Werner, Gröger Harald, Schwaneberg Ulrich
Institute of Biotechnology, RWTH Aachen, Worringerweg 3, 52074 Aachen, Germany.
Institute of Molecular Enzyme Technology at the Heinrich-Heine-University of Düsseldorf, Research Centre Jülich, Stetternicher Forst, 52426 Jülich, Germany.
J Biotechnol. 2014 Dec 10;191:196-204. doi: 10.1016/j.jbiotec.2014.06.001. Epub 2014 Jun 9.
Biocascades allow one-pot synthesis of chemical building blocks omitting purification of reaction intermediates and expenses for downstream processing. Here we show the first whole cell double oxidation of n-heptane to produce chiral alcohols and heptanones. The concept of an artificial operon for co-expression of a monooxygenase from Bacillus megaterium (P450 BM3) and an alcohol dehydrogenase (RE-ADH) from Rhodococcus erythropolis is reported and compared to the widely used two-plasmid or Duet-vector expression systems. Both catalysts are co-expressed on a polycistronic constructs (single mRNA) that reduces recombinant DNA content and metabolic burden for the host cell, therefore increasing growth rate and expression level. Using the artificial operon system, the expression of P450 BM3 reached 81mgg(-1) cell dry weight. In addition, in situ cofactor regeneration through the P450 BM3/RE-ADH couple was enhanced by coupling to glucose oxidation by E. coli. Under optimized reaction conditions the artificial operon system displayed a product formation of 656mgL(-1) (5.7mM) of reaction products (heptanols+heptanones), which is 3-fold higher than the previously reported values for an in vitro oxidation cascade. In conjunction with the high product concentrations it was possible to obtain ee values of >99% for (S)-3-heptanol. Coexpression of a third alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) in the same host yielded complete oxidation of all heptanol isomers. Introduction of a second ADH enabled further to utilize both cofactors in the host cell (NADH and NADPH) which illustrates the simplicity and modular character of the whole cell oxidation concept employing an artificial operon system.
生物级联反应可实现一锅法合成化学结构单元，省去了反应中间体的纯化步骤以及下游加工的费用。在此，我们展示了首例将正庚烷全细胞双氧化以生产手性醇和庚酮的过程。报道了用于共表达巨大芽孢杆菌的单加氧酶（P450 BM3）和红平红球菌的醇脱氢酶（RE-ADH）的人工操纵子概念，并将其与广泛使用的双质粒或双顺反子载体表达系统进行了比较。两种催化剂在多顺反子构建体（单个mRNA）上共表达，这降低了重组DNA含量和宿主细胞的代谢负担，从而提高了生长速率和表达水平。使用人工操纵子系统，P450 BM3的表达量达到81mg g(-1)细胞干重。此外，通过大肠杆菌的葡萄糖氧化偶联，P450 BM3/RE-ADH对的原位辅因子再生得到增强。在优化的反应条件下，人工操纵子系统的反应产物（庚醇+庚酮）生成量为656mg L(-1)（5.7mM），比先前报道的体外氧化级联反应值高3倍。结合高产物浓度，(S)-3-庚醇的对映体过量值（ee值）可达到>99%。在同一宿主中共同表达来自短乳杆菌的第三种醇脱氢酶（Lb-ADH）可使所有庚醇异构体完全氧化。引入第二种ADH还能使宿主细胞利用两种辅因子（NADH和NADPH），这说明了采用人工操纵子系统的全细胞氧化概念的简单性和模块化特点。
