Dietrich Jeffrey A, Shis David L, Alikhani Azadeh, Keasling Jay D
UCSF-UCB Joint Graduate Group in Bioengineering, Berkeley, CA 94720, USA.
ACS Synth Biol. 2013 Jan 18;2(1):47-58. doi: 10.1021/sb300091d. Epub 2012 Nov 14.
Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3' of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.
代谢工程的持续进步使得越来越多的小分子成为微生物生产的目标。然而，途径产量和生产率往往不尽人意，由于大多数小分子难以筛选且其生物合成无法提高宿主适应性，菌株改良仍然是一个长期挑战。在这项工作中，我们开发了一种通用方法，利用基于转录因子的生物传感器来筛选或选择改良的小分子生物合成。利用小分子诱导型启动子下游的四环素抗性基因，宿主的抗生素抗性以及生长速率与生长培养基中的小分子浓度或小分子生产表型相关联。使用源自恶臭假单胞菌、丁烷嗜硫菌或大肠杆菌的转录因子 - 启动子对，构建了针对两种重要化学类别（二羧酸和醇）的生物传感器。选择的转录因子可被琥珀酸、己二酸或1 - 丁醇特异性激活，并且我们证明了使用这三种化合物时大肠杆菌中产物依赖性生长。1 - 丁醇生物传感器被应用于原理验证的液体培养筛选中，以优化工程大肠杆菌中的1 - 丁醇生物合成，通过优化酶表达水平鉴定出一种途径变体，其1 - 丁醇比生产率提高了35%。最后，为了证明选择酶活性的能力，1 - 丁醇生物传感器被用作合成选择，将体内1 - 丁醇生物合成与大肠杆菌适应性相关联，并且在一轮正向选择后观察到1 - 丁醇生产表型富集了120倍。
