Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida.

恶臭假单胞菌KT2440 作为一种重要的生物催化剂，可将多种碳源转化为多种产品，包括烯二酸、顺式、顺式粘康酸（粘康酸盐），因此受到越来越多的关注。恶臭假单胞菌之前已被设计为从葡萄糖产生粘康酸盐。然而，葡萄糖的周质氧化会导致大量的 2-酮葡糖酸盐积累，从而降低产品产量和选择性。葡萄糖脱氢酶基因 ( gcd ) 的缺失) 防止 2-酮葡萄糖酸积累，但显着减慢生长和粘糖酸的产生。在这项工作中，我们采用适应性实验室进化来提高无法生产 2-酮葡萄糖酸盐的菌株的粘糖酸盐产量。基于生长的选择改善了生长，但降低了粘酸滴度。因此，开发了一种新的粘糖响应生物传感器，以使用荧光激活细胞分选实现基于粘糖的筛选。分选的克隆表现出改善的生长和粘蛋白的产生。通过对这些分离株进行全基因组重测序鉴定的突变表明，葡萄糖代谢可能在缺乏gcd的菌株中失调。利用这些信息，我们使用有针对性的工程来概括通过进化实现的改进。转录抑制基因的缺失hexR改善了菌株的生长并提高了粘康酸的产生率，并使用转录组学研究了这种缺失的影响。基因gntZ和gacS在几个进化的克隆中也被破坏，这些基因的缺失进一步改善了菌株的生长和粘糖体的产生。总之，这些目标提供了一套修饰，可在gcd缺失的情况下改善恶臭假单胞菌的葡萄糖转化为粘糖酸盐。在这项工作之前，我们的缺乏gcd的工程菌株在补料分批生物反应器中以 0.07 g/L/h 的生产率和 38% 的产率 (mol/mol) 产生 7.0 g/L 粘康酸盐。在这里，产生的菌株删除了hexR、gntZ和gacS在 0.21 g/L/h 时达到 22.0 g/L，在类似条件下葡萄糖的产率为 35.6% (mol/mol)。这些策略能够增强粘康酸的产生，并且还可以改善恶臭假单胞菌中葡萄糖的其他目标分子的产生。