Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, Nebraska, USA.
Department of Bioengineering, University of California, San Diego, California, USA.
Biotechnol Bioeng. 2022 Sep;119(9):2541-2550. doi: 10.1002/bit.28131. Epub 2022 May 16.
Lignin is a largely untapped source for the bioproduction of value-added chemicals. Pseudomonas putida KT2440 has emerged as a strong candidate for bioprocessing of lignin feedstocks due to its resistance to several industrial solvents, broad metabolic capabilities, and genetic amenability. Here we demonstrate the engineering of P. putida for the ability to metabolize syringic acid, one of the major products that comes from the breakdown of the syringyl component of lignin. The rational design was first applied for the construction of strain Sy-1 by overexpressing a native vanillate demethylase. Subsequent adaptive laboratory evolution (ALE) led to the generation of mutations that achieved robust growth on syringic acid as a sole carbon source. The best mutant showed a 30% increase in the growth rate over the original engineered strain. Genomic sequencing revealed multiple mutations repeated in separate evolved replicates. Reverse engineering of mutations identified in agmR, gbdR, fleQ, and the intergenic region of gstB and yadG into the parental strain recaptured the improved growth of the evolved strains to varied extent. These findings thus reveal the ability of P. putida to utilize lignin more fully as a feedstock and make it a more economically viable chassis for chemical production.
木质素是生物生产高附加值化学品的一个未充分开发的来源。由于其对几种工业溶剂的抗性、广泛的代谢能力和遗传可操作性，恶臭假单胞菌 KT2440 已成为木质素原料生物加工的有力候选者。在这里，我们展示了对恶臭假单胞菌进行工程改造的能力，使其能够代谢丁香酸，这是木质素的丁香基成分分解后的主要产物之一。首先通过过表达天然香草酸脱甲基酶，对菌株 Sy-1 进行了合理设计。随后的适应性实验室进化 (ALE) 导致产生了突变，使菌株能够以丁香酸作为唯一碳源进行稳健生长。最佳突变体的生长速度比原始工程菌株提高了 30%。基因组测序揭示了多个在不同进化重复中重复的突变。将 agmR、gbdR、fleQ 以及 gstB 和 yadG 之间的基因间区中的突变反向工程引入亲本菌株，在不同程度上重新获得了进化菌株的生长改善。这些发现揭示了恶臭假单胞菌利用木质素作为原料的能力更强，使其成为更具经济可行性的化学生产底盘。
