Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, c/Profesor Albareda 1, Granada, 18008, Spain.
BioEnterprise Master Program, School of Pharmacy, University of Granada, Granada, Spain.
Microb Biotechnol. 2021 Sep;14(5):1931-1943. doi: 10.1111/1751-7915.13844. Epub 2021 Aug 17.
Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner-Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.
铜绿假单胞菌是一种高度耐溶剂的微生物，是从木质纤维素残渣中生产增值化合物的有用底盘，特别是从苯丙氨酸生产的芳香族化合物。使用这些农业残渣需要两步处理，以将纤维素和半纤维素的多糖成分释放为单体糖，最丰富的单体糖是葡萄糖和木糖。泛基因组研究表明，铜绿假单胞菌通过三种收敛途径代谢葡萄糖，生成 6-磷酸葡萄糖酸，然后通过 Entner-Doudoroff 途径代谢它，但该菌株不能降解木糖。从该过程的经济可行性的角度来看，两种糖的增值都是至关重要的。出于这个原因，通过掺入将这种 C5 糖转化为戊糖磷酸循环中间体的异源分解代谢基因，赋予了一株铜绿假单胞菌通过木糖异构酶途径代谢木糖的能力。此外，敲除编码葡萄糖脱氢酶的 T1E_2822 开放阅读框，以避免产生末端产物木酸盐。我们生成了一组源自 DOT-T1E 的菌株，这些菌株可以在培养基中同时代谢葡萄糖和木糖，并且在葡萄糖培养中达到约 100 分钟的代时，在木糖培养中达到约 300 分钟的代时达到高细胞密度。这些菌株在稀酸预处理和蒸汽爆破预处理的玉米秸秆和甘蔗秸秆的 2G 水解物中生长。在生长过程中，这些菌株代谢了>98%的葡萄糖、>96%的木糖和>85%的乙酸。在 2G 水解物中，携带多达五个独立突变以避免苯丙氨酸代谢的 DOT-T1E 衍生菌株 P. putida 5PL 在培养基中积累了这种氨基酸。我们构建了 P. putida 5PLΔgcd(xylABE)，当在 2G 预处理的玉米秸秆或甘蔗秸秆中生长时，它可以产生高达 250mg/L 的苯丙氨酸。这些结果支持了铜绿假单胞菌作为 2G 过程底盘的潜力。
