Fig. 5: One-step engineering of P. putida for glucose-dependent production of PCA. | Nature Communications

Fig. 5: One-step engineering of P. putida for glucose-dependent production of PCA.

From: Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Fig. 5: One-step engineering of P. putida for glucose-dependent production of PCA.The alternative text for this image may have been generated using AI.

A Simplified metabolic map indicating key reactions eliminated or modified towards protocatechuate (PCA) overproduction. Editing pcaG (PP_4655), pcaH (PP_4656), ppc (PP_1505), pykA (PP_1362), and pyk (PP_4301) blocked the cognate biochemical reactions, while aroF-I (PP_2324) was modified to remove feedback inhibition on phospho-2-dehydro-3-deoxyheptonate aldolase. All of these modifications are encoded in a single, multiplex editing module, containing nine gRNAs. Additionally, quiC, aroQ and tktA were overexpressed as a synthetic operon under ChnR/PchnB transcriptional regulation. Genetic elements not drawn to scale. 6PG, 6-phosphogluconate; PEP, phosphoenolpyruvate; Pyr, pyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; CMA, β-carboxy-cis,cis-muconate; EDP, Entner-Doudoroff pathway; PPP, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle. B Growth and substrate consumption and C product formation in shaken-flask fermentations of the edited strain. CDW; cell dry weight; 2KG, 2-ketogluconate; qPCA, PCA volumetric productivity; YPCA/S, PCA yield on substrate. Data represent mean values ± standard deviation of three independent biological experiments. Source data underlying panels B, C are provided as a Source Data file.

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