Fig. 2: Characterization of the multi-gene engineered strain via RNAseq and proteomics.

a–c P. putida harboring a genomically integrated indigoidine expression cassette and either an empty vector (control strain) or a dCpf1/CRISPRi targeting array examined for gene knockdown efficiency. a RNAseq analysis of plasmid-borne gRNA array targeting 14 genes (Fig. 1a, and Supplementary Data 1) in P. putida. b Knockdown efficiency of a representative gene locus PP_1444 (gcd) targeted for inhibition over a 72-h time course. RNA expression levels (right hand panel) were validated with targeted proteomic analysis (left hand panel). Proteomic samples were analyzed with n = 3 for control samples and n = 6 for the engineered strain. For the RNAseq analysis for the control sample and n = 2, n = 4 for the engineered strain. For the proteomics sample, all data points are shown. Transcripts per kilobase million (TPM) counts from a representative RNAseq time course are shown. c dCpf1/CRISPR interference causes global RNA expression level changes. Volcano plot of mRNA expression levels compared at t = 0 h and t = 24 h between multi-gene engineered and control strains. 184 data points (0 h) and 391 data points (24 h) out of 5369 data points are outliers some are displayed on the edge of the axes. The y-axis indicates the expression value of log₁₀(q-value), and the x-axis displays the log₂fold change. The blue dots represent gene expression levels that were significantly different, and the dotted blue line indicates the threshold where P = 0.01. P-values were calculated using Fisher’s exact test. Gray dots indicate transcripts that were not statistically significant. d Validation of carbon source rewiring. Genome-scale modeling predicts that glucose/indigoidine rewiring blocks growth of engineered strains on lysine or para-coumarate as a carbon source. A representative set of plates is shown from three biologically independent experiments. Source data are provided as a Source Data file.