Optimizing a High Performing Multiplex-CRISPRi P. putida Strain with Integrated Metabolomics and ¹³C-Metabolic Flux Analyses

Abstract

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

“…Consequently, CRISPRi systems can reversibly and dynamically modulate expression of both non-essential and essential genes to elucidate their relative impacts on a given production pathway. Downregulation of selected genes using CRISPRi has contributed to significant bioproduct titer improvements, including dyes (Kim et al, 2016;Czajka et al, 2022;Cho et al, 2023), flavonoids (Tao et al, 2018), nutraceuticals (Wu et al, 2017), and biofuels (Tian et al, 2019;Wang et al, 2022).…”

Citrate Synthase Overexpression of Pseudomonas putida Increases Succinate Production from Acetate in Microaerobic Cultivation

“… 19 In particular, succinate production from glucose and glycerol under nitrogen-limited conditions was observed in P. putida. ( 20 , 21 ) With the characteristics listed above, this strain could be a potential host for succinate production from acetate.…”

Photobiological production of high-value pigments via compartmentalized co-cultures using Ca-alginate hydrogels

“…In this study, we used a Ca-alginate hydrogel con guration for heterotrophic production. P. putida was used as a model bacterial factory 19,20 and Y. lipolytica was used as a model yeast factory 21 due to their genetic tractability, rapid growth rate, and ability to grow on diverse feedstock. Complementing these workhorse microbes, Synechococcus 2973 has the fastest sucrose secretion capability (1.9g/L/day) compared to other cyanobacterial hosts.…”