Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Abstract

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts. − , m k + ) supE44 thi −1 gyrA996 relA1 phoA Hanahan (1983) E. coli BW29427 A conjugal donor strain to transfer plasmids, auxotrophic to diaminopimelic acid Wang et al. (2019) P. putida KT2440 Wild type ATCC 12633 P. putida M2 Accession number JADOUD010000001 Park, Fong, et al. (2022) P. putida PPK1 (PP1; JPUB_014489) P. putida KT2440 carrying pRGPDuo2; Kan R Gauttam et al. (2020) P. putida PPK2 (PP2; JPUB_014491) P. putida KT2440 carrying pRGPDuo2-sfGFPtac RBS0; Kan R Gauttam et al. (2020) P. putida PPK3 (PP3; JPUB_014493) P. putida KT2440 carrying pRGPDuo2-sfGFPtet RBS0; Kan R Gauttam et al. (2020) P. putida PPK7 (PP7; JPUB_014775) P. putida KT2440 carrying pRGPDuo1; Gent R Gauttam et al. (2021) P. putida PPK9 (PP9; JPUB_014779) P. putida KT2440 carrying pRGPDuo1-sfGFPtac RBS0; Gent R Gauttam et al. (2021) P. putida PPK12 (PP12; JPUB_014785) P. putida KT2440 carrying pRGPDuo3; Gent R Gauttam et al. (2021) P. putida PPK13 (PP13; JPUB_014787) P. putida KT2440 carrying pRGPDuo4; Kan R Gauttam et al. (2021) P. putida PPK14 (PP14; JPUB_014810) P. putida KT2440 carrying pRGPDuo3-sfGFPbad RBS0; Gent R Gauttam et al. (2021) P. putida PPK16 (PP16; JPUB_014791) P. putida KT2440 carrying pRGPDuo4-sfGFPbad RBS0; Kan R Gauttam et al. (2021) P. putida PP2M226 (JPUB_020213) P. putida M2 carrying pRGPDuo1; Gent R This study P. putida PP2M227 (JPUB_020214) P. putida M2 carrying pRGPDuo2; Kan R This study P. putida PP2M229 (JPUB_020215) P. putida M2 carrying pRGPDuo1-sfGFPtac RBS0; Gent R This study P. putida PP2M230 P. putida M2 carrying pRGPDuo2-sfGFPtet RBS0; Kan R This study P. putida PP2M231 (JPUB_020216) P. putida M2 carrying pRGPDuo2-sfGFPtac RBS0; Kan R ...

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

“…Simultaneous and complete utilization of sugar and aromatic compound could also be beneficial for bioproduct formation, one substrate could be used for energy for cell growth, while the other substrate for product formation, a strategy already being used in KT2440 ( 14 ). As M2 is amenable to genetic manipulation via CRISPRi as shown by our study and previous publications ( 10 , 25 ), this study provides a framework for the use of metabolic engineering tools to alleviate CCR without causing other metabolism imbalances.…”

“…As Crc has been identified as the main regulatory protein governing CCR in Pseudomonads ( 12 ), the crc gene expression was reduced using a previously developed dual-inducible CRISPR/dCas9-based gene repression system to determine if the aromatic and sugar metabolism in M2 are subject to Crc regulation ( 24 , 25 ). The functionality of the arabinose-inducible pRGPspdCas9bad-edd plasmid (the backbone plasmid used to design CRISPRi strains) was confirmed to verify sgRNA expression (Fig.…”

“…The CRISPR interference system utilizing the type II dCas9 homologs from Streptococcus pyogenes (pRGPspdCas9bad, Table 1 ) previously developed for KT2440 and M2 ( 24 , 25 ) was used as the backbone plasmid to develop a dual inducible CRISPR/dCas9 interference-based gene repression system in M2. Expression of spdCas9 was under the control of the P tac [Isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible] promoter, while sgRNA expression was under the control of the P araC/bad (arabinose-inducible) promoter.…”

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization inPseudomonas putidaM2

“…Simultaneous and complete utilization of sugar and aromatics could also be beneficial for bioproduct formation, one substrate could be used for energy for cell growth while the other substrate for product formation, a strategy already being used in KT2440 (14). As M2 is amenable to genetic manipulation via CRISPRi as shown by our study and previous publications (10,25), this study provides a framework for use of metabolic engineering tools to alleviate CCR without causing other metabolism imbalances.…”

“…As Crc has been identified as the main regulatory protein governing CCR in Pseudomonads (12), the crc gene was downregulated using a previously developed dual-inducible CRISPR/dCas9based gene repression system to determine if the aromatic and sugar metabolism in M2 are subject to Crc regulation (24,25). The functionality of the arabinose-inducible pRGPspdCas9bad-edd plasmid (the backbone plasmid used to design CRISPRi strains) was confirmed to verify sgRNA expression (Figure S5).…”