Metabolic engineering of genome-streamlined strain Pseudomonas putida KTU-U27 for medium-chain-length polyhydroxyalkanoate production from xylose and cellobiose.

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-Ptac-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.

Creating an efficient 1,2-dichloroethane-mineralizing bacterium by a combination of pathway engineering and promoter engineering.

Currently, 1,2-dichloroethane (DCA) is frequently detected in groundwater and has been listed as a potential human carcinogen by the U.S. EPA. Owing to its toxicity and recalcitrant nature, inefficient DCA mineralization has become a bottleneck of DCA bioremediation. In this study, the first engineered DCA-mineralizing strain KTU-P8DCA was constructed by functional assembly of DCA degradation pathway and enhancing pathway expression with a strong promoter P8 in the biosafety strain Pseudomonas putida KT2440. Strain KTU-P8DCA can metabolize DCA to produce CO2 and utilize DCA as the sole carbon source for cell growth by quantifying 13C stable isotope ratios in collected CO2 and in lyophilized cells. Strain KTU-P8DCA exhibited superior tolerance to high concentrations of DCA. Excellent genetic stability was also observed in continuous passage culture. Therefore, strain KTU-P8DCA has enormous potential for use in bioremediation of sites heavily contaminated with DCA. In the future, our strategy for pathway construction and optimization is expected to be developed as a standard pipeline for creating a wide variety of new contaminants-mineralizing microorganisms. The present study also highlights the power of synthetic biology in creating novel degraders for environmental remediation.

Unsterile production of a polyhydroxyalkanoate copolymer by Halomonas cupida J9.

Microbial production of bioplastics polyhydroxyalkanoates (PHA) has opened new avenues to resolve "white pollution" caused by petroleum-based plastics. PHAs consisting of short- and medium-chain-length monomers, designated as SCL-co-MCL PHAs, exhibit much better thermal and mechanical properties than PHA homopolymers. In this study, a halophilic bacterium Halomonas cupida J9 was isolated from highly saline wastewater and proven to produce SCL-co-MCL PHA consisting of 3-hydroxybutyrate (3HB) and 3-hydroxydodecanoate (3HDD) from glucose and glycerol. Whole-genome sequencing and functional annotation suggest that H. cupida J9 may possess three putative PHA biosynthesis pathways and a class I PHA synthase (PhaCJ9). Interestingly, the purified His6-tagged PhaCJ9 from E. coli BL21 (DE3) showed polymerizing activity towards 3HDD-CoA and a phaCJ9-deficient mutant was unable to produce PHA, which indicated that a low-substrate-specificity PhaCJ9 was exclusively responsible for PHA polymerization in H. cupida J9. Docking simulation demonstrated higher binding affinity between 3HB-CoA and PhaCJ9 and identified the key residues involved in hydrogen bonds formation between 3-hydroxyacyl-CoA and PhaCJ9. Furthermore, His489 was identified by site-specific mutagenesis as the key residue for the interaction of 3HDD-CoA with PhaCJ9. Finally, PHA was produced by H. cupida J9 from glucose and glycerol in shake flasks and a 5-L fermentor under unsterile conditions. The open fermentation mode makes this strain a promising candidate for low-cost production of SCL-co-MCL PHAs. Especially, the low-specificity PhaCJ9 has great potential to be engineered for an enlarged substrate range to synthesize tailor-made novel SCL-co-MCL PHAs.

Development of a novel promoter engineering-based strategy for creating an efficient para-nitrophenol-mineralizing bacterium.

A toxic and persistent pollutant para-nitrophenol (PNP) enters into the environment through improper industrial waste treatment and agricultural usage of chemical pesticides, leading to a potential risk to humans. Although a variety of PNP-degrading bacteria have been isolated, their application in bioremediation has been precluded due to unknown biosafety, poor PNP-mineralizing capacity, and lack of genome editing tools. In this study, a novel promoter engineering-based strategy is developed for creating efficient PNP-mineralizing bacteria. Initially, a complete PNP biodegradation pathway from Pseudomonas sp. strain WBC-3 was introduced into the genome of a biosafety and soil-dwelling bacterium Pseudomonas putida KT2440. Subsequently, five strong promoters were identified from P. putida KT2440 by transcriptome analysis and strength characterization, and each of the five promoters was independently inserted into upstream of the pnp operon in the KT2440 genome. Consequently, a P8 promoter-substituted mutant strain showed the highest PNP degradation rate and strong tolerance against high concentrations of PNP. Furthermore, when using P8 promoter to regulate the transcription of all PNP degradation genes pnpABCDEF, the complete and efficient PNP mineralization was demonstrated by stable isotope 13C-labeled PNP transformation assay. Additionally, the finally constructed KTU-P8pnp can be monitored using integrated GFP on chromosome. This strategy of a combination of pathway construction and promoter engineering should open new avenues for creating efficient degraders for bioremediation.

A promoter engineering-based strategy enhances polyhydroxyalkanoate production in Pseudomonas putida KT2440.

Polyhydroxyalkanoate (PHA), a class of biopolyester synthesized by various bacteria, is considered as an alternative to petroleum-based plastics because of its excellent physochemical and material properties. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from glucose, fatty acid and glycerol, and its whole-genome sequences and cellular metabolic networks have been intensively researched. In this study, we aim to improve the PHA yield of P. putida KT2440 using a novel promoter engineering-based strategy. Unlike previous studies, endogenous strong promoters screening from P. putida KT2440 instead of synthetic or exogenous promoters was applied to the optimization of PHA biosynthesis pathway. Based on RNA-seq and promoter prediction, 30 putative strong promoters from P. putida KT2440 were identified. Subsequently, the strengths of these promoters were characterized by reporter gene assays. Furthermore, each of 10 strong promoters screened by transcriptional level and GFP fluorescence was independently inserted into upstream of PHA synthase gene (phaC1) on chromosome. As a result, the transcriptional levels of the phaC1 and phaC2 genes in almost all of the promoter-substituted strains were improved, and the relative PHA yields of the three promoter-substituted strains KTU-P1C1, KTU-P46C1 and KTU-P51C1 were improved obviously, reaching 30.62 wt%, 33.24 wt% and 33.29 wt% [the ratio of PHA weight to cell dry weight (CDW)], respectively. By further deletion of the glucose dehydrogenase gene in KTU-P1C1, KTU-P46C1 and KTU-P51C1, the relative PHA yield of the resulting mutant strain KTU-P46C1-∆gcd increased by 5.29% from 33.24% to 38.53%. Finally, by inserting P46 into upstream of pyruvate dehydrogenase gene in the genome of KTU-P46C1-∆gcd, the relative PHA yield and CDW of the resulting strain KTU-P46C1A-∆gcd reached nearly 42 wt% and 4.06 g/l, respectively, which increased by 90% and 40%, respectively, compared with the starting strain KTU. In particular, the absolute PHA yield of KTU-P46C1A-∆gcd reached 1.7 g/l, with a 165% improvement compared with the strain KTU. Herein, we report the highest PHA yield obtained by P. putida KT2440 in shake-flask fermentation to date. We demonstrate for the first time the effectiveness of endogenous strong promoters for improving the PHA yield and biomass of P. putida KT2440. More importantly, our findings highlight great potential of this strategy for enhanced production of secondary metabolites and heterologous proteins in P. putida KT2440.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production.

BACKGROUND: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. RESULTS: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. CONCLUSIONS: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Morphology engineering: a new strategy to construct microbial cell factories.

Currently, synthetic biology approaches have been developed for constructing microbial cell factories capable of efficient synthesis of high value-added products. Most studies have focused on the construction of novel biosynthetic pathways and their regulatory processes. Morphology engineering has recently been proposed as a novel strategy for constructing efficient microbial cell factories, which aims at controlling cell shape and cell division pattern by manipulating the cell morphology-related genes. Morphology engineering strategies have been exploited for improving bacterial growth rate, enlarging cell volume and simplifying downstream separation. This mini-review summarizes cell morphology-related proteins and their function, current advances in manipulation tools and strategies of morphology engineering, and practical applications of morphology engineering for enhanced production of intracellular product polyhydroxyalkanoate and extracellular products. Furthermore, current limitations and the future development direction using morphology engineering are proposed.