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Development of genetically stable Escherichia coli strains for poly(3-hydroxypropionate) production.

ABSTRACT: Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In our previous study, a pathway for P3HP production was constructed in recombinant Esecherichia coli. Seven exogenous genes in P3HP synthesis pathway were carried by two plasmid vectors. However, the P3HP production was severely suppressed by strain instability due to plasmid loss. In this paper, two strategies, chromosomal gene integration and plasmid addiction system (PAS) based on amino acid anabolism, were applied to construct a genetically stable strain. Finally, a combination of those two methods resulted in the best results. The resultant strain carried a portion of P3HP synthesis genes on chromosome and the others on plasmid, and also brought a tyrosine-auxotrophy based PAS. In aerobic fed-batch fermentation, this strain produced 25.7 g/L P3HP from glycerol, about 2.5-time higher than the previous strain with two plasmids. To the best of our knowledge, this is the highest P3HP production from inexpensive carbon sources.

SUBMITTER: Gao Y

PROVIDER: S-EPMC4023983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant Escherichia coli.

Project description:Poly(3-hydroxypropionate) (P3HP) is a thermoplastic with great compostability and biocompatibility, and can be produced through several biosynthetic pathways, in which the glycerol pathway achieved the highest P3HP production. However, exogenous supply of vitamin B12 was required to maintain the activity of glycerol dehydratase, resulting in high production cost. To avoid the addition of VB12, we have previously constructed a P3HP biosynthetic route with β-alanine as intermediate, and the present study aimed to improve the P3HP production of this pathway. L-aspartate decarboxylase PanD was found to be the rate-limiting enzyme in the β-alanine pathway firstly. To improve the pathway efficiency, PanD was screened from four different sources (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Corynebacterium glutamicum). And PanD from C. glutamicum was found to have the highest activity, the P3HP production was improved in flask cultivation with this enzyme. To further improve the production, the host strain was screened and the culture condition was optimized. Under optimal conditions, production and content of P3HP reached to 10.2 g/L and 39.1% (wt/wt [cell dry weight]) in an aerobic fed-batch fermentation. To date, this is the highest P3HP production without VB12.

| S-EPMC5333900 | biostudies-literature

Conversion of glycerol to poly(3-hydroxypropionate) in recombinant Escherichia coli.

Project description:We have developed the conversion of glycerol into thermoplastic poly(3-hydroxypropionate) [poly(3HP)]. For this, the genes for glycerol dehydratase (dhaB1) of Clostridium butyricum, propionaldehyde dehydrogenase (pduP) of Salmonella enterica serovar Typhimurium LT2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha were expressed in recombinant Escherichia coli. Poly(3HP) was accumulated up to 11.98% (wt/wt [cell dry weight]) in a two-step, fed-batch fermentation. The present study shows an interesting application to engineer a poly(3HP) synthesis pathway in bacteria.

| S-EPMC2805219 | biostudies-literature

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Project description:3-hydroxypropionate (3HP) is an important platform chemical, and its biosynthesis is severely restricted by the toxicity of 3HP on cell growth and survival. To improve Escherichia coli resistance to 3HP and reduce the total production cost in industrial applications, we have identified variations in protein expression level exposed to sub-lethal concentration of this chemical using 2-dimensional gel electrophoresis. Under 3HP stress, the amount of 46 proteins was increased while the amount of 23 proteins was reduced. According to the proteomic results, overexpression of some identified proteins significantly increased the E. coli survival rate under 3HP stress. This study shed light on clues for developing E. coli strains with higher resistance to 3HP toxicity and lower production cost for industrial applications.

| S-EPMC4878269 | biostudies-literature

A forward-design approach to increase the production of poly-3-hydroxybutyrate in genetically engineered Escherichia coli.

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| S-EPMC4336316 | biostudies-literature

Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli.

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| S-EPMC92453 | biostudies-literature

Systems evaluation reveals novel transporter YohJK renders 3-hydroxypropionate tolerance in Escherichia coli.

Project description:Previously, we have reported that 3-hydroxypropionate (3-HP) tolerance in Escherichia coli W is improved by deletion of yieP, a less-studied transcription factor. Here, through systems analyses along with physiological and functional studies, we suggest that the yieP deletion improves 3-HP tolerance by upregulation of yohJK, encoding putative 3-HP transporter(s). The tolerance improvement by yieP deletion was highly specific to 3-HP, among various C2-C4 organic acids. Mapping of YieP binding sites (ChIP-exo) coupled with transcriptomic profiling (RNA-seq) advocated seven potential genes/operons for further functional analysis. Among them, the yohJK operon, encoding for novel transmembrane proteins, was the most responsible for the improved 3-HP tolerance; deletion of yohJK reduced 3-HP tolerance regardless of yieP deletion, and their subsequent complementation fully restored the tolerance in both the wild-type and yieP deletion mutant. When determined by 3-HP-responsive biosensor, a drastic reduction of intracellular 3-HP was observed upon yieP deletion or yohJK overexpression, suggesting that yohJK encodes for novel 3-HP exporter(s).

| S-EPMC7642389 | biostudies-literature

Engineering Escherichia coli for Poly-β-hydroxybutyrate Production from Methanol.

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| S-EPMC10135841 | biostudies-literature

Project description:BackgroundComparatively little is known about the prevalence or the molecular characteristics of the zoonotic pathogen E. coli O157:H7 in the sheep reservoir. To investigate this and determine the host specificity of subclones of the bacterium, we have conducted a slaughterhouse prevalence study in sheep and compared the collected isolates to O157:H7 previously isolated from cattle and human patients.ResultsVerotoxin-producing O157:H7 was found in 11/597 (1.8%) of samples from sheep in Swedish slaughterhouses, 9/492 faecal (1.8%) and 2/105 ear samples (1.9%). All positive sheep were < 6 months old. Pulsed field gel electrophoresis typing revealed exact matches between isolates from the sheep prevalence study and human patients as well as between isolates from sheep and cattle. In one case, matching isolates were found in sheep, cattle, and a human patient in the same municipality. Identical PFGE profiles generally corresponded to similar but non-identical multi-locus VNTR profiles. In one sheep sample, SNP-typing found the highly virulent clade 8 variant of O157:H7. The virulence gene profiles of sheep isolates from the prevalence study and three sheep farms linked to cases of human illness were investigated by PCR detection (eaeA, hlyA, cdtV-B, vtx1), and partial sequencing of vtx2. The observed profiles were similar to those of cattle strains investigated previously.ConclusionsThe same pathogenic subtypes of VTEC O157:H7, including the highly virulent clade 8, appear to be present in both sheep and cattle in Sweden, suggesting strains can circulate freely between ruminant reservoirs.

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| S-EPMC6385071 | biostudies-literature

Styrene Production in Genetically Engineered Escherichia coli in a Two-Phase Culture.

Project description:Styrene is an important industrial chemical. Although several studies have reported microbial styrene production, the amount of styrene produced in batch cultures can be increased. In this study, styrene was produced using genetically engineered Escherichia coli. First, we evaluated five types of phenylalanine ammonia lyases (PALs) from Arabidopsis thaliana (AtPAL) and Brachypodium distachyon (BdPAL) for their ability to produce trans-cinnamic acid (Cin), a styrene precursor. AtPAL2-expressing E. coli produced approximately 700 mg/L of Cin and we found that BdPALs could convert Cin into styrene. To assess styrene production, we constructed an E. coli strain that co-expressed AtPAL2 and ferulic acid decarboxylase from Saccharomyces cerevisiae. After a biphasic culture with oleyl alcohol, styrene production and yield from glucose were 3.1 g/L and 26.7% (mol/mol), respectively, which, to the best of our knowledge, are the highest values obtained in batch cultivation. Thus, this strain can be applied to the large-scale industrial production of styrene.

| S-EPMC10801462 | biostudies-literature

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