Enhanced poly(3-hydroxypropionate) production via β-alanine pathway in recombinant <i>Escherichia coli</i>

<div><p>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 (<i>Escherichia coli</i>, <i>Bacillus subtilis</i>, <i>Pseudomonas fluorescens</i>, and <i>Corynebacterium glutamicum</i>). And PanD from <i>C</i>. <i>glutamicum</i> 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.</p></div

Time profiles for CDW, P3HP production and substrate consumption during an aerobic fed-batch fermentation of <i>E</i>. <i>coli</i> JM109(DE3)/pHP302/pFS03.

<p>The content of P3HP was calculated using the ratio of P3HP weight to cell dry weight.</p

Effect of the host strain on P3HP production.

<p>Different host strains carrying plasmids pHP302 and pFS03 were grown in minimal medium, and the P3HP production (white), CDW (light grey), and P3HP content (heavy grey) were presented. The experiments were perfomed in triplicate in shake-flask cultures.</p

Bacterial strains, plasmids, and primers used in this study.

<p>Bacterial strains, plasmids, and primers used in this study.</p

Effect of IPTG on the biomass and P3HP production.

<p>Effect of IPTG on the biomass and P3HP production.</p

Effect of β-alanine and L-aspartate on the P3HP production.

<p>The strain Q2153 was grown in minimal medium with supplement of 5 g/L β-alanine (β-Ala) or L-aspartate (L-Asp), cultivation without amino acid was used as the control (CK), and the P3HP production (white), CDW (light grey), and P3HP content (heavy grey) were presented. The experiment was carried out in shaking flask in triplicate. All shake-flask experiments were incubated for 48 h after induction.</p

β-Alanine pathway used in this study.

<p>Four L-aspartate decarboxylases (PanD) from <i>E</i>. <i>coli</i>, <i>B</i>. <i>subtilis</i>, P. <i>fluorescens</i>, and <i>C</i>. <i>glutamicum</i> were tested. PP0596, β-alanine-pyruvate transaminase from <i>Pseudomonas putida</i>; YdfG, 3-hydroxyacid dehydrogenase from <i>E</i>. <i>coli</i>; PrpE, propionyl-CoA synthase rom <i>E</i>. <i>coli</i>; PhaC1, polyhydroxyalkanoate synthase from <i>Cupriavidus necator</i>.</p

Screening of L-aspartate decarboxylase for P3HP production.

<p>P3HP-producing strains with different <i>panD</i> gene were grown in minimal medium, and the P3HP production (white), CDW (light grey), and P3HP content (heavy grey) were presented. The experiment was carried out in shaking flask in triplicate. <i>Ec</i>, <i>Escherichia coli</i>; <i>Bs</i>, <i>Bacillus subtilis</i>; <i>Pf</i>, <i>Pseudomonas fluorescens</i>; <i>Cg</i>, <i>Corynebacteria glutamicum</i>.</p