Construction and optimization of trans‐4‐hydroxy‐L‐proline production recombinant E. coli strain taking the glycerol as carbon source

Abstract

BACKGROUND: Trans-4-hydroxy-L-proline (Hyp) is a value-added amino acid that is an applicable chiral building block in some industries, such as the pharmaceutical, cosmetic and healthcare industries. Given that glycerol is a good carbon source for microbial fermentation and a large amount of glycerol is produced by the biodiesel industry, this study investigated a value-added Hyp fermentation from glycerol using an engineered Escherichia coli strain. RESULTS: By optimizing the proline-4-hydroxylase (P4H) gene codons and vectors, a constructed recombinant Escherichia coliBL21 strain successfully converted L-proline to Hyp. With a chemo-physical combination mutagenesis, a high Hyp production strain that can grow with glycerol as a sole carbon source, NA45 was isolated, yielding 1.24 g L −1 from 20 g L −1 glycerol at 12 h in a 30 mL/250 mL conical flask fermentation. With further systemic optimization of nutritional elements, the output was enhanced to 1.62 g L −1 . In a 5 L fermentator, the strain achieved a surprisingly high output of 25.4 g L −1 at 48 h in fed batch mode. CONCLUSION: A high Hyp production strain growing well on glycerol as carbon source was successfully engineered, which is a promising candidate for the industrial Hyp production.

Metabolic engineering strategy for synthetizing trans-4-hydroxy-l-proline in microorganisms

“…The highest t 4Hyp production to date was 54.8 g/L at 60 h using integrated system engineering in E. coli without L-proline addition in a 7.5-L bioreactor , in which the deletion of putA , proP , putP , and aceA , and the mutation of proB contributed to l -proline and relieved its feedback inhibition, and a tunable circuit based on quorum sensing for the dynamic regulation of ODHC activity and rationally designed l -proline hydroxylase [ 69 ]. t 4Hyp production using proline 4-hydroxylase with chemo-physical combination mutagenesis, taking glycerol as the carbon source, and optimizing the fermentation medium(tryptone, FeSO 4 , l -proline) with single-factor, Plackett–Burman, steepest ascent, and a central composite design experiment, was achieved at 25.4 g/L of t 4Hyp [ 81 ]. Subsequently, Zhao et al attained a t 4Hyp concentration of 14.4 g/L by integrating the Vitreoscilla hemoglobin gene ( vgb ) into recombinant E. coli expressing proline 4-hydroxylase in fed-batch fermentation in a 1.4-L bioreactor after 57 h of cultivation [ 75 ].…”

Bioprocessing of tea oil fruit hull with acetic acid organosolv pretreatment in combination with alkaline H2O2

“…Given the influence of such variables as pretreatment temperature, pretreatment time, acetic acid concentration (v/v), and H 2 SO 4 addition (w/v), the AAO pretreatment was optimized with a series of experiments of a Plackett–Burman design (PBD), a steepest ascent design and a central composite design [39, 40]. The most prominent parameters and the optimal concentrations domain were tested using a Plackett–Burman design (Additional file 1: Table S1) and a steepest ascent experiment, respectively.…”

Significantly enhancing production of trans -4-hydroxy- l -proline by integrated system engineering in Escherichia coli

“…l-Pro production in HYP2M was eventually decreased to a final concentration of 0.2 g liter −1 in 60 hours. Park et al (5,30) reported that recombinant E. coli with datp4h catalyzed 4-Hyp with l-Pro as substrate in a yield of 25 g liter −1 , with glycerol and glucose (41 g liter −1 ) as carbon sources, respectively. Compared with previous results, our reported 4-Hyp production method gave the highest yield.…”