Long-chain α,ω-dicarboxylic acids (LDCAs, ≥ C12) are widely used as a raw material for preparing various commodities and polymers. In this study, a CYP450-monooxygenase-mediated ω-oxidation pathway system with high ω-regioselectivity was heterologously expressed in Escherichia coli to produce DCAs from fatty acids. The resulting engineered E. coli produced a maximum of 41 mg/L of C12 DCA and 163 mg/L of C14 DCA from fatty acids (1 g/L), following 20 h of whole cell biotransformation. Addition of a heme precursor and the hydroxyl radical scavenger, thiourea, increased product concentration (159 mg/L of C12 DCA and 410 mg/L of C14 DCA) in a shorter culture duration than that of the corresponding controls. DCAs of various chain lengths were synthesized from coconut oil hydrolysate using the engineered E. coli. This novel synthetic biocatalytic system could be applied to produce high value DCAs in a cost-effective manner from renewable plant oils.
Monomeric
4-hydroxyvalerate is a versatile chemical used to produce
various commodities and fine chemicals. In the present study, the lvaAB gene was deleted from the lva operon
in Pseudomonas putida KT2440 and tesB, obtained from Escherichia coli, was overexpressed
under the control of the lva operon system, which
is induced by the substrate levulinic acid and the product 4-hydroxyvalerate
to produce 4-hydroxyvalerate from levulinic acid. The lvaAB-deleted strain showed almost complete conversion of levulinic acid
to 4-hydroxyvalerate, compared with 24% conversion in the wild-type
strain. In addition, under optimized culture conditions, the final
engineered strain produced a maximum of 50 g/L 4-hydroxyvalerate with
97% conversion from levulinic acid. The system presented here could
be applied to produce high titers of 4-hydroxyvalerate in a cost-effective
manner at a large scale from renewable cellulosic biomass.
Inducible and tunable expression systems are essential for the microbial production of biochemicals. Five different carbon source- and substrate-inducible promoter systems were developed and further evaluated in Pseudomonas putida KT2440 by analyzing the expression of green fluorescent protein (GFP) as a reporter protein. These systems can be induced by low-cost compounds such as glucose, 3-hydroxypropionic acid (3HP), levulinic acid (LA), and xylose. 3HP-inducible HpdR/PhpdH was also efficiently induced by LA. LvaR/PlvaA and XutR/PxutA systems were induced even at low concentrations of LA (0.1 mM) and xylose (0.5 mM), respectively. Glucose-inducible HexR/Pzwf1 showed weak GFP expression. These inducer agents can be used as potent starting materials for both cell growth and the production of a wide range of biochemicals. The efficiency of the reported systems was comparable to that of conventional chemical-inducible systems. Hence, the newly investigated promoter systems are highly useful for the expression of target genes in the widely used synthetic biology chassis P. putida KT2440 for industrial and medical applications.
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