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A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

ABSTRACT: Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied in vitro Among the ?-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would be supported by the viability of the ?fabH mutant. In this study, we show that the ?fabH and ?yiiD mutations were synthetically lethal and that ?fabH ?relA ?spoT and ?fabH ?dksA synthetic lethality was rescued by the heterologous expression of yiiD In the ?fabH mutant, the expression of yiiD was positively regulated by (p)ppGpp. The growth defect, reduced cell size, and altered fatty acid profile of the ?fabH mutant and the growth defect of the ?fabH ?fabF fabB15(Ts) mutant in oleate- and palmitate-supplemented medium at 42°C were rescued by the expression of yiiD from a multicopy plasmid. Together, these results indicate that the yiiD-encoded function supported initiation of fatty acid biosynthesis in the absence of FabH. We have renamed yiiD as fabY IMPORTANCE Fatty acid biosynthesis is an essential process conserved across life forms. ?-Ketoacyl-ACP synthases are essential for fatty acid biosynthesis. FabH is a ?-ketoacyl-ACP synthase that contributes to the initiation of fatty acid biosynthesis in Escherichia coli In this study, we present genetic and biochemical evidence that the yiiD (renamed fabY)-encoded function contributes to the biosynthesis of fatty acid in the absence of FabH activity and that under these conditions, the expression of FabY was regulated by the stringent response factors (p)ppGpp and DksA. Combined inactivation of FabH and FabY resulted in growth arrest, possibly due to the loss of fatty acid biosynthesis. A molecule(s) that inhibits the two activities can be an effective microbicide.

SUBMITTER: Sanyal R

PROVIDER: S-EPMC6755751 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

Sanyal Rajeshree R Singh Vani V Harinarayanan Rajendran R

Journal of bacteriology 20190906 19

Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied <i>in vitro</i> Among the β-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would ...[more]

PMID: 31331975

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Project description:Background:As a versatile platform chemical, construction of microbial catalysts for free octanoic acid production from biorenewable feedstocks is a promising alternative to existing petroleum-based methods. However, the bio-production strategy has been restricted by the low capacity of E. coli inherent fatty acid biosynthesis. In this study, a combination of integrated computational and experimental approach was performed to manipulate the E. coli existing metabolic network, with the objective of improving bio-octanoic acid production. Results:First, a customized OptForce methodology was run to predict a set of four genetic interventions required for production of octanoic acid at 90% of the theoretical yield. Subsequently, all the ten candidate proteins associated with the predicted interventions were regulated individually, as well as in contrast to the combination of interventions as suggested by the OptForce strategy. Among these enzymes, increased production of 3-hydroxy-acyl-ACP dehydratase (FabZ) resulted in the highest increase (+ 45%) in octanoic acid titer. But importantly, the combinatorial application of FabZ with the other interventions as suggested by OptForce further improved octanoic acid production, resulting in a high octanoic acid-producing E. coli strain +fabZ ΔfadE ΔfumAC ΔackA (TE10) (+ 61%). Optimization of TE10 expression, medium pH, and C:N ratio resulted in the identified strain producing 500 mg/L of C8 and 805 mg/L of total FAs, an 82 and 155% increase relative to wild-type MG1655 (TE10) in shake flasks. The best engineered strain produced with high selectivity (> 70%) and extracellularly (> 90%) up to 1 g/L free octanoic acid in minimal medium fed-batch culture. Conclusions:This work demonstrates the effectiveness of integration of computational strain design and experimental characterization as a starting point in rewiring metabolism for octanoic acid production. This result in conjunction with the results of other studies using OptForce in strain design demonstrates that this strategy may be also applicable to engineering E. coli for other customized bioproducts.

Dataset Information

A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

Publications

A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

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