Elimination of d-Lactate Synthesis Increases Poly(3-Hydroxybutyrate) and Ethanol Synthesis from Glycerol and Affects Cofactor Distribution in Recombinant Escherichia coli

The effect of eliminating d-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP⁺ ratio was higher than the NADH/NAD⁺ ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP⁺. In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.Poly(3-hydroxybutyrate) (PHB) is the best-known and most common polyhydroxyalkanoate (PHA). PHAs are polymers with thermoplastic properties that are totally biodegradable by microorganisms present in most environments and that can be produced from different renewable carbon sources (38). Accumulated as intracellular granules by many bacteria under unfavorable conditions (1, 21), PHAs are carbon and energy reserves and also act as electron sinks, enhancing the fitness and stress resistance of bacteria and contributing to redox balance (12, 30). Escherichia coli offers a well-defined physiological environment for the construction and manipulation of various metabolic pathways to produce different bioproducts, such as PHB, from cost-effective carbon sources.In recent years, a significant increase in the production of biodiesel has caused a sharp fall in the cost of glycerol, the main by-product of biodiesel synthesis. As a result, glycerol has become a very attractive substrate for bacterial fermentations (10), specially for reduced products, such as PHB (36). The E. coli strain used in this work, K24K, carries phaBAC, the structural genes responsible for PHB synthesis, from Azotobacter sp. strain FA8 (23) (Table ​(Table1).1). The pha genes in K24K are expressed from a chimeric promoter and consequently are not subject to the genetic regulatory systems present in natural PHA producers. Because of this, it can be assumed that regulation of PHA synthesis in the recombinants is restricted by enzyme activity levels, modulated principally by substrate availability. In most natural producers, and also in PHB-producing E. coli recombinants, PHB is synthesized through the condensation of two molecules of acetyl-coenzyme A (acetyl-CoA), catalyzed by an acetoacetyl-CoA transferase or 3-ketothiolase, resulting in acetoacetyl-CoA. This compound is subsequently reduced by an NAD(P)H-dependent acetoacetyl-CoA reductase to R-(−)-3-hydroxybutyryl-CoA, which is then polymerized by a specific PHA synthase (34).

TABLE 1.

E. coli strains, plasmids, and oligonucleotides used in this study

Strain, plasmid, or oligonucleotide	Relevant characteristicsb	Reference or source
E. coli strains
K1060a	F−fadE62 lacI60 tyrT58(AS) fabB5 mel-1	29
K24	Same as K1060, carrying pJP24; Apr	23
K24K	Same as K1060, carrying pJP24K; Apr Kmr	23
ALS786a	F− λ−rph-1 ΔldhA::kan; Kmr	14
K24LT	Same as K1060 but ΔldhA::kan by K1060 × P1(ALS786), carrying pJP24; Apr Kmr	This work
K24KL	Same as K1060 but ΔldhA by allelic replacement, carrying pJP24K; Kmr	This work
TA3522a	F− λ− Δ(his-gnd)861 hisJo-701	2
TA3514a	Same as TA3522 but pta-200	19
TA3522L	Same as TA3522 but ΔldhA::kan by TA3522 × P1(ALS786); Kmr	This work
TA3514L	Same as TA3514 but ΔldhA::kan by TA3514 × P1(ALS786); Kmr	This work
Plasmids
pQE32	Expression vector, ColE1 ori; Apr	Qiagen GmbH, Hilden, Germany
pJP24	pQE32 derivative expressing a 4.3-kb BamHI-HindIII insert containing the phaBAC genes from Azotobacter sp. strain FA8 under the control of a T5 promoter/lac operator element; Apr	23
pJP24K	pJP24 derivative; Apr Kmr	23
pCP20	Helper plasmid used for kan excision; Saccharomyces cerevisiae FLP λ cI857 λ PRrepA(Ts); Apr Cmr	7
Oligonucleotides
ΔldhA-F	5′-TAT TTT TAG TAG CTT AAA TGT GAT TCA ACA TCA CTG GAG AAA GTC TTA TGG TGT AGG CTG GAG CTG CTT C-3′	This work
ΔldhA-R	5′-CTC CCC TGG AAT GCA GGG GAG CGG CAA GAT TAA ACC AGT TCG TTC GGG CAC ATA TGA ATA TCC TCC TTA G-3′	This work

Open in a separate window^aStrain obtained through the E. coli Genetic Stock Center, Yale University, New Haven, CT.^bFor oligonucleotides, the ATG codon of ldhA is underlined and the sequences with homology to FRT-kan-FRT in the template plasmid pKD4 (11) are shown in boldface.Cells growing on glycerol are in a more reduced intracellular state than cells grown on glucose under similar conditions of oxygen availability. This has a significant effect on the intracellular redox state, which causes the cells to direct carbon flow toward the synthesis of more-reduced products when glycerol is used than when glucose is used in order to achieve redox balance (31). When metabolic product distribution was analyzed in bioreactor cultures of K24K using glucose or glycerol as the substrate, product distributions with the two substrates were found to be different, as glycerol-grown cultures produced smaller amounts of acetate, lactate, and formate and more ethanol than those grown on glucose. However, PHB production from glycerol was lower than that from glucose, except under conditions of low oxygen availability (13).Manipulations to enhance the synthesis of a metabolic product include several approaches to increase the availability of the substrates needed for its formation or to inhibit competing pathways. The effect of eliminating competing pathways on PHB production from glucose has been investigated through the inactivation of different genes, such as those encoding enzymes participating in the synthesis of acetate (ackA, pta, and poxB) or d-lactate (ldhA). A pta mutant, which produces very little acetate (6), and an frdA ldhA double mutant (40) had increased PHB accumulation from glucose. A recent report using an ackA pta poxB ldhA adhE mutant under microaerobic conditions attained similar results (17). The inactivation of ldhA has also been shown to have an important effect on the metabolic product distribution in recombinant E. coli with glycerol as the carbon source, promoting ethanol synthesis (28). In the present work we analyzed the effect of ldhA inactivation in strain K24K using glycerol as the carbon source, with special emphasis on changes in carbon distribution and in the intracellular redox state, determined through cofactor levels.