Engineering of primary carbon metabolism for improved antibiotic production in Streptomyces lividans

Abstract

Deletions were made in Streptomyces lividans in either of two genes (zwf1 and zwf2) encoding isozymes of glucose-6-phosphate dehydrogenase, the first enzyme in the oxidative pentose phosphate pathway (PPP). Each mutation reduced the level of Zwf activity to approximately one-half that observed in the wild-type strain. When the mutants were transformed with multicopy plasmids carrying the pathway-specific transcriptional activator genes for either the actinorhodin (ACT) or undecylprodigiosin (RED) biosynthetic pathway, they produced higher levels of antibiotic than the corresponding wild-type control strains. The presumed lower flux of carbon through the PPP in each of the Deltazwf mutants may allow more efficient glucose utilization via glycolysis, resulting in higher levels of antibiotic production. This appears to occur without lowering the concentration of NADPH (the major biochemical product of the oxidative PPP activity) to a level that would limit antibiotic biosynthesis. Consistent with this hypothesis, deletion of the gene (devB) encoding the enzyme that catalyzes the next step in the oxidative PPP (6-phosphogluconolactonase) also resulted in increased antibiotic production. However, deletion of both zwf genes from the devB mutant resulted in reduced levels of ACT and RED production, suggesting that some of the NADPH made by the PPP is utilized, directly or indirectly, for antibiotic biosynthesis. Although applied here to the model antibiotics ACT and RED, such mutations may prove to be useful for improving the yield of commercially important secondary metabolites.

FIG. 1.

Overview of central carbon metabolism, showing the early steps of the PPP. Genes encoding the enzymes relevant to this work are shown. 6PGA, 6-phosphogluconate; ksp, spontaneous hydrolysis of 6-phospho-δ-gluconolactone (6PGL); G6P, glucose-6-phosphate; TCA, tricarboxylic acid; CoA, coenzyme A.

FIG. 2.

(a) Map of cosmid SC5A7 containing a cluster of PPP-related genes. The open arrows indicate the directions and extents of the predicted protein-encoding regions. Genes encoding predicted proteins showing strong homology to proteins having known metabolic functions are indicated by boldface italic type. Genes encoding predicted proteins having unknown functions are designated hyp. Segments of DNA that were used to make the in-frame zwf1 deletion are indicated by lines terminating in solid circles. The extent and location of the in-frame deletion in the deletion plasmid pIJ8722 and the Δzwf1 deletion strain M704 are indicated by a dotted line. The fragments (from pIJ8707 and pIJ8719) used to produce pIJ8715 are indicated by shaded boxes. The region of the zwf1 cluster cloned in pIJ8715 and used to complement the Δzwf1 mutation is indicated by a line terminating in solid diamonds. Naturally occurring restriction enzyme sites are indicated by boldface type, while sites introduced by PCR or from vector polylinkers are indicated by lightface type in parentheses. (b) Map of cosmid StC22 containing a second cluster of PPP-related genes. The restriction enzyme sites used to join the various fragments were added by using PCR primers. The box labeled hyg^R shows the position of the insertion of the hygromycin resistance gene in the ΔdevB mutant strain. The extents of deleted DNA in the deletion plasmids and strains are indicated by dotted lines.

FIG. 3.

RED production in fermentors by wild-type and zwf mutant S. lividans strains carrying multiple copies of the pathway-specific activator gene for RED biosynthesis. Symbols: ▵, ◊, and +, dry weight of cells of Δzwf1 mutant M704, wild-type strain 1326, and Δzwf1 Δzwf2-opc2-devB mutant M706, respectively; ○, □, and ×, RED production by zwf1, wild-type, and Δzwf1 Δzwf2-opc2-devB strains, respectively.

FIG. 4.

Antibiotic production in shake flask fermentations with wild-type and mutant S. lividans strains. (a) Mean maximum specific ACT production (in micrograms of ACT per milligram [dry weight] of cells [DCW]) for each strain carrying pIJ68. The error bars indicate the standard errors of the means. The strains used and their genetic lesions are indicated. The strain in which Δzwf1 was complemented with wild-type DNA was designated M704/pIJ8715. (b) RED production by strains carrying pIJ6014. Symbols: ▵, wild-type strain; ⋄, Δzwf1 Δzwf2-opc2 devB strain; and ○, Δzwf1 strain.

FIG. 5.

Increased diamide sensitivity of S. lividans mutant strains. The sensitivities of the strains to inhibition of growth by diamide are indicated by zones of clearing (around the diamide-containing discs) on lawns of growth. The larger zones were observed around the discs containing the higher concentrations of diamide (0.5 M compared to 0.1 M).