Directed accumulation of less toxic pimaricin derivatives by improving the efficiency of a polyketide synthase dehydratase domain

Abstract

Pimaricin is an important polyene antifungal antibiotic that binds ergosterol and extracts it from fungal membranes. In previous work, two pimaricin derivatives (1 and 2) with improved pharmacological activities and another derivative (3) that showed no antifungal activity were produced by the mutant strain of Streptomyces chattanoogensis, in which the P450 monooxygenase gene scnG has been inactivated. Furthermore, inactivation of the DH12 dehydratase domain of the pimaricin polyketide synthases (PKSs) resulted in specific accumulation of the undesired metabolite 3, suggesting that improvement of the corresponding dehydratase activity may reduce or eliminate the accumulation of 3. Accordingly, the DH12-KR12 didomain within the pimaricin PKS was swapped with the fully active DH11-KR11 didomain. As predicted, the mutant was not able to produce 3 but accumulated 1 and 2 in high yields. Moreover, the effect of the flanking linker regions on domain swapping was evaluated. It was found that retention of the DH12-KR12 linker regions was more critical for the processivity of hybrid PKSs. Subsequently, high-yield production of 1 or 2 was obtained by overexpressing the scnD gene and its partner scnF and by disrupting the scnD gene, respectively. To our knowledge, this is the first report on the elimination of a polyketide undesired metabolite along with overproduction of desired product by improving the catalytic efficiency of a DH domain using a domain swapping technology.

Engineered polyketides: Synergy between protein and host level engineering

“…In addition to full reductive loop swaps, other examples of reductive domain replacements have been published. A swap of the DH-KR didomain from module 11 to module 12 of the pimaricin PKS improved activity of the marginally active module 12 DH [106] . The most successful mutant retained the linkers flanking the native DH-KR, while replacing simply the DH domain proved ineffective.…”

Structural and Mechanistic Insights into Chain Release of the Polyene PKS Thioesterase Domain

“…21 Similarly, three decarboxylated pimaricin derivatives (1, 2, and 3; Figures 1 and S1) were obtained by inactivating the P450 monooxygenase gene pimG/scnG in the pimaricin producer Strepto-myces chattanoogensis L10. 28,29 The total amount of these derivatives (1, 2, and 3) in mutant QZ01 was up to 25% of the pimaricin titer in the wild-type strain. 28 Considering that the titers of all of the decarboxylated polyene derivatives are reduced, we hypothesize that the low titer of these polyene derivatives is caused by inefficient recognition and processing of downstream enzymes toward the unnatural intermediates, especially the TE domain, which acts…”

“…However, the titers of these unnatural decarboxylated polyene derivatives in AmphN-null and NysN-null strains were less than 2% of their parent antibiotic titers . Similarly, three decarboxylated pimaricin derivatives ( 1 , 2 , and 3 ; Figures and S1) were obtained by inactivating the P450 monooxygenase gene pimG / scnG in the pimaricin producer Streptomyces chattanoogensis L10. , The total amount of these derivatives ( 1 , 2 , and 3 ) in mutant QZ01 was up to 25% of the pimaricin titer in the wild-type strain …”

Enhanced Natamycin production by Streptomyces natalensis in shake-flasks and stirred tank bioreactor under batch and fed-batch conditions

“…Different strategies have been used to maximize natamycin production. These include optimization of medium composition and cultivation parameters [2, 5, 15, 16], engineering of the biosynthetic pathway [17], random mutation and selection [18] and genome shuffling and chromosomal integration [19]. Furthermore, addition of short-chain carboxylic or fatty acids has been found to improve natamycin production [5, 20].…”

“…Fed-batch is characterized by feeding one or more of the components of the production medium, usually the limiting components, to avoid problems associated with their depletion from medium. Pathway engineering and genetic manipulation greatly improved natamycin production, where the improved strains produced about about 3.3 and 8.2 g/L natamycin in shake-flask and bioreactor levels, respectively [17, 19]. On the other hand, optimization of medium composition and cultivation parameters in shake-flask level resulted in the production of 1.5–1.6 g/L natamycin [15, 16].…”