Boosting titers of engineered triketide and tetraketide synthases to record levels through T7 promoter tuning

Abstract

Modular polyketide synthases (PKS's) are promising platforms for the rational engineering of designer polyketides and commodity chemicals, yet their low productivities are a barrier to the practical biosynthesis of these compounds. Previously, we engineered triketide lactone synthases such as Pik167 using the recently updated module definition and showed they generate hundreds of milligrams of product per liter of Escherichia coli K207-3 shake flask culture. As the molar ratio between the 2 polypeptides of Pik167 is highly skewed, we sought to attenuate the strength of the T7 promoter controlling the production of the smaller, better-expressing polypeptide and thereby increase production of the first polypeptide under the control of an unoptimized T7 promoter. Through this strategy, a 1.8-fold boost in titer was obtained. After a further 1.5-fold boost obtained by increasing the propionate concentration in the media from 20 to 80 mM, a record titer of 791 mg L^-1 (627 mg L^-1 isolated) was achieved, a 2.6-fold increase overall. Spurred on by this result, the tetraketide synthase Pik1567 was engineered and the T7 promoter attenuation strategy was applied to its second and third genes. A 5-fold boost, from 20 mg L^-1 to 100 mg L^-1, in the titer of its tetraketide product was achieved.

Keywords: Modular polyketide synthase; Promoter tuning; T7 promoter; Tetraketide lactone; Triketide lactone.

Fig. 1.

Natural and engineered modular PKS’s. a) The Pikromyein PKS, colored both with the updated and traditional module boundary, biosynthesizes the Pikromycin precursor Narbonolide. Unlabeled circles and half-circles represent acyl carrier protein (ACP) domains and docking domain (DD) motifs, respectively, b) The engineered synthases Pik127 and Pik167, each comprised of 2 polypeptides, produce their anticipated triketide lactones(Miyazawa et al., 2021). The PikAIII/PikAIV DD motifs are present in both engineered synthases.

Fig. 2.

The 2-polypeptide Pik167 triketide lactone synthase, a) The Pik167 polypeptides, 16^N and ^C67, are encoded by pTM4 and pTM5, respectively, b) An SDS-PAGE gel shows polypeptide levels in the soluble lysate from E. coli K207-3 transformed with pTM4 and pTM5 from the 6th day of polyketide production, c) The sequence of the T7 promoter regulating the expression of 16^N and ^C67 is shown, d) A time course of triketide lactone production by Pik167 reveals that levels plateau on the 6th day (30 mL scale with 20 mM propionate), e) An anti-His₆ Western blot of soluble lysate from E. coli K207-3 transformed with pTM4 (Lane 1), with empty vectors (Lane 2), and with pTM5 (diluted 80-fold) (Lane 3) shows the levels of 16^N and ^C67.

Fig. 3.

Optimizing Pik167 titers through T7 promoter attenuation. Triketide lactone production and polypeptide expression levels for the Pik167 variants are shown (30 mL scale with 20 mM propionate). Each variant contains a different mutation to the T7 promoter (measured strengths in parentheses, see Table 1) controlling the expression of the smaller polypeptide, ^C67. Measurements of polyketide production were obtained from biological triplicates (Data S1, error bars show standard deviation), and the corresponding SDS-PAGE gels were also analyzed in triplicate (Data S2).

Fig. 4.

The effect of propionate concentration on triketide lactone titers, a) The concentration of propionate in the media (30 mL scale) affects the productivity of optimized Pik167 (with 40% strength promoter), b) Production by unoptimized Pik167 slightly decreases with increasing concentrations of propionate (30 mL scale), c) A time course of production by unoptimized Pik167 in media containing 20 mM propionate (300 mL scale) shows that levels plateau on the 10th day. d) The productivities of unoptimized and optimized Pik167 with either 20 or 80 mM propionate are shown (300 mL scale). Triplicate measurements were made, and error bars show standard deviations (Data S1).

Fig. 5.

Promoter optimization for the 3-polypeptide Pik1567. a) The 15^N, ^C56^N, and ^C67 polypeptides are encoded by pRB1, pRB2, and pTM5, respectively, b) A schematic shows tetraketide lactone synthesis by Pik1567. c) The tetraketide lactone titers and protein expression levels of the 3 Pik1567 polypeptides resulting from mutations of the T7 promoter regulating the expression of ^C67 are compared (30 mL scale with 80 mM propionate), d) The tetraketide lactone titers of Pik1567 variants in which the 37% strength promoter controls the expression of ^C67 and the strength of the promoter controlling the expression of ^C56^N is varied are compared (30 mL scale with 80 mM sodium propionate), e) The tetraketide lactone titers of unoptimized and optimized Pik1567 (37% strength promoter controlling ^C67 expression) are compared (300 mL scale with 80 mM propionate). Triplicate measurements were made. Error bars show standard deviations (Data S1).