Development of a versatile chassis for the efficient production of diverse type II polyketides

Abstract

Type II polyketides (T2PKs) exhibit a wide range of structural diversity and potent pharmacological activities. However, the optimal chassis for the synthesis of T2PKs remains elusive, impeding the effective mining and production of these compounds. In this study, we identify Streptomyces aureofaciens J1-022, a high-yield producer of chlortetracycline, as a promising chassis for T2PKs synthesis. To mitigate precursor competition, we execute an in-frame deletion of two endogenous T2PKs gene clusters, resulting in a pigmented-faded host, designated Chassis2.0. Compared to conventional Streptomyces chassis, Chassis2.0 demonstrates enhanced efficiency in the production of oxytetracycline, achieving a 370% increase relative to commercial production strains. Additionally, the tri-ring type T2PKs, which includes actinorhodin and flavokermesic acid, are synthesized in Chassis2.0 with high efficiency. Furthermore, an unidentified biosynthetic gene cluster (BGC) associated with pentangular T2PKs is directly activated, leading to the production of a structurally distinct TLN-1. In conclusion, we successfully achieve the efficient synthesis of tri-ring type pigmented products, the overproduction of tetra-ring antibiotics, and the discovery of penta-ring type polyketides in Chassis2.0. These findings underscore the potential of Chassis2.0 as an optimal platform for the discovery and overproduction of T2PKs.

Fig. 1. Examples of T2PKs classified based on the number of backbone cyclization and variations in polyketide chain length.

The products related to this study are highlighted in their respective colors.

Fig. 2. The selection of T2PKs heterologous expression host.

a The genetic architecture of the complete OTC BGC in S. rimosus ATCC10970. b The high-performance liquid chromatography (HPLC) results of the OTC heterologous expression strains. OTC, TC, and CTC standards serve as positive controls. The J1-022 serves as control. c Comparative analysis of colony phenotypes between S. aureofaciens and S. rimosus. d Relative acetyl-CoA accumulation levels per unit cell dry weight (CDW) in three strains across distinct growth phases. e Relative malonyl-CoA accumulation levels per CDW in three strains across distinct growth phases. f Relative NADP⁺ levels per unit CDW in three strains across distinct growth phases. g Relative NADPH levels per unit CDW in three strains across distinct growth phases. Bar figures show mean values with error bars indicating s.d. (standard deviations, n = 3 biological replicates). Statistical significance was denoted as follows: ns (not significant), *P < 0.05, **P < 0.001 (two-tailed unpaired t-test, uncorrected for multiple comparisons). Source data for this figure can be found in the source data file.

Fig. 3. The Construction of a T2PKs BGC-free chassis.

a The genetic architecture of the complete CTC BGC in S. aureofaciens J1-022. b The results of the HPLC comparing the Chassis1.0 and Chassis1.1. J1-022 serves as control; CTC standard serves as positive control. The colony phenotypes observed on the culture plate of mutants were presented. c S. aureofaciens chassis were constructed by executing iterative in-frame knockout. d Relative acetyl-CoA accumulation levels per unit CDW in J1-022 and three variant chassis across distinct growth phases. e Relative malonyl-CoA accumulation levels per CDW in J1-022 and three variant chassis across distinct growth phases. f Relative NADP⁺ levels per unit CDW in J1-022 and three variant chassis across distinct growth phases. g Relative NADPH levels per unit CDW in J1-022 and three variant chassis across distinct growth phases. Bar figures show mean values with error bars indicating s.d. (n = 3 biological replicates). Statistical significance was denoted as follows: ns (not significant), *P < 0.05, **P < 0.001 (two-tailed unpaired t-test, uncorrected for multiple comparisons). Source data for this figure can be found in the source data file.

Fig. 4. Efficient production of OTC in S. aureofaciens Chassis2.0.

a HPLC results of metabolite accumulation in different OTC heterologous expression chassis. OTC, TC and CTC standards serve as positive controls. b Quantification of the OTC titer in different OTC heterologous expression chassis. c The depiction of the downstream biosynthetic pathways for OTC and CTC in Chassis2.0_otc. d Comparison of minimal PKS expression levels per unit CDW between J1-22_otc and the variant chassis heterologously expressing OTC BGC across distinct growth phases. Bar figures show mean values with error bars indicating s.d. (n = 3 biological replicates). Statistical significance was denoted as follows: ns (not significant), *P < 0.05, **P < 0.001, ****P < 0.0001 (two-tailed unpaired t-test, uncorrected for multiple comparisons). Source data for this figure can be found in the source data file.

Fig. 5. The distinct outcomes in S. cerevisiae and Chassis2.0 utilizing identical downstream biosynthetic pathways of OTC.

In vitro and native OTC host results are based on previous research^,,.

Fig. 6. Efficient activation of tri-ring type pigmented T2PKs BGCs in Chassis2.0.

a The genetic architecture of the complete ACT BGC in S. coelicolor A3 (2). b Different coloration of phenotype on agar plates. c The coloration of crude extract in different pH environments. d The HRMS-based per-unit CDW quantification result of ACT and its derivatives in Chassis2.0_ACT versus S. coelicolor A3(2). e The color comparison of crude extract and the Q-TOF LC/MS analysis of primary accumulated products in Chassis2.0_FK. f The quantification result of FK in Chassis2.0_FK. The Chassis2.0 served as the blank control check. Bar figures show mean values with error bars indicating s.d. (n = 3 biological replicates). Statistical significance was denoted as follows: ns (not significant), *P < 0.05, **P < 0.001 (two-tailed unpaired t-test, uncorrected for multiple comparisons). Source data for this figure can be found in the source data file.

Fig. 7. The efficient activation of unidentified penta-ring type T2PKs BGC in Chassis 2.0.

a The genetic architecture of the unknown pentangular polyketide BGC in M. echinospora J1-020. b The color comparison of crude extract and the LC-UV/Q-TOF MS analysis of primary accumulated products in Chassis2.0_FK. c The product ion profiles of the activated products are presented. The red arrow indicates the precursor ion. d The quantification result of activated products in Chassis2.0_TLN. e The appearance of the purified products for compounds 1–3 is presented. The structure of TLN-1 (1) has been confirmed through NMR analysis and HRMS/MS analysis. Bar figures show mean values with error bars indicating s.d. (n = 3 biological replicates). Statistical significance was denoted as follows: ns (not significant), **P < 0.001 (two-tailed unpaired t-test, uncorrected for multiple comparisons). Source data for this figure can be found in the source data file.