Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2S)-Naringenin Production from l-Tyrosine in Escherichia coli

Abstract

Malonyl coenzyme A (malonyl-CoA) is an important precursor for the synthesis of natural products, such as polyketides and flavonoids. The majority of this cofactor often is consumed for producing fatty acids and phospholipids, leaving only a small amount of cellular malonyl-CoA available for producing the target compound. The tuning of malonyl-CoA into heterologous pathways yields significant phenotypic effects, such as growth retardation and even cell death. In this study, fine-tuning of the fatty acid pathway in Escherichia coli with antisense RNA (asRNA) to balance the demands on malonyl-CoA for target-product synthesis and cell health was proposed. To establish an efficient asRNA system, the relationship between sequence and function for asRNA was explored. It was demonstrated that the gene-silencing effect of asRNA could be tuned by directing asRNA to different positions in the 5'-UTR (untranslated region) of the target gene. Based on this principle, the activity of asRNA was quantitatively tailored to balance the need for malonyl-CoA in cell growth and the production of the main flavonoid precursor, (2S)-naringenin. Appropriate inhibitory efficiency of the anti-fabB/fabF asRNA improved the production titer by 431% (391 mg/liter). Therefore, the strategy presented in this study provided a useful tool for the fine-tuning of endogenous gene expression in bacteria.

FIG 1

Heterologous biosynthesis of (2S)-naringenin from l-tyrosine in E. coli. The overall metabolic pathway performed heterologous biosynthesis of (2S)-naringenin from l-tyrosine in E. coli. TAL, tyrosine ammonia lyase; 4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; MatB, R. trifolii malonate synthetase; MatC, R. trifolii malonate carrier protein; FabD, malonyl-CoA-acyl carrier protein transacylase; FabB, β-ketoacyl-acyl carrier protein synthase (KAS) I; and FabF, β-ketoacyl-acyl carrier protein synthase (KAS) II. The overall biosynthetic pathway was constructed using premade modules from a plasmid toolbox; FabB and FabF were repressed by asRNAs.

FIG 2

Construction of initial asRNA expression system. (A) The growth patterns of asRNA-regulated strains compared to the control were measured. (B) pRSF-PTasRNA is derived from pRSFDuet-1(Mut). (C) The transcriptional levels of fabB mRNA from asRNA-regulated strains also were calculated relative to the control. pRSFDuet-1(Mut), E. coli BL21(DE3) strains with pRSFDuet-1(Mut); pCDF-asRNA(147), E. coli BL21(DE3) strains with pCDF-asRNA(147); pRSF-asRNA(147), E. coli BL21(DE3) strains with pRSF-asRNA(147); pRSF-PTasRNA(147), E. coli BL21(DE3) strains with pRSF-PTasRNA(147). (D) Predicted secondary structure of the PTasRNA (13). Ptrc, trc promoter; lac^O, lac operator; PT, 38-bp paired-terminal sequence; rrnB, stem-loop structure of the rrnB terminator. Error bars are standard deviations from biological triplicates.

FIG 3

Effect of binding position of asRNAs on inhibition efficiency. (A) The binding sequences of six anti-fabB asRNA variants. The length of the 5′-UTR was set as 158 bp (R11-R18), 128 bp (R21-R28), 98 bp (R31-R38), 68 bp (R41-R48), 38 bp (R51-R58), or 0 bp (R61-R68). In each set, the length of the fabB gene coding region was set as 0 to 847 bp, 0 to 747 bp, 0 to 647 bp, 0 to 547 bp, 0 to 447 bp, 0 to 347 bp, 0 to 247 bp, or 0 to 147 bp (e.g., R11-R18). (B) The binding sequences of four anti-fabF asRNA variants. The length of the 5′-UTR was set as 87 bp (R11-R18), 57 bp (R21-R28), 27 bp (R31-R38), or 0 bp (R41-R48). In each set, the length of the fabF gene coding region was set as 0 to 813 bp, 0 to 713 bp, 0 to 613 bp, 0 to 513 bp, 0 to 413 bp, 0 to 313 bp, 0 to 213 bp, and 0 to 113 bp (e.g., R11-R18). (C) The growth patterns of E. coli strains with anti-fabB asRNA sets. The control was E. coli BL21 strains with pRSFDuet-1(Mut). (D) qPCR analysis for downregulation of fabB gene transcription by asRNA. (E) qPCR analysis for downregulation of fabF gene transcription by asRNA. Error bars are standard deviations from biological triplicates.

FIG 4

Effect of an asRNA system on fatty acid contents in E. coli. (A) The concentration of saturated fatty acids (myristic acid, palmitic acid, and stearic acid) and unsaturated fatty acids (palmitoleic acid and cis-11-octadecenoic acid) in E. coli strains with anti-fabB asRNA sets. (B) The concentration of saturated fatty acids (myristic acid, palmitic acid, and stearic acid) and unsaturated fatty acids (palmitoleic acid and cis-11-octadecenoic acid) in E. coli strains with anti-fabF sets. The control was E. coli BL21 strains with pRSFDuet-1(Mut). Error bars are standard deviations from biological triplicates.

FIG 5

Simultaneously repressing fabB and fabF by asRNA system. The transcriptional levels of target gene mRNA from asRNA-regulated strains also were calculated relative to the control. Control indicates BL21(DE3) strains transformed with pRSFDuet-1(Mut). Designations using the format fabF-RX+fabB-RY were BL21(DE3) strains transformed with plasmids expressing fabF asRNA sequence (RX) and fabB asRNA sequence (RY) simultaneously, where X is 18, 28, or 38 and Y is 18, 28, 38, 48, or 58. Error bars are standard deviations from biological triplicates.

FIG 6

Effect of asRNA system on the intracellular malonyl-CoA concentration. Control indicates BL21(DE3) strains transformed with pRSFDuet-1(Mut). Designations using the format fabF-RX+fabB-RY are BL21(DE3) strains transformed with plasmids expressing fabF asRNA sequence (RX) and fabB asRNA sequence (RY) simultaneously, where X is 18, 28, or 38 and Y is 18, 28, 38, 48, or 58. The effects of asRNA systems on malonyl-CoA concentration and the final OD₆₀₀ of engineered strains were investigated. Error bars are standard deviations from biological triplicates.

FIG 7

Effect of various asRNA systems on (2S)-naringenin production. Control, strains containing the (2S)-naringenin heterologous pathway without an asRNA system. Designations using the format fabF-RX+fabB-RY indicate the control strain transformed with plasmids expressing fabF asRNA sequence (RX) and fabB asRNA sequence (RY) simultaneously, where X is 18, 28, and 38 and Y is 38, 48, and 58. The final OD₆₀₀ of cultures and concentrations of malonyl-CoA, p-coumaric acid, and (2S)-naringenin were measured from production strains after a total fermentation time of 48 h. Error bars are standard deviations from biological triplicates.