Enhancement of Patchoulol Production in Escherichia coli via Multiple Engineering Strategies

Abstract

As a natural sesquiterpene compound with numerous biological activities, patchoulol has extensive applications in the cosmetic industry and potential usage in pharmaceuticals. Although several patchoulol-producing microbial strains have been constructed, the low productivity still hampers large-scale fermentation. Escherichia coli possesses the ease of genetic manipulation and simple nutritional requirements and does not comprise competing pathways for the farnesyl diphosphate (FPP) precursor, showing its potential for patchoulol biosynthesis. Here, combinatorial strategies were applied to produce patchoulol in E. coli. The initial strain was constructed, and it produced 14 mg/L patchoulol after fermentation optimization. Patchoulol synthase (PTS) was engineered by semirational design, resulting in improved substrate binding affinity and a patchoulol titer of 40.3 mg/L; the patchoulol titer reached 66.2 mg/L after fusing of PTS with FPP synthase. To further improve the patchoulol production, the genome of an efficient chassis strain was engineered by deleting the competitive routes for acetate, lactate, ethanol, and succinate synthesis and cumulatively enhancing the expression of efflux transporters, which improved patchoulol production to 338.6 mg/L. When tested in a bioreactor, the patchoulol titer and productivity were further improved to 970.1 mg/L and 199 mg/L/d, respectively, and were among the highest levels reported using mineral salt medium.

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

“…Since peptide linker composition and length in enzyme fusions was previously reported to affect terpene production [4][5][6]21 , we designed the FPPS-NES fusion constructs with four different peptide linkers: flexible linkers AGGGGTGGA and (AGGGGTGGA)2, helical linker (EAAAK)2, and rigid linker (PT)4P (Figure 1b). The fusions were compared against a 'free' enzyme control that expresses FPPS and NES as separate proteins.…”

“…Furthermore, an in vitro study with purified enzymes found that fusion constructs of FPPS and epi-aristolochene synthase outperformed a mixture of the two individual enzymes in the coupled reaction 17 . However, this does not explain why the opposite was observed in other terpenoid bioproduction studies 5,6 , and the fact that the performance of enzyme fusions can vary when the order of the two fusion partners is swapped 6,16,[18][19][20][21] . Despite the popularity of the enzyme fusion approach, limited work has been done to interrogate the mechanism(s) of titre enhancement from enzyme fusion in detail.…”

“…The translational fusion of enzymes that catalyse successive steps in a reaction pathway has been widely adopted as a straightforward, orthogonal, and host-agnostic metabolic engineering strategy. This strategy has proven particularly effective in the case of isoprenoid (terpenoid) production, where the fusion of a prenyl diphosphate synthase (such as farnesyl diphosphate synthase, FPPS) with the corresponding terpene synthase improved the production of diverse products such as patchoulol 4,5 , linalool 6 , sabinene 7 , and sclareol 8 .…”

Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia coli

“…TPs application is usually limited by their catalytic efficiency, substrate specificity, and stability. Previous reports have shown that plant TPs have such a strong functional plasticity and that minor modifications can significantly alter the properties of the enzyme. − In recent years, many efforts have been made to analyze the catalytic specificity and the associated residues of TPs through enzyme mutagenesis. The methods are focused on the following areas: (1) targeted mutation of conserved structural domains, (2) alignment of active residues of TPs, (3) analysis of a large number of homologous gene sequences followed by the most conserved amino acid residues as candidates for targeted mutation, and (4) error-prone PCR combined with high-throughput screening.…”

Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae

“…Successful examples include the production of geraniol, limonene, linalool, (+)-borneol, citronellol, and nerol. Efforts to improve the efficiency of monoterpene production mainly focus on: (1) improving the geranyl diphosphate (GPP) pool by overexpressing the upstream pathway genes; (2) selecting for monoterpene synthases with higher activity and the same catalytic function; (3) improving the expression level or catalytic activities of monoterpene synthases by truncating the N-terminal transit peptide; and (4) constructing genetic fusions to bring enzymes together for the catalysis of a cascade of reactions [ [9] , [10] , [11] , [12] , [13] ]. It is highly possible that high-titer production of (-)-borneol could be achieved by integrating these strategies to maximize flux toward target products.…”