Systematic engineering for high-yield production of viridiflorol and amorphadiene in auxotrophic Escherichia coli

Shukal, Sudha; Chen, Xixian; Zhang, Congqiang

doi:10.1016/j.ymben.2019.07.007

Cited by 83 publications

(84 citation statements)

References 30 publications

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“…In order to test the effectiveness and robustness of the system at larger scale, we first constructed a single-plasmid strain by grafting gene age and neuB from plasmid pP43NMK to pBUA-P 224 (Fig. 5c) 41 . At the same time, seven different promoters were tested to express gene age and neuB on pBUA-P 224 .…”

Section: Resultsmentioning

confidence: 99%

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

et al. 2020

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Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature.

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Section: Resultsmentioning

confidence: 99%

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

et al. 2020

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“…IP03/ pAaLINS-ispA* and IP04/pAaLINS-ispA* were unable to completely consume the initial glucose in 48 h (43 ± 0.6 and 36 ± 0.6 g/L), unlike SWITCH-PphoC Δgcd/pAaL-INS-ispA*, and consequently produced only 0.8 ± 0.0 and 1.0 ± 0.0 g/L of (S)-linalool, respectively (Table 1). This result indicates that their lower sugar consumptions may result from intracellular accumulation of cytotoxic intermediates such as IPP and DMAPP [7,33]. This assumption was supported by the observation that SWITCH-PphoC Δgcd and IP04 harboring the empty vector (pACYC177), which are likely to accumulate IPP/ DMAPP intracellularly because of the lack of linalool synthase, were unable to completely consume all initial glucose in 48 h (43 ± 3.3 and 38 ± 1.7 g/L, Table 1).…”

Section: Search For Putative Rate-limiting Reaction To Increase (S)-lmentioning

confidence: 96%

“…Several TPSs have been identified as primary bottlenecks in terpene biosynthesis because of their poor in vivo properties [35,36]. In the experiments described above, AaLINS and ispA*, which were codon-optimized for Synechocystis [15], were used even in P. ananatis; however, the codon usages of heterologous eukaryotic genes are commonly optimized for the prokaryotic host [6,7,31,36] in order to improve their translation rate or efficiency. Therefore, the plasmid pAaLINS_pa-ispA*_pa expressing AaLINS_pa and ispA*_pa which were optimized to match the codonpreference of P. ananatis was constructed to increase AaLINS production or activity (Additional file 1: Figure S1 and S2).…”

Section: Approaches To Improve Intracellular Aalins Activitymentioning

confidence: 99%

“…Additionally, microbial production allows for enantioselective production of terpenes, which is difficult via chemical synthesis, by exploiting the stereoselectivity (enantioselectivity) of enzymatic reactions such as enantiopure production of (R)-α-ionone [5] and ( −)-α-bisabolol [6] in Escherichia coli. However, examples of fermentative terpene production reaching 10 g/L titer have been confined to four molecules, viridiflorol, amorpha-4,11-diene [7], β-farnesene [8] (sesquiterpenes), and isoprene (a hemiterpene) [9], according to our literature search [10].…”

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confidence: 99%

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Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

et al. 2021

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Background Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. Results Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic β-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in “dual-phase” fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. Conclusions This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

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“…Drawbacks that include the instability and the necessity of use of antibiotics in plasmid expression systems limit the industrial use of plasmid expression systems. To overcome these drawbacks associated with plasmid expression systems, a chemically inducible chromosomal evolution (CIChE) system (Tyo et al, 2009) and an auxotrophic system (Shukal et al, 2019) were developed to achieve high gene copy expression. However, the constructed CIChE strains contained an antibiotic resistance marker (chloramphenicol resistance) (Tyo et al, 2009).…”

Section: Concluding Remarks and Future Prospectsmentioning

confidence: 99%

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

Shen

Niu

Yan

et al. 2020

Front. Bioeng. Biotechnol.

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Aromatic compounds derived from aromatic amino acids are an important class of diverse chemicals with a wide range of industrial and commercial applications. They are currently produced via petrochemical processes, which are not sustainable and eco-friendly. In the past decades, significant progress has been made in the construction of microbial cell factories capable of effectively converting renewable carbon sources into value-added aromatics. Here, we systematically and comprehensively review the recent advancements in metabolic engineering and synthetic biology in the microbial production of aromatic amino acid derivatives, stilbenes, and benzylisoquinoline alkaloids. The future outlook concerning the engineering of microbial cell factories for the production of aromatic compounds is also discussed.

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Systematic engineering for high-yield production of viridiflorol and amorphadiene in auxotrophic Escherichia coli

Cited by 83 publications

References 30 publications

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids

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