Engineering Vibrio sp. SP1 for the production of carotenoids directly from brown macroalgae

Park, Sungwoo; Cho, Sung Won; Lee, Yungyu; Choi, Mincheol; Yang, Joseph; Lee, Hojun; Seo, Sang Woo

doi:10.1016/j.csbj.2021.03.007

Cited by 9 publications

(11 citation statements)

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“…In this host, previous studies indicated that the tacI promoter produced GFP upon activation with IPTG, while induction of the tet promoter resulted in low GFP yields (Tschirhart et al, 2019 ). The constitutive and inducible versions of the synthetic lac and tet promoters were evaluated in the pColE1‐derived plasmid with ~300 copies per cell (Tschirhart et al, 2019 ), except for the constitutive V2Lac , which was evaluated in pCloDF13‐derived vector with ~64 copies per cell in this strain (Park et al, 2021 ). The constitutive V2Lac (Figure 7Ai , green triangles) and V4Tc (Figure 7Bi , purple circles) promoters outperformed tacI (open squares) by ~16 and ~2‐fold respectively, while V3Lac (Figure 7Ai , blue diamonds ) , V1Tc (Figure 7Bi , red triangles) and V2Tc (Figure 7Bi , green triangles) produced similar levels as tacI .…”

Section: Resultsmentioning

confidence: 99%

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Rincón

Farny

2023

Microbial Biotechnology

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Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the −10 and −35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus −10 and −35 sequence boxes strongly targeted by σ 70 , incorporation of a strong ribosome binding site recognized broadly by Gram-negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which significantly improves the signal-to-background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregateprone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.

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Section: Resultsmentioning

confidence: 99%

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Rincón

Farny

2023

Microbial Biotechnology

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“…In this host, previous studies indicated that the tacI promoter produced GFP upon activation with IPTG, while induction of the tet promoter resulted in low GFP yields [21]. The constitutive and inducible versions of the synthetic lac and tet promoters were evaluated in the pColE1 derived plasmid with ∼300 copies per cell [21], except for the constitutive V2Lac , which was evaluated in pCloDF13 derived vector with ∼64 copies per cell in this strain [42]. The constitutive V2Lac (Fig.…”

Section: Resultsmentioning

confidence: 99%

Unlocking the strength of inducible promoters in gram-negative bacteria

Rincón

Farny

2022

Preprint

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Inducible promoters, such as the lac and tet promoters, are ubiquitous biotechnology tools. Inducible bacterial promoters have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulator proteins (e.g., LacI and TetR, and the -10 and -35 consensus sequences required to recruit the sigma (σ) subunits of RNA polymerase to initiate transcription. Despite their widespread use in molecular biology, there remain problems with current inducible expression systems. Leaky transcription in the OFF state remains a particular challenge. Here we have updated the architecture of the lac and tet expression systems to improve their strength, control, and portability. We modified the genetic architecture of the lac and tet expression systems to contain consensus -10 and -35 sequence boxes to be strongly targeted by σ70, to incorporate of a strong ribosome binding site recognized broadly by gram-negative bacteria, and to independently control of the transcriptional regulators by optimized constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which we demonstrate significantly improves the signal-to-background ratio of promoter measurement assays over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.SignificanceMany of the latest advances in pharmaceuticals, materials, and foods involve the production of recombinant proteins from bacterial hosts. However, the regulated production of enzymes and functional protein products that are toxic to their microbial hosts remains a challenge. Our work provides new tools that enable tight control over expression of protein products in bacterial host strains. We show that our tools function not only in the broadly utilized Escherichia coli, but also in other gram-negative bacteria including the soil organism Pseudomonas putida and the marine bacterium Vibrio natriegens. Our technology will facilitate more efficient production of a broader range of protein products in diverse microbial hosts.

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“…SP1 with brown algae as the substrate (2.98 mg/L) was approximately 6 times higher than that with P. atlantica T6c. 17 The high yield of β-carotene in Vibrio sp. SP1 could be due to the difference in construction of the β-carotene biosynthetic pathway.…”

Section: Discussionmentioning

confidence: 99%

“…dhg were reported as effective metabolic chassis for the bioconversion of brown algal feedstock into value-added chemicals. 17,18 However, none of these microbes were used for the deconstruction and metabolic conversion of red algal galactans such as agar and carrageenan. MHBs including Saccharophagus degradans 2−40, 19 Zobellia galactanivorans Dsij T , 20 Colwellia echini A3 T , 21,22 Pseudoalteromonas atlantica T6c, 23 and Paraglaciecola hydrolytica S66 T24 have been extensively studied for the deconstruction and metabolism of agar and carrageenan.…”

Section: Introductionmentioning

confidence: 99%

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Constructing Marine Bacterial Metabolic Chassis for Potential Biorefinery of Red Algal Biomass and Agaropectin Wastes

et al. 2023

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Marine red algal biomass is a promising feedstock for sustainable production of value-added chemicals. However, the major constituents of red algal biomass, such as agar and carrageenan, are not easily assimilated by most industrial metabolic chassis developed to date. Synthetic biology offers a solution by utilizing nonmodel organisms as metabolic chassis for consolidated biological processes. In this study, the marine heterotrophic bacterium Pseudoalteromonas atlantica T6c was harnessed as a metabolic chassis to produce value-added chemicals from the affordable red algal galactans or agaropectin, a byproduct of industrial agarose production. To construct a heterologous gene expression device in P. atlantica T6c, promoters related to agar metabolism were screened from the differentially expressed genes using RNA-Seq analysis. The expression device was built and tested with selected promoters fused to a reporter gene and tuned by incorporation of a cognate repressor predicted from the agar-specific polysaccharide utilization locus. The feasibility of the marine bacterial metabolic chassis was examined by introducing the biosynthetic gene clusters of β-carotene and violacein. Our results demonstrate that the metabolic chassis platform enables direct conversion of low-cost red algal galactans or industrial waste agaropectin into valuable bioactive pigments without any pretreatment of biomass. The developed marine bacterial chassis could potentially be used in a biorefinery framework to produce value-added chemicals from marine algal galactans.

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Engineering Vibrio sp. SP1 for the production of carotenoids directly from brown macroalgae

Abstract: Graphical abstract

Cited by 9 publications

References 50 publications

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Unlocking the strength of inducible promoters in gram-negative bacteria

Constructing Marine Bacterial Metabolic Chassis for Potential Biorefinery of Red Algal Biomass and Agaropectin Wastes

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