TY - JOUR
T1 - Hybrid phenolic-inducible promoters towards construction of self-inducible systems for microbial lignin valorization
AU - Varman, Arul
AU - Follenfant, Rhiannon
AU - Liu, Fang
AU - Davis, Ryan W.
AU - Lin, Yone K.
AU - Singh, Seema
N1 - Funding Information:
This research has been supported by the National Science Centre, Poland, under grant No. 2015/19/P/ST3/03541, 2015/19/B/ST4/02721, and 2017/26/D/NZ1/00466. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 665778. This research was supported in part by PLGrid Infrastructure.
Funding Information:
This project was supported by Laboratory Directed Research and Development Program 16-0758 of Sandia National Laboratories to PI Seema Singh. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the US Department of Energy’s National Nuclear Security Administration under Contract DE-AC04-94AL85000. PI Singh acknowledges partial funding support during manuscript writing by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (DE-AC02-05CH11231), Office of Basic Energy Sciences and Division of Chemical Sciences, Geosciences and Biosciences.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/6/28
Y1 - 2018/6/28
N2 - Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, P emrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters P vtac, P vtrc, and P vtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, P emrR. In the presence of vanillic acid, P vtac, P vtrc, and P vtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with P emrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.
AB - Background: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. Results: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, P emrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters P vtac, P vtrc, and P vtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, P emrR. In the presence of vanillic acid, P vtac, P vtrc, and P vtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with P emrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. Conclusions: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.
KW - Biochemicals
KW - Hybrid promoters
KW - Lignin valorization
KW - Phenolic inducers
KW - Promoter engineering
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U2 - 10.1186/s13068-018-1179-8
DO - 10.1186/s13068-018-1179-8
M3 - Article
AN - SCOPUS:85049173847
VL - 11
SP - 1
EP - 13
JO - Biotechnology for Biofuels
JF - Biotechnology for Biofuels
SN - 1754-6834
IS - 1
ER -