Dataset Information

Engineering Escherichia coli to convert acetic acid to ?-caryophyllene.

ABSTRACT: Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce ?-caryophyllene by the engineered E. coli using acetic acid as the only carbon source.Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to ?-caryophyllene, acetyl-CoA synthase (ACS), ?-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance ?-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of ?-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and ?-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to ?-caryophyllene (gram to gram) reached 2.1%. The yield of ?-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield.In the present study, a novel biosynthetic pathway for ?-caryophyllene has been investigated by means of conversion of acetic acid to ?-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in ?-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for ?-caryophyllene synthesis.

SUBMITTER: Yang J

PROVIDER: S-EPMC4857421 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering Escherichia coli to convert acetic acid to β-caryophyllene.

Yang Jianming J Nie Qingjuan Q

Microbial cell factories 20160505

<h4>Background</h4>Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce β-caryophyllene by the engineered E. coli using acetic acid as the only carbon source.<h4>Results</h4>Firstly, to constr ...[more]

PMID: 27149950

Similar Datasets

Project description:BackgroundThe production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes.ResultsA set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities.ConclusionsThe use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

Project description:BackgroundIndole pyruvic acid (IPA) is a versatile platform intermediate and building block for a number of high-value products in the pharmaceutical and food industries. It also has a wide range of applications, such as drugs for the nervous system, cosmetics, and luminophores. Chemical synthesis of IPA is a complicated and costly process. Moreover, through the biosynthesis route employing L-amino acid oxidase, the byproduct hydrogen peroxide leads the degradation of IPA. TdiD, identified as a specific tryptophan aminotransferase, could be an alternative solution for efficient IPA biosynthesis.ResultsEscherichia coli strain W3110, which demonstrates basic production when supplied with tryptophan, was engineered for IPA biosynthesis. Several strategies were implemented to improve IPA production. First, through incorporating the codon-optimized tdiD into W3110, IPA levels increased from 41.54 ± 1.26 to 52.54 ± 2.08 mg/L. Second, after verifying the benefit of an increased phenylpyruvate pool, a YL03 strain was constructed based on a previously reported mutant strain of W3110 with a plasmid carrying aroF fbr and pheA fbr to further improve IPA production. The recombinant YL03 strain accumulated IPA at 158.85 ± 5.36 mg/L, which was 3.82-fold higher than that of the wild-type W3110 strain. Third, optimization of tdiD co expression was carried out by replacing the Trc promoter with a series of constitutively active promoters along with increasing the plasmid copy numbers. The highest IPA production was observed in YL08, which achieved 236.42 ± 17.66 mg/L and represented a greater than 5-fold increase as compared to W3110. Finally, the effects of deletion and overexpression of tnaA on IPA biosynthesis were evaluated. The removal of tnaA led to slightly reduced IPA levels, whereas the overexpression of tnaA resulted in a considerable decline in production.ConclusionsThis study illustrates the feasibility of IPA biosynthesis in E. coli through tdiD. An efficient IPA producing strain, YL08, was developed, which provides a new possibility for biosynthesis of IPA. Although the final production was limited, this study demonstrates a convenient method of IPA synthesis.

Project description:Fermentative production of many attractive biorenewable fuels and chemicals is limited by product toxicity in the form of damage to the microbial cell membrane. Metabolic engineering of the production organism can help mitigate this problem, but there is a need for identification and prioritization of the most effective engineering targets. Here, we use a set of previously characterized environmental Escherichia coli isolates with high tolerance and production of octanoic acid, a model membrane-damaging biorenewable product, as a case study for identifying and prioritizing membrane engineering strategies. This characterization identified differences in the membrane lipid composition, fluidity, integrity, and cell surface hydrophobicity from those of the lab strain MG1655. Consistent with previous publications, decreased membrane fluidity was associated with increased fatty acid production ability. Maintenance of high membrane integrity or longer membrane lipids seemed to be of less importance than fluidity. Cell surface hydrophobicity was also directly associated with fatty acid production titers, with the strength of this association demonstrated by plasmid-based expression of the multiple stress resistance outer membrane protein BhsA. This expression of bhsA was effective in altering hydrophobicity, but the direction and magnitude of the change differed between strains. Thus, additional strategies are needed to reliably engineer cell surface hydrophobicity. This work demonstrates the ability of environmental microbiological studies to impact the metabolic engineering design-build-test-learn cycle and possibly increase the economic viability of fermentative bioprocesses.IMPORTANCE The production of bulk fuels and chemicals in a bio-based fermentation process requires high product titers. This is often difficult to achieve, because many of the target molecules damage the membrane of the microbial cell factory. Engineering the composition of the membrane in order to decrease its vulnerability to this damage has proven to be an effective strategy for improving bioproduction, but additional strategies and engineering targets are needed. Here, we studied a small set of environmental Escherichia coli isolates that have higher production titers of octanoic acid, a model biorenewable chemical, than those of the lab strain MG1655. We found that membrane fluidity and cell surface hydrophobicity are strongly associated with improved octanoic acid production. Fewer genetic modification strategies have been demonstrated for tuning hydrophobicity relative to fluidity, leading to the conclusion that there is a need for expanding hydrophobicity engineering strategies in E. coli.

Dataset Information

Engineering Escherichia coli to convert acetic acid to ?-caryophyllene.

Publications

Engineering Escherichia coli to convert acetic acid to β-caryophyllene.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets