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Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid.

ABSTRACT: p-Coumaric acid is the precursor of phenylpropanoids, which are plant secondary metabolites that are beneficial to human health. Tyrosine ammonia lyase catalyzes the production of p-coumaric acid from tyrosine. Because of their photosynthetic ability and biosynthetic versatility, cyanobacteria are promising candidates for the production of certain plant metabolites, including phenylpropanoids. Here, we produced p-coumaric acid in a strain of transgenic cyanobacterium Synechocystis sp. Pasteur Culture Collection 6803 (hereafter Synechocystis 6803). Whereas a strain of Synechocystis 6803 genetically engineered to express sam8, a tyrosine ammonia lyase gene from the actinomycete Saccharothrix espanaensis, accumulated little or no p-coumaric acid, a strain that both expressed sam8 and lacked slr1573, a native hypothetical gene shown here to encode a laccase that oxidizes polyphenols, produced ?82.6 mg/L p-coumaric acid, which was readily purified from the growth medium.

SUBMITTER: Xue Y

PROVIDER: S-EPMC4084433 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid.

Xue Yong Y Zhang Yan Y Cheng Dan D Daddy Soumana S He Qingfang Q

Proceedings of the National Academy of Sciences of the United States of America 20140609 26

p-Coumaric acid is the precursor of phenylpropanoids, which are plant secondary metabolites that are beneficial to human health. Tyrosine ammonia lyase catalyzes the production of p-coumaric acid from tyrosine. Because of their photosynthetic ability and biosynthetic versatility, cyanobacteria are promising candidates for the production of certain plant metabolites, including phenylpropanoids. Here, we produced p-coumaric acid in a strain of transgenic cyanobacterium Synechocystis sp. Pasteur Cu ...[more]

PMID: 24927550

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Project description:BackgroundPhenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis.ResultsWe eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout.ConclusionsHPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

Project description:BackgroundPhenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis).ResultsA selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Δslr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L-1, respectively, after 4 days.ConclusionsProduction of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids.

Project description:Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroGfbr and trpEfbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroGfbr and trpEfbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2 We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.IMPORTANCE Aromatic amino acids such as tryptophan are primarily used as additives in the animal feed industry and are typically produced using genetically engineered heterotrophic organisms such as Escherichia coli This involves a two-step process, where the substrate such as molasses is first obtained from plants followed by fermentation by heterotrophic organisms. We have engineered photoautotrophic cyanobacterial strains by a combination of random mutagenesis and metabolic engineering. These strains grow on CO2 as the sole carbon source and utilize light as the sole energy source to produce tryptophan, thus converting the two-step process into a single step. Our results show that combining random mutagenesis and metabolic engineering was superior to either approach alone. This study also builds a foundation for further engineering of cyanobacteria for industrial tryptophan production.

Dataset Information

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid.

Publications

Genetically engineering Synechocystis sp. Pasteur Culture Collection 6803 for the sustainable production of the plant secondary metabolite p-coumaric acid.

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