Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

333

298

Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDPglycosyltransferase, converting vanillin into vanillin ␤-D-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity.

Section: Resultsmentioning

confidence: 99%

De Novo Biosynthesis of Vanillin in Fission Yeast ( Schizosaccharomyces pombe ) and Baker's Yeast ( Saccharomyces cerevisiae )

Hansen¹,

Møller

Kock³

et al. 2009

333

298

“…Carboxylic acids are found throughout cellular metabolism, and many can be converted to aldehydes with the aid of a single enzyme. Prior to the detailed characterization and cloning of enzymes capable of broadly catalyzing aldehyde formation, various natural organisms ranging from actinomycetes to white rot fungi were tested for the innate ability to convert carboxylic acids into their corresponding aldehydes or alcohols (17)(18)(19)(20)(21). A significant advance occurred roughly 1 decade ago, when a carboxylic acid reductase (Car Ni ) from Nocardia iowensis was cloned into Escherichia coli and shown to be active on several aromatic carboxylic acids in vitro (22).…”

Section: Engineering Aldehyde Biosynthetic Reactions and Pathwaysmentioning

confidence: 99%

Microbial Engineering for Aldehyde Synthesis

Kunjapur

Prather

2015

145

129

Aldehydes are a class of chemicals with many industrial uses. Several aldehydes are responsible for flavors and fragrances present in plants, but aldehydes are not known to accumulate in most natural microorganisms. In many cases, microbial production of aldehydes presents an attractive alternative to extraction from plants or chemical synthesis. During the past 2 decades, a variety of aldehyde biosynthetic enzymes have undergone detailed characterization. Although metabolic pathways that result in alcohol synthesis via aldehyde intermediates were long known, only recent investigations in model microbes such as Escherichia coli have succeeded in minimizing the rapid endogenous conversion of aldehydes into their corresponding alcohols. Such efforts have provided a foundation for microbial aldehyde synthesis and broader utilization of aldehydes as intermediates for other synthetically challenging biochemical classes. However, aldehyde toxicity imposes a practical limit on achievable aldehyde titers and remains an issue of academic and commercial interest. In this minireview, we summarize published efforts of microbial engineering for aldehyde synthesis, with an emphasis on de novo synthesis, engineered aldehyde accumulation in E. coli, and the challenge of aldehyde toxicity.

“…Chemical methods for carboxylic acid reductions are limited, and they usually require prior derivatization and product deblocking with reactants containing competing functional groups. Biocatalytic reductions of carboxylic acids are attractive because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantiospecific, and the scope of the reaction is very broad (24, 32).Although microbial reductions of carboxylic acids, usually producing the acids' corresponding aldehydes or alcohols, have been observed with whole-cell reactions of bacteria and fungi (3,4,6,8,20,22,24,25,30,(36)(37)(38)40), enzymatic reductions of carboxylic acids are relatively new and unexploited biocatalytic reactions of great potential value in organic synthesis (12).Aldehyde oxidoreductases, also known as carboxylic acid reductases (CAR), require ATP, Mg 2ϩ , and NADPH as cofactors (16,17,18,21,24). The reduction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme in order to form mixed 5Ј-adenylic acid-carbonyl anhydride intermediates (9,15,25,27,39) that are subsequently reduced by hydride delivery from NADPH to form aldehyde products (16,25) (Fig.…”

mentioning

confidence: 99%

“…Aldehyde oxidoreductases, also known as carboxylic acid reductases (CAR), require ATP, Mg 2ϩ , and NADPH as cofactors (16,17,18,21,24). The reduction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme in order to form mixed 5Ј-adenylic acid-carbonyl anhydride intermediates (9,15,25,27,39) that are subsequently reduced by hydride delivery from NADPH to form aldehyde products (16,25) (Fig.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

He¹,

Li²,

Daniels

et al. 2004

107

100

We have cloned, sequenced, and expressed the gene for a unique ATP-and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K m values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.Aromatic, aliphatic, and alicyclic aldehydes and alcohols are useful intermediates in the chemical, pharmaceutical, and food industries. Chemical methods for carboxylic acid reductions are limited, and they usually require prior derivatization and product deblocking with reactants containing competing functional groups. Biocatalytic reductions of carboxylic acids are attractive because the substrates are water soluble, blocking chemistry is not necessary, reductions are enantiospecific, and the scope of the reaction is very broad (24, 32).Although microbial reductions of carboxylic acids, usually producing the acids' corresponding aldehydes or alcohols, have been observed with whole-cell reactions of bacteria and fungi (3,4,6,8,20,22,24,25,30,(36)(37)(38)40), enzymatic reductions of carboxylic acids are relatively new and unexploited biocatalytic reactions of great potential value in organic synthesis (12).Aldehyde oxidoreductases, also known as carboxylic acid reductases (CAR), require ATP, Mg 2ϩ , and NADPH as cofactors (16,17,18,21,24). The reduction is a stepwise process involving initial binding of both ATP and the carboxylic acid to the enzyme in order to form mixed 5Ј-adenylic acid-carbonyl anhydride intermediates (9,15,25,27,39) that are subsequently reduced by hydride delivery from NADPH to form aldehyde products (16,25) (Fig. 1). Aromatic CARs have been purified to homogeneity only from Neurospora (17) and Nocardia (21, 24) species. Although N-terminal and internal amino acid sequences were reported for our Nocardia enzyme (24), sequences have never been determined for any gene coding for CARs. CAR from Nocardia sp. strain NRRL 5646 has an extremely wide substrate range, and it enantiospecifically reduces carboxylic acids (8,24,32). We report here the cloning and expression of the first CAR gene, car, and the use of cloned enzyme in vitro and in vivo to reduce carboxylic acid substrates.Strains, plasmids, media, and growth. Bacteria and plasmids used in this study are listed in Table 1. Nocardia sp. strain NRRL 5646 (14) was grown at 30°C in Luria-Bertani (LB) medium containing 0.05% Tween 80 (vol/vol [liquid medium only]). With Escherichia coli as the recombinant host for pHAT-based vectors, cells were grown at 37°C on solid or in liquid LB medium. Ampicillin (100 g/ml) was incorporated into LB medium to select for recombinants, and isopropyl-␤-D-thiogalactopyranoside (IPTG) (1 mM) and/or 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranosi...