Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin.

UNLABELLED: The Gram-positive bacterium Amycolatopsis sp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of the vdh gene, which codes for the central vanillin catabolism enzyme, vanillin dehydrogenase. This mutation resulted in improvement of the final concentration of vanillin by more than 2.2 g/liter, with a molar yield of 80.9%. Further improvement was achieved with constitutive expression of the vanillin anabolism genes ech and fcs, coding for the enzymes feruloyl-coenzyme A (CoA) synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech). The transcription of both genes was shown to be induced by ferulic acid, which explains the unwanted adaptation phase in the fermentation process before vanillin was efficiently produced by the wild-type cells. Through the constitutive and enhanced expression of the two genes, the adaptation phase was eliminated and a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. Moreover, an even higher final vanillin concentration of 22.3 g/liter was achieved, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest reported vanillin concentration reached in microbial fermentation processes without extraction of the product. Furthermore, the vanillin was produced almost without by-products, with a molar yield that nearly approached the theoretical maximum. IMPORTANCE: Much effort has been put into optimization of the biotechnological production of natural vanillin. The demand for this compound is growing due to increased consumer concerns regarding chemically produced food additives. Since this compound is toxic to most organisms, it has proven quite difficult to reach high concentrations and molar yields. This study shows that improvements in the final vanillin concentrations and molar yields can be made through a combination of modification of the fermentation parameters and molecular strain engineering, without the need for methods such as continuous extraction from the fermentation broth. Using this approach, we were able to reach a final vanillin concentration of 22.3 g/liter, which is the highest vanillin concentration reported to date that was generated with Amycolatopsis sp. ATCC 39116 without additional extraction of the toxic product.

Construction of expression vectors for metabolic engineering of the vanillin-producing actinomycete Amycolatopsis sp. ATCC 39116.

Amycolatopsis sp. ATCC 39116 is able to synthesize the important flavoring agent vanillin from cheap natural substrates. The bacterium is therefore of great interest for the industry and used for the fermentative production of vanillin. In order to improve the production of natural vanillin with Amycolatopsis sp. ATCC 39116, the strain has been genetically engineered to optimize the metabolic flux towards the desired product. Extensive metabolic engineering was hitherto hampered, due to the lack of genetic tools like functional promoters and expression vectors. In this study, we report the establishment of a plasmid-based gene expression system for Amycolatopsis sp. ATCC 39116 that allows a further manipulation of the genotype. Four new Escherichia coli-Amycolatopsis shuttle vectors harboring different promoter elements were constructed, and the functionality of these regulatory elements was proven by the expression of the reporter gene gusA, encoding a ß-glucuronidase. Glucuronidase activity was detected in all plasmid-harboring strains, and remarkable differences in the expression strength of the reporter gene depending on the used promoter were observed. The new expression vectors will promote the further genetic engineering of Amycolatopsis sp. ATCC 39116 to get insight into the metabolic network and to improve the strain for a more efficient industrial use.

Investigation of the Amycolatopsis sp. strain ATCC 39116 vanillin dehydrogenase and its impact on the biotechnical production of vanillin.

The actinomycete Amycolatopsis sp. strain ATCC 39116 is capable of synthesizing large amounts of vanillin from ferulic acid, which is a natural cell wall component of higher plants. The desired intermediate vanillin is subject to undesired catabolism caused by the metabolic activity of a hitherto unknown vanillin dehydrogenase (VDH(ATCC 39116)). In order to prevent the oxidation of vanillin to vanillic acid and thereby to obtain higher yields and concentrations of vanillin, the responsible vanillin dehydrogenase in Amycolatopsis sp. ATCC 39116 was investigated for the first time by using data from our genome sequence analysis and further bioinformatic approaches. The vdh gene was heterologously expressed in Escherichia coli, and the encoded vanillin dehydrogenase was characterized in detail. VDH(ATCC 39116) was purified to apparent electrophoretic homogeneity and exhibited NAD(+)-dependent activity toward vanillin, coniferylaldehyde, cinnamaldehyde, and benzaldehyde. The enzyme showed its highest level of activity toward vanillin at pH 8.0 and at a temperature of 44°C. In a next step, a precise vdh deletion mutant of Amycolatopsis sp. ATCC 39116 was generated. The mutant lost its ability to grow on vanillin and did not show vanillin dehydrogenase activity. A 2.3-times-higher vanillin concentration and a substantially reduced amount of vanillic acid occurred with the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant when ferulic acid was provided for biotransformation in a cultivation experiment on a 2-liter-bioreactor scale. Based on these results and taking further metabolic engineering into account, the Amycolatopsis sp. ATCC 39116 Δvdh::Km(r) mutant represents an optimized and industrially applicable platform for the biotechnological production of natural vanillin.

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16.

The ß-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner-Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929-2936, 2001), was expressed in R. eutropha H16 PHB(-)4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB(-)4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.