Development of an Improved System for the Generation of Knockout Mutants of Amycolatopsis sp. Strain ATCC 39116

Abstract

The Gram-positive actinomycete Amycolatopsis sp. strain ATCC 39116 is used for the industrial production of natural vanillin. Previously, the only gene deletion performed in this strain targeted the gene vdh, coding for a vanillin dehydrogenase. The generation of this mutant suffered from a high number of illegitimate recombinations and a low rate of homologous recombination. To alleviate this, we constructed an optimized deletion system based on a modified suicide vector. Thereby, we were able to increase the rate of homologous integration from less than 1% of the analyzed clones to 20% or 50%, depending on the targeted gene. We were furthermore able to reduce the screening effort needed to identify homogenotes through the use of the rpsL gene from Saccharopolyspora erythraea, which confers streptomycin sensitivity on clones still carrying the suicide vector. The new suicide vector is p6SUI5ERPSL, and its applicability was demonstrated by the deletion of three Amycolatopsis gene clusters. The deletion of the first of the gene clusters, coding for an aldehyde oxidase (yagRST), led to no altered phenotype compared to the parent strain; deletion of the second, coding for a vanillic acid decarboxylase (vdcBCD), led to a phenotype that was strongly impaired in its growth with vanillic acid as the sole carbon source and also unable to form guaiacol; and deletion of the third, coding for a vanillate demethylase (vanAB), led to only a negligible impact in comparison. Therefore, we showed that decarboxylation of vanillic acid is the main degradation pathway in Amycolatopsis sp. ATCC 39116 while the demethylation plays only a minor role and does not compensate the deletion of vdcBCD.IMPORTANCE Amycolatopsis sp. ATCC 39116 is an important microorganism used for the production of natural vanillin from ferulic acid. In contrast to this importance, it has previously been shown that this strain is hard to manipulate on a genetic level. We therefore generated an optimized system to facilitate the deletion of genes in this strain. This allowed us to greatly reduce the time and work requirements for generating deletions. This could allow the improvement of vanillin production in the future and also the elucidation of metabolic pathways. To test our deletion system, we deleted three gene clusters in Amycolatopsis sp. ATCC 39116. One showed no involvement in the metabolism of vanillin, while the second proved to be the main pathway of vanillic acid degradation and completely stopped the formation of the off-flavor guaiacol. The third appeared to have only a negligible impact on the degradation of vanillic acid.

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin

“…In the cases where bacterial strains have been engineered and optimized to produce bioproducts from lignin-derived compounds from G-and S-type lignin, aromatic O-demethylation is a rate-limiting step (14)(15)(16). Accordingly, there has been significant interest in the mechanistic understanding of the three enzyme types known to catalyze aromatic O-demethylation: tetrahydrofolate-dependent O-demethylases (17,18), Rieske-type nonheme iron oxygenases (19)(20)(21), and cytochromes P450 (22)(23)(24). A critical unknown for all three enzyme paradigms is their structure-activity relationship on the various G-and S-type lignin-derived compounds, which exhibit…”

Microbial hosts for metabolic engineering of lignin bioconversion to renewable chemicals

“…Efforts have been made to improve methods for making gene deletions in Amycolatopsis via the use of suicide plasmids and homologous recombination. Meyer et al improved off target integration by redesigning the vector, and improved the screening for second cross events by use of rpsL from Saccharopolyspora erythraea, which confers sensitivity to streptomycin in strains that already have resistance [114]. Barton et al used the introduction of β-glucuronidase to produce a blue-white screen that could be used for the detection of integration and second crossovers, without the need of a strain that was resistant to streptomycin.…”

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

“…remains difficult as many of the established methods and tools fail in genetic manipulation of these strains ( Figure 1 ). A new suicide vector p6SUI5ERPSL was constructed and optimized by Meyer et al [ 206 ] to facilitate the deletion of genes in Amycolatopsis sp. The backbone of p6SUI5ERPSL includes an apramycin resistance gene ( aacC ), pBR origin of replication for E. coli ( oriV ) and the erythromycin promoter ( ermE *p) upstream of the gene coding for 30S ribosomal subunit protein S12 from Saccharopolyspora erythraea ( rpsL ).…”