Stefan Haefner

Biotechnological production and applications of phytases

Phytases decompose phytate, which is the primary storage form of phosphate in plants. More than 10 years ago, the first commercial phytase product became available on the market. It offered to help farmers reduce phosphorus excretion of monogastric animals by replacing inorganic phosphates by microbial phytase in the animal diet. Phytase application can reduce phosphorus excretion by up to 50%, a feat that would contribute significantly toward environmental protection. Furthermore, phytase supplementation leads to improved availability of minerals and trace elements. In addition to its major application in animal nutrition, phytase is also used for processing of human food. Research in this field focuses on better mineral absorption and technical improvement of food processing. All commercial phytase preparations contain microbial enzymes produced by fermentation. A wide variety of phytases were discovered and characterized in the last 10 years. Initial steps to produce phytase in transgenic plants were also undertaken. A crucial role for its commercial success relates to the formulation of the enzyme solution delivered from fermentation. For liquid enzyme products, a long shelf life is achieved by the addition of stabilizing agents. More comfortable for many customers is the use of dry enzyme preparations. Different formulation technologies are used to produce enzyme powders that retain enzyme activity, are stable in application, resistant against high temperatures, dust-free, and easy to handle.

Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen

Gram-negative, coccoid, non-motile bacteria that are catalase-, urease- and indole-negative, facultatively anaerobic and oxidase-positive were isolated from the bovine rumen using an improved selective medium for members of the Pasteurellaceae. All strains produced significant amounts of succinic acid under anaerobic conditions with glucose as substrate. Phenotypic characterization and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, infB and recN genes were performed on seven independent isolates. All four genes showed high sequence similarity to their counterparts in the genome sequence of the patent strain MBEL55E, but less than 95 % 16S rRNA gene sequence similarity to any other species of the Pasteurellaceae. Genetically these strains form a very homogeneous group in individual as well as combined phylogenetic trees, clearly separated from other genera of the family from which they can also be separated based on phenotypic markers. Genome relatedness as deduced from the recN gene showed high interspecies similarities, but again low similarity to any of the established genera of the family. No toxicity towards bovine, human or fish cells was observed and no RTX toxin genes were detected in members of the new taxon. Based on phylogenetic clustering in the MLSA analysis, the low genetic similarity to other genera and the phenotypic distinction, we suggest to classify these bovine rumen isolates as Basfia succiniciproducens gen. nov., sp. nov. The type strain is JF4016(T) (=DSM 22022(T) =CCUG 57335(T)).

Systems‐wide analysis and engineering of metabolic pathway fluxes in bio‐succinate producing Basfia succiniciproducens

Basfia succiniciproducens has been recently isolated as novel producer for succinate, an important platform chemical. In batch culture, the wild type exhibited a high natural yield of 0.75 mol succinate (mol glucose)−1. Systems‐wide 13C metabolic flux analysis identified undesired fluxes through pyruvate‐formate lyase (PflD) and lactate dehydrogenase (LdhA). The double deletion strain B. succiniciproducens ΔldhA ΔpflD revealed a 45% improved product yield of 1.08 mol mol−1. In addition, metabolic flux analysis unraveled the parallel in vivo activity of the oxidative and reductive branch of the TCA cycle in B. succiniciproducens, whereby the oxidative part mainly served for anabolism. The wild type re‐directed surplus NADH via a cycle involving malic enzyme or via transhydrogenase, respectively, to supply NADPH for anabolism, because the fluxes through the oxidative PPP and isocitrate dehydrogenase, that also provide this cofactor, were not sufficient. This was not observed for the deletion mutants, B. succiniciproducens ΔpflD and ΔldhA ΔpflD, where PPP and isocitrate dehydrogenase flux alone matched with the reduced anabolic NADPH demand. The integration of the production performance into the theoretical flux space, computed by elementary flux mode analysis, revealed that B. succiniciproducens ΔldhA ΔpflD reached 62% of the theoretical maximum yield. Biotechnol. Bioeng. 2013;110: 3013–3023.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite d,l-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of l-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using 13C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol-1 and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71gL-1 succinate and a yield of 2.5molmol-1 from sucrose.

Improved riboflavin production with Ashbya gossypii from vegetable oil based on 13 C metabolic network analysis with combined labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR

The fungus Ashbya gossypii is an important industrial producer of riboflavin, i.e. vitamin B2. In order to meet the constantly increasing demands for improved production processes, it appears essential to better understand the underlying metabolic pathways of the vitamin. Here, we used a highly sophisticated set-up of parallel 13C tracer studies with labeling analysis by GC/MS, LC/MS, 1D, and 2D NMR to resolve carbon fluxes in the overproducing strain A. gossypii B2 during growth and subsequent riboflavin production from vegetable oil as carbon source, yeast extract, and supplemented glycine. The studies provided a detailed picture of the underlying metabolism. Glycine was exclusively used as carbon-two donor of the vitamins pyrimidine ring, which is part of its isoalloxazine ring structure, but did not contribute to the carbon-one metabolism due to the proven absence of a functional glycine cleavage system. The pools of serine and glycine were closely connected due to a highly reversible serine hydroxymethyltransferase. Transmembrane formate flux simulations revealed that the one-carbon metabolism displayed a severe bottleneck during initial riboflavin production, which was overcome in later phases of the cultivation by intrinsic formate accumulation. The transiently limiting carbon-one pool was successfully replenished by time-resolved feeding of small amounts of formate and serine, respectively. This increased the intracellular availability of glycine, serine, and formate and resulted in a final riboflavin titer increase of 45%.

Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the techniques utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzymes specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.