Jan Marienhagen

Metabolic engineering of microorganisms for the synthesis of plant natural products.

Of more than 200,000 plant natural products known to date, many demonstrate important pharmacological activities or are of biotechnological significance. However, isolation from natural sources is usually limited by low abundance and environmental, seasonal as well as regional variation, whereas total chemical synthesis is typically commercially unfeasible considering the complex structures of most plant natural products. With advances in DNA sequencing and recombinant DNA technology many of the biosynthetic pathways responsible for the production of these valuable compounds have been elucidated, offering the opportunity of a functional integration of biosynthetic pathways in suitable microorganisms. This approach offers promise to provide sufficient quantities of the desired plant natural products from inexpensive renewable resources. This review covers recent advancements in the metabolic engineering of microorganisms for the production of plant natural products such as isoprenoids, phenylpropanoids and alkaloids, and highlights general approaches and strategies to gain access to the rich biochemical diversity of plants by employing the biosynthetic power of microorganisms.

Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate l-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause l-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different l-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques.

Looking for the pick of the bunch: high-throughput screening of producing microorganisms with biosensors

The engineering of microbial strains for the production of small molecules of biotechnological interest is a time-consuming, laborious and expensive process. This can be mostly attributed to the fact that good producers cannot be readily obtained by high-throughput screening approaches since increased product formation usually does not confer a clear phenotype to producing strain variants. Recently, advances were made in the design and construction of genetically encoded RNA aptamer-based or transcription factor-based biosensors for detecting small molecules at the single-cell level. The first promising examples for the application of these molecular biosensors in combination with fluorescent-activated cell sorting as a high-throughput screening device demonstrated the value and potential of these new tools for microbial strain development.

Novel screening methods--biosensors.

Biosensors offer exciting possibilities for improving cells or enzymes as biocatalysts for the synthesis of small molecules. We here review recent progress in the development and the screening applications of transcription-factor-based biosensors. An example is a cofactor-dependent biosensor which provides a generalizable screen for NADPH-dependent enzymes. Another example is the use of a biosensor in combination with recombineering for strain development, thereby expanding the genome engineering techniques to deliver directly bacteria producing small molecules of interest. Biosensor-based techniques in combination with fluorescence-activated cell sorting demonstrate that the gap regarding throughput capabilities of existing methods for the generation of genetic diversity and methods for the subsequent screening can be closed.

OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons

Focused mutant library generation methods have been developed to improve mainly “localizable” enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

Metabolic Function of Corynebacterium glutamicum Aminotransferases AlaT and AvtA and Impact on l-Valine Production

ABSTRACT Aminotransferases (ATs) interacting with l-alanine are the least studied bacterial ATs. Whereas AlaT converts pyruvate to l-alanine in a glutamate-dependent reaction, AvtA is able to convert pyruvate to l-alanine in an l-valine-dependent manner. We show here that the wild type of Corynebacterium glutamicum with a deletion of either of the corresponding genes does not exhibit an explicit growth deficiency. However, a double mutant was auxotrophic for l-alanine, showing that both ATs can provide l-alanine and that they are the only ATs involved. Kinetic studies with isolated enzymes demonstrate that the catalytic efficiency, kcat/Km, of AlaT is higher than 1 order of magnitude in the direction of l-alanine formation (3.5 × 104 M−1 s−1), but no preference was apparent for AvtA, suggesting that AlaT is the principal l-alanine-supplying enzyme. This is in line with the cytosolic l-alanine concentration, which is reduced in the exponential growth phase from 95 mM to 18 mM by a deletion of alaT, whereas avtA deletion decreases the l-alanine concentration only to 76 mM. The combined data show that the presence of both ATs has subtle but obvious consequences on balancing intracellular amino acid pools in the wild type. The consequences are more obvious in an l-valine production strain where a high intracellular drain-off of the l-alanine precursor pyruvate prevails. We therefore used deletion of alaT to successfully reduce the contaminating l-alanine in extracellular accumulated l-valine by 80%.

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35mg/L naringenin and 37mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.

Stereoselective epoxidation of the last double bond of polyunsaturated fatty acids by human cytochromes P450.

Cytochromes P450 (CYPs) metabolize polyunsaturated long-chain fatty acids (PUFA-LC) to several classes of oxygenated metabolites. Through use of human recombinant CYPs, we recently showed that CYP1A1, -2C19, -2D6, -2E1, and -3A4 are mainly hydroxylases, whereas CYP1A2, -2C8, -2C9, and -2J2 are mainly epoxygenases of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. It is worth noting that the last double bond of these PUFAs, i.e., ω6 in AA or ω3 in EPA and DHA, respectively, was preferentially epoxidized. In this study, we have characterized the stereoselectivity of this epoxidation reaction by comparison with the PUFA-LC epoxide stereoisomers obtained from the enantioselective bacterial CYP102A1 F87V. The stereoselectivity of the epoxidation of the last olefin of AA (ω6), EPA (ω3), or DHA (ω3) differed between the CYP isoforms but was similar for EPA and DHA. These data give additional insight into the PUFA-LC epoxide enantiomers generated by the hepatic CYPs.

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

ABSTRACT Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum Δald ΔadhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.