Claudia Schmidt-Dannert

Molecular breeding of carotenoid biosynthetic pathways.

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3,4,3′,4′-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.

Metabolic engineering towards biotechnological production of carotenoids in microorganisms

Abstract. Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.

Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties

An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha. Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E. coli DH5alpha. A subclone with a 2.2 kb insert was sequenced. The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa. As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases. The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E. coli DH5alpha. The expressed lipase was isolated and purified 312-fold to homogeneity. N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E. coli DH5alpha. Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates. The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents.

Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus

By screening of 15 thermophilic Bacillus strains, five strains exhibiting lipase activity were found. Among these the strain Bacillus thermocatenulatus (DSM 730) produced the highest lipase activity. The lipase proved to be inducible and extracellular and was purified 67-fold to homogenous state by hexane extraction, methanol precipitation and ion-exchange chromatography on Q-Sepharose. The molecular weight of the lipase determined by SDS-PAGE is 16 kDa. However, the lipase forms very large aggregates (> 750 kDa) as observed after native PAGE, which makes handling of the lipase very difficult. The lipase binds almost irreversibly on different chromatography matrices, e.g., Amberlite and Serolite, and is very stable in the immobilised form. The N-terminal sequence consists of 53% apolar amino acids and shows no significant homology towards other known lipase sequences. Maximum activity was found at pH 7.5-8.0 and 60-70 degrees C with pNPP and olive oil as substrates.

Directed evolution of industrial enzymes

Directed evolution has emerged in just a few years as one of the most effective approaches to adapting biocatalysts to the performance requirements of industrial and medical applications. Directed evolution mimics the processes of Darwinian evolution in a test tube, combining random mutagenesis and/or recombination with screening or selection for enzyme variants that have the desired properties1. A recent conference* that convened practitioners from industry and academia surveyed current efforts and highlighted the rapid progress the field has enjoyed. In addition to impressive demonstrations of enzyme engineering, there were a variety of challenging problems and approaches to laboratory evolution.

Thermoalkalophilic lipase of Bacillus thermocatenulatus large-scale production, purification and properties: aggregation behaviour and its effect on activity.

Escherichia coli BL321 was transformed with the expression plasmid pCYTEXP1 carrying the BTL2 gene from Bacillus thermocatenulatus under the control of the strong temperature-inducible lambda pL promoter and was cultivated in a 100 1 bioreactor. The mature lipase was produced in large quantities (54,000 U g-1 wet cells) and further purified to homogeneity by a two-step purification protocol (hydrophobic chromatography and gel filtration chromatography). The pure enzyme was characterized and its physicochemical properties compared to those of the BTL2 lipase which had previously been weakly expressed in E. coli under the control of its native promoter on pUC18, yielding 600 U g-1 wet cells. The specific activity of the overexpressed enzyme was approx. 5-fold higher than that of the weakly expressed enzyme. The two proteins showed the same pI and N-terminal sequence and had very similar thermostability, pH stability, optimum pH and temperature activity, and substrate specificity. Both enzymes were extremely stable in the presence of several organic solvents and detergents. With trioleylglycerol as a substrate, the overexpressed lipase cleaves each of the three ester bonds. The purified BTL2 lipase shows a strong tendency to aggregate. Direct evidence for changes in the aggregation state was obtained by gel filtration chromatography. The effect of aggregation on lipase activity was strongly dependent on both substrate and temperature during the assay. Under certain conditions, a direct relationship was found between the molecular mass of the lipase aggregates and the increase in activity upon the addition of 1% (w/v) sodium cholate.

Multi-enzymatic synthesis

Biocatalytic conversions can involve one enzyme that carries out one specific reaction at a time, or multiple enzymes that carry out a series of conversions to yield a desired product. The use of several enzymes allows the realization of much more complex synthetic schemes. Multi-step synthesis can be carried out in biological systems by utilizing or engineering their metabolic networks for catalysis. Alternatively, multi-enzymatic catalysis can be carried out in vitro using isolated biocatalysts. Both approaches, in vivo or in vitro, have their specific advantages, problems, and challenges that will be illustrated using recent examples.

Total synthesis and functional overexpression of a candida rugosa lip1 gene coding for a major industrial lipase

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip1 gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site‐directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pasto is cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lip1 of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.

Biosynthesis of plant-specific stilbene polyketides in metabolically engineered Escherichia coli

BackgroundPhenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells.ResultsA pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure.ConclusionPhenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Exploring Recombinant Flavonoid Biosynthesis in Metabolically Engineered Escherichia coli

Flavonoids are important plant‐specific secondary metabolites synthesized from 4‐coumaroyl coenzyme A (CoA), derived from the general phenylpropanoid pathway, and three malonyl‐CoAs. The synthesis involves a plant type III polyketide synthase, chalcone synthase. We report the cloning and coexpression in Escherichia coli of phenylalanine ammonia lyase, cinnamate‐4‐hydroxylase, 4‐coumarate:CoA ligase, and chalcone synthase from the model plant Arabidopsis thaliana. Simultaneous expression of all four genes resulted in a blockage after the first enzymatic step caused by the presence of nonfunctional cinnamate‐4‐hydroxylase. To overcome this problem we fed exogenous 4‐coumaric acid to induced cultures. We observed high‐level production of the flavanone naringenin as a result. We were also able to produce phloretin by feeding cultures with 3‐(4‐hydroxyphenyl)propionic acid. Feeding with ferulic or caffeic acid did not yield the corresponding flavanones. We have also cloned and partially characterized a new tyrosine ammonia lyase from Rhodobacter sphaeroides. Tyrosine ammonia lyase was substituted for phenylalanine ammonia lyase and cinnamate‐4‐hydroxylase in our E. coli clones and three different growth media were tested. After 48 h induction, high‐level production (20.8 mg L−1) of naringenin in metabolically engineered E. coli was observed for the first time.