Improving Biocatalyst Performance by Integrating Statistical Methods into Protein Engineering

Directed evolution and rational design were used to generate active variants of toluene-4-monooxygenase (T4MO) on 2-phenylethanol (PEA), with the aim of producing hydroxytyrosol, a potent antioxidant. Due to the complexity of the enzymatic system—four proteins encoded by six genes—mutagenesis is labor-intensive and time-consuming. Therefore, the statistical model of Nov and Wein (J. Comput. Biol. 12:247-282) was used to reduce the number of variants produced and evaluated in a lab. From an initial data set of 24 variants, with mutations at nine positions, seven double or triple mutants were identified through statistical analysis. The average activity of these mutants was 4.6-fold higher than the average activity of the initial data set. In an attempt to further improve the enzyme activity to obtain PEA hydroxylation, a second round of statistical analysis was performed. Nine variants were considered, with 3, 4, and 5 point mutations. The average activity of the variants obtained in the second statistical round was 1.6-fold higher than in the first round and 7.3-fold higher than that of the initial data set. The best variant discovered, TmoA I100A E214G D285Q, exhibited an initial oxidation rate of 4.4 ± 0.3 nmol/min/mg protein, which is 190-fold higher than the rate obtained by the wild type. This rate was also 2.6-fold higher than the activity of the wild type on the natural substrate toluene. By considering only 16 preselected mutants (out of ∼13,000 possible combinations), a highly active variant was discovered with minimum time and effort.Enzymes are remarkable biocatalysts that perform numerous chemical reactions. They have evolved in nature to do their task in an efficient and specific way, mostly under aqueous physiological conditions (12). However, the term “biocatalysis” refers to the use of enzymes as process catalysts under artificial conditions, and a major challenge today is to render biocatalysts suitable for the tough reaction conditions of an industrial process (11).A widely used approach for improving enzyme function is directed evolution, whereby protein sequences are repeatedly selected, mutated, or recombined in a process that mimics natural evolution to produce better and better “generations” of protein variants. Directed evolution has been successfully used in numerous studies, but since it requires generation, purification, and screening of large numbers of variants, it is typically expensive and labor-intensive (28, 35). An alternative to directed evolution is an approach termed rational design, whereby predictions are made as to how mutations in a protein will affect its structure and hence its interaction with the target molecule. Unfortunately, both the sequence-structure and the structure-activity relationships are extremely intricate, and while this approach proved to be fruitful in some cases, its practical use is still limited (15). The rational-design approach also requires knowledge of the three-dimensional structure of the protein, which, unlike with the protein''s sequence, is costly and time-consuming to decipher. It has been suggested lately that a combination of both methods may be the best tactic to obtain enzymes with desired activities and selectivities (15, 23).Yet a third approach for protein improvement, which is less often used, is based on statistical analysis. According to this approach, the activity of any protein variant is viewed as a random quantity, and statistical methods are used to predict from activity data which mutation combinations are likely to improve activity. The statistical approach does not require structural knowledge about the protein at hand and allows one to focus screening efforts on a few promising variants, thus reducing labor, time, and expenses. Earlier statistical models for the sequence-activity relationship include Kauffman''s NK model (14) and the “rough Mt. Fuji” model of Aita and Husimi (1). More recently, Fox et al. combined a machine learning technique termed ProSAR with directed evolution and rational design to significantly increase the catalytic function of a halohydrin dehalogenase in the production of the cholesterol-lowering drug atorvastatin (Lipitor) (9, 10). Liao et al. (18) employed eight machine learning algorithms to improve 20-fold the ability of proteinase K to hydrolyze a tetrapeptide substrate.The statistical model that lies at the center of this work is that of Nov and Wein (22). Unlike the statistical algorithms used in the work of Fox et al. (9, 10) and Liao et al. (18), which are generic methods from the machine learning literature, this model was devised specifically for the protein design problem to capture characteristics of the protein sequence-activity relationship. Barak et al. (3) employed a variation of this model in conjunction with directed evolution to greatly improve the oxidoreductase ChrR in reducing chromate and uranyl.In this work, we combine all three approaches—directed evolution, rational design, and statistical methods—to improve the capacity of toluene 4-monooxygenase (T4MO) to produce an important antioxidant, hydroxytyrosol (6). This phenol, which is naturally present in olives and olive oil, has the highest free radical scavenging capacity and has been shown to be beneficial in preventing various diseases, such as diabetes, atherosclerosis, and cancer (13, 19, 34). Developing a biotechnological process for the synthesis of this antioxidant is of interest to the food and cosmetics industries.T4MO from Pseudomonas mendocina KR1 is a soluble four-component enzyme belonging to the toluene monooxygenase family. T4MO is composed of six genes, designated tmoABCDEF, which are essential for the efficient catalysis and high regiospecificity of the enzyme. Genes tmoA, tmoB, and tmoE encode the α, β, and γ subunits, respectively, that comprise the (αβγ)₂ quaternary structure of the 212-kDa hydroxylase component. The α hydroxylase subunit contains the catalytically active diiron center (2, 8). The tmoC, tmoD, and tmoF genes encode the 12.5-kDa Rieske-type [2Fe-2S] ferredoxin, the 11.6-kDa effector protein, and the 36-kDa NADH oxidoreductase, respectively (2, 16, 21).Previously, a 35-fold improvement in T4MO activity on 2-phenylethanol (PEA) for the synthesis of hydroxytyrosol was reported for the TmoA I100A mutant (5). The goal of the present work was to further generate a better T4MO variant for the production of hydroxytyrosol. As the cloning steps associated with producing double and triple mutants of this enzyme are very laborious and time-consuming (e.g., QuikChange mutagenesis cannot be applied due to the large plasmid involved [9 kb], and a three-step PCR is needed for each mutant [5, 6]), the integration of the Nov and Wein statistical model was evaluated.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Temporal dynamics of mating and predation in mosquito swarms

We determined the numbers of copulations and predatory attacks in swarms ofAnopheles freeborni (Diptera: Culicidae), and the distribution of these events throughout the duration of the swarming period each day. On 19 evenings of observation, we recorded 2724 copulating pairs leaving swarms and 1351 dragonfly (Pantala hymenaea andErythemis collocata) attacks. Mating activity partially coincided with predator activity. Most copulations occurred between 10 and 20 min after the swarms formed, while predation events were most frequent during the initial 15 min of the swarm. We calculated the ratio of copulations to predatory attacks during the swarming period. This ratio was significantly higher in an area sheltered by trees than it was in the open. We suggest that physiological and ecological constraints other than predation operate on the mating system of this anopheline to affect the timing of swarm initiation and swarm site selection.     

Effects of vasopressin-mastoparan chimeric peptides on insulin release and G-protein activity.

Two chimeric peptides, consisting of the linear vasopressin receptor V1 antagonist PhAc-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr, in the N-terminus and mastoparan in the C-terminus connected directly (M375) or via 6-aminohexanoic acid (M391), have been synthesised. At 10 microM concentration, these novel peptides increased insulin secretion from isolated rat pancreatic islet cells 18-26-fold at 3.3 mM glucose and 3.5-5-fold at 16.7 mM glucose. PTX pretreatment of the islets decreased the peptide-induced insulin release. M375 and M391 bind to V1a vasopressin receptors with affinities lower than the unmodified vasopressin antagonist, but with K(D) values of 3.76 nM and 9.02 nM, respectively, both chimeras are high affinity ligands. The GTPase activity and GTPgammaS binding in the presence of these peptides has been characterised in Rin m5F cells. Comparison of the influence of the peptides M375 and M391 on GTPase activity in native and pertussis toxin-treated cells suggests a selective activation of G alpha(i)/G alpha(o) subunits, combined with a suppression of other GTPases, primarily G alpha(s). However, the GTPgammaS binding data show that the peptides retain some of the activating property even in PTX-treated cell membranes. In conclusion, the conjugation of mastoparan with the V1a receptor antagonists produce peptides with properties different from the parent peptides that could be used to elucidate the role of different G proteins in insulin release.     

Mediterranean Fruit Fly leks: factors affecting male location

1. The mating system of Mediterranean Fruit Flies ( Ceratitis capitata , Diptera: Tephritidae) is based on male leks which form within the canopies of certain trees. In this study the following hypotheses are tested: (a) fly location depends on microclimate and illumination and (b) larger and heavier males occupy preferred locations in leks. Accordingly, systematic quantitative observations of diel three-dimensional (3-D) locations of lekking C. capitata males were performed in field and field cage studies.
2. Fly locations were found to vary significantly during the activity hours.
3. Medflies were generally found calling from highest and most exterior locations during early morning and late afternoon hours. During the hottest hours (1200–1500) flies occupied lower locations within the tree canopy, and interior locations from 1000 to 1500.
4. Fly location (from 1000 to 1700) was correlated with the azimuth of the sun. However, the mean azimuth range of fly location was limited to 85° (59·45°–143·94°).
5. Both in the field and in the field cage the temperature, relative humidity and light intensity beneath the leaves on which the males perched were closer to the microclimate beneath fully shaded leaves than to microclimates beneath leaves exposed to direct sunlight.
6. It is concluded that male medflies occupy locations which confer suitable microclimates for calling and copulating, and suitable 3-D locations within the canopy which provide protection from predators, wind, direct sunlight and water loss.     

Herders of Indian and European cattle share their predominant allele for lactase persistence

Milk consumption and lactose digestion after weaning are exclusively human traits made possible by the continued production of the enzyme lactase in adulthood. Multiple independent mutations in a 100-bp region--part of an enhancer--approximately 14-kb upstream of the LCT gene are associated with this trait in Europeans and pastoralists from Saudi Arabia and Africa. However, a single mutation of purported western Eurasian origin accounts for much of observed lactase persistence outside Africa. Given the high levels of present-day milk consumption in India, together with archaeological and genetic evidence for the independent domestication of cattle in the Indus valley roughly 7,000 years ago, we sought to determine whether lactase persistence has evolved independently in the subcontinent. Here, we present the results of the first comprehensive survey of the LCT enhancer region in south Asia. Having genotyped 2,284 DNA samples from across the Indian subcontinent, we find that the previously described west Eurasian -13910 C>T mutation accounts for nearly all the genetic variation we observed in the 400- to 700-bp LCT regulatory region that we sequenced. Geography is a significant predictor of -13910*T allele frequency, and consistent with other genomic loci, its distribution in India follows a general northwest to southeast declining pattern, although frequencies among certain neighboring populations vary substantially. We confirm that the mutation is identical by descent to the European allele and is associated with the same>1 Mb extended haplotype in both populations.     

Pharmacologic modulation of RNA splicing enhances anti-tumor immunity

1. Download : Download high-res image (217KB)
2. Download : Download full-size image